Development of new methodologies for the mass spectrometry study of bioorganic macromolecules

DEVELOPMENT OF NEW METHODOLOGIES FOR THE MASSSPECTROMETRY STUDY OF BIOORGANIC MACROMOLECULES

Simone Cristoni* and Luigi Rossi BernardiUniversita degli Studi di Milano, Centro Interdisciplinare StudiBio-molecolari e Applicazioni Industriali CISI, Via Fratelli Cervi 93, 20090Segrate Milano, Italy

Received 3 March 2003; accepted 20 May 2003

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370

II. Techniques That Are Usually Employed in the Study of Bioorganic Macromolecules . . . . . . . . . . . . . . . . . . . . . 371A. Proteins and Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371B. Oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374C. Oligosaccharides and Glycoconjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375D. Various Complexes (DNA–Protein, Antigen–Antibody, Macromolecules–Metals) . . . . . . . . . . . . . . . . . . . . 377

III. Common Problems and Developments in the Analysis of Bioorganic Macromolecules by Mass Spectrometry . . . . 377A. Ionization Source Problems and Developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377

1. Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3772. Problems with Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3793. Multicharge Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381

B. Mass Analyzer Problems and Developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3811. Linear Dynamic Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3812. Tandem Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3823. Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3824. Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3835. Mass Accuracy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383

IV. New Promising Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384A. Interfaces and Ionization Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384

1. Improvements in the Coupling of Liquid-Phase Separation Systems to Mass Spectrometry . . . . . . . . . . . 3842. AP-MALDI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3843. Deprotonant Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3854. Sonic Spray Ionization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3885. APCI without Corona Discharge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391

B. Mass Analyzers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3931. Linear Quadrupole Ion Trap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3942. TOF Analyzers with New Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3953. Multiple Mass Analyzers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396

V. Software for Data Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397

VI. Conclusions and Future Developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400

In recent years, mass spectrometry has been increasingly usedfor the analysis of various macromolecules of biological, bio-medical, and biochemical interest. This increase has been made

possible by two key developments: the advent of electrosprayionization (ESI) and matrix-assisted laser desorption ionization(MALDI) sources. The two new techniques produce a signi-ficant increase in mass range and in sensitivity that led to thedevelopment of new applications and of new analyzer designs,software, and robotics. This review, apart from the descriptionof the status of mass spectrometry in the analysis of bioorganic

Mass Spectrometry Reviews, 2003, 22, 369– 406# 2003 by Wiley Periodicals, Inc.

————*Correspondence to: Dr. Simone Cristoni, Universita degli studi di

Milano (CISI), Via Fratelli Cervi 93, 20090 Segrate, Milano, Italy.

E-mail: [email protected]

macromolecules, is mainly devoted to the illustration of themore recent promising techniques and on their possible futureevolution. # 2003 Wiley Periodicals, Inc., Mass Spec Rev22:369–406, 2003; Published online in Wiley InterScience(www.interscience.wiley.com).DOI 10.1002/mas.10062Keywords: macromolecules; mass spectrometry; bioorganicmacromolecules; ESI; MALDI; no-discharge APCI; SSI

I. INTRODUCTION

A mass spectrometric system is mainly characterized byits ion source, analyzer, and sample injection system.With suitable choices of those elements, mass spectro-metry has become a very powerful tool for the analysis ofbioorganic macromolecules mainly due to the develop-ment of new ionization methods; i.e., fast atom bombard-ment (FAB) (Barber et al., 1981; Morris et al., 1981;Nichols & McMeekin, 2002), electrospray ionization (ESI)(Whitehouse et al., 1985), and matrix-assisted laserdesorption ionization (MALDI) (Karas & Hillekamp,1988). Those methods have been widely described in theliterature (Li et al., 1999; Basile et al., 2001; Cunsolo et al.,2001; Cozzolino et al., 2001; Fierens et al., 2001a,b;Galvani, Hamdan, & Rigetti, 2001; Madonna et al.,2001; Ogorzalek Loo et al., 2001; Wall et al., 2001), and

their mechanisms have been amply discussed; thus, wereport here only on the main points of their analyticalpower.

MALDI uses a pulsed laser in order to ionize thesample that has been cocrystallized with matrix molecules.The ions obtained are usually analyzed by a time-of-flightanalyzer. Recently, interesting results have been achievedwith ion traps (either FTMS systems or Paul ion traps).Using this method, mainly low-charge (usually singlycharged) ions are produced. For example, Figure 1(Onnerfjord et al., 1998) shows a typical MALDI spectrumof a protein mixture that contained cytochrome c,ribonuclease A, lysosyme, and myoglobin. As can be seen,for all of the analyzed proteins the singly charged ion isclearly detected. It must be emphasized that, in order toincrease the resolution and mass accuracy of the MALDI-TOF instrument, two techniques—named delayed extrac-tion (DE) (Takach et al., 1997) and reflectron (Mamyrin,1994; Fancher, Woods, & Cotter, 2000)—are usuallyemployed. An interesting phenomenon observed withMALDI is the unimolecular metastable decomposition ofmolecular ions during their flight through the analyzer.That phenomenon can be used to obtain structural detailsby collecting data with the TOF analyzer in the reflectronmode. That technique has been named post-source decay(PSD) (Kaufmann, Spengler, & Lutzenkirchen, 1993;Hellman & Bhikhabhai, 2002).

FIGURE 1. MALDI mass spectrum of a protein mixture containing cytochrome c, ribonuclease A,

lysozime, and myoglobin. (Reproduced from Onnerfjord et al. (1998) with permission from American

Chemical Society, copyright 1998.)

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The ESI source operates, coupled with various in-troduction systems [e.g., syringe, liquid chromatography(LC), capillary electrophoresis (CE)] to spray the samplesolution. In that case, multi-charged ion distributions areusually produced in the 100–2000 Th range for proteinmolecules. Figure 2 (Grandori, 2002) shows some typicalmulti-charge spectra of Cytochrome C obtained with anESI ionization source by varying the H2O/CH3OH molarratio; the multi-charge distribution changes with the con-centration of organic solvent.

That effect can be reasonably explained by the fact thatthe solvent induces protein denaturation, and that a highnumber of basic sites become exposed to the solvent andare thus protonated. The ESI source can be coupled withvarious mass analyzers [e.g., ion trap, TOF, quadrupole,triple quadrupole (Q1Q2Q3), FT-ICR]. The ESI-ion trap(ESI-IT) systems are widely employed in the analysis ofbiological macromolecules. Despite their low-mass accu-racy, due either to the operating conditions of the ion trap orto possible space-charge effects, they can perform MS/MS and MSn experiments, leading to detailed structuralinformation. This information makes it possible, forexample, to obtain the protein sequence by first digestingthe protein of interest with an enzyme (usually trypsin) andobtain the sequence of the peptides with the MS/MSapproach. Another non-negligible positive attribute of theESI-IT instruments is their relatively low cost. The ESI-FTICR instruments can provide high mass accuracy andresolution, which are strongly dependent on the intensity ofthe magnetic field that is applied to produce the cyclotronion motion. It can also use various excitation methods inorder to fragment selected ions [sustained off-resonanceirradiation (SORI) (Cody & Freiser, 1982; Mirgorodskaya,O’Connor, & Costello, 2002), infrared multi-photondissociation (IRMPD) (Little et al., 1994; Flora &Muddiman, 2001), electron-capture dissociation (ECD)(Zubarev, Kelleher, & McLafferty, 1998; Horn, Zubarev,& McLafferty, 2000)]. Those techniques, described indetail by Williams (1998), have been successfully used todecompose high-mass ions inside the cyclotron cell. In thatcase, the fragmentation process of multi-charged ions ofbioorganic macromolecules created by the ESI process canbe studied. In fact, it is possible to determine the chargestate of the different ions by analyzing their isotopicdistribution. Figure 3 (Horn, Zubarev, & McLafferty, 2000)shows a typical isotopic distribution obtained by using theECD technique on the 15þ ion of Cytochrome C, and bymonitoring the isotopic distribution of the ions in the rangem/z 987–995. The isotopic distribution is clearly dis-played, and allows the unequivocal identification of thecharge state (value of z), and thus of the related molecularmass. Another interesting approach to analyze bioorganicmacromolecules is ESI-TOF (Krutchinsky et al., 1998;Whitehouse, Dresch, & Andrien, 2000; Hang, Lewis, &

Majidi, 2003). In the past, it has not been possible to usecontinuous sources, such as ESI, and pulsed sources, suchas MALDI, in the same instrument, which would havesignificant advantages. The use of orthogonal injection,with or without collision damping, leads to compatibilitywith the short pulse requirements of TOF (Krutchinskyet al., 1998). In that technique, a continuous flow of ions(either from a static source or from a flowing system suchas capillary electrophoresis) is gently accelerated in onedirection, and results in a densely packed, but slowlymoving, analyte ion stream. A second acceleration mech-anism that pulses at right angles to this ion stream pushes awell-defined packet of ions toward a TOF mass analyzer.

Even though the commercially available mass spectro-meters are currently very powerful for macromoleculeanalysis, new developments focused on some specificproblems are in continuous progress.

The analytical power of the techniques usuallyemployed to analyze bioorganic macromolecules will bediscussed in this review. Furthermore, the commonproblems and recent developments of the most commonlyused mass spectrometry techniques will also be discussed.Finally, some new promising techniques and their possibleapplications will be disclosed.

II. TECHNIQUES THAT ARE USUALLYEMPLOYED IN THE STUDY OFBIOORGANIC MACROMOLECULES

Various bioorganic macromolecules have been analyzedwith mass spectrometry techniques. Major applicationsdiscussed in the last 2 years include the determination ofbiopolymer structure and of macromolecular assemblies.Proteins have received priority, but progress in other areas,for example in the understanding of interactions within andamong biopolymers, has been very fast. In this section, thetechniques that are usually employed for the analysis ofvarious kinds of macromolecules will be discussed.

A. Proteins and Peptides

A large number of studies have recently appeared in thisfield, with a correspondingly large literature. The ap-proaches usually employed for protein analysis by massspectrometry are mainly concerned with intact protein MSanalysis and with the digestion of the protein before MSanalysis. In the study of intact proteins, the molecular massis obtained with MALDI or ESI (see, e.g., Figs. 1 and 2). Itis possible to combine the data obtained by 2D-GELseparation [on the basis of molecular weight (MW) andisoelectric point (pI)] with the accurate mass values obtain-ed by mass spectrometry. In fact, the mass values obtainedby mass spectrometry are more accurate and precise than

ANALYSIS OF MACROMOLECULES &

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FIGURE 2. Direct infusion ESI multicharge mass spectra of cytochrome c. Methanol-induced partially

folded states. Nano-ESI-MS spectra of 5 mM solution of cytochrome c in water/acetate. (A) pH 3.0, 0%

methanol; (B) pH 2.8, 0% methanol; (C) pH 2.6, 0% methanol; (D) pH 3.0, 25% methanol; (E) pH 2.8, 25%

methanol; (F) pH 2.6, 25% methanol. Peaks are labeled according to the corresponding charge states.

(Reproduced from Grandori (2002) with permission from Protein Science, copyright 2002.)


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those obtained by the 2D-GEL approach or by otherelectrophoretic approaches [isoelectric focusing (IEF) andSDS–PAGE]. Furthermore, in the case of FT-ICR, thehigh resolution available leads to high confidence in thedetermination of the charge state and of the masses ofmolecular and fragment ions. The SORI collision-induceddecomposition technique, when applied to protein multi-charged molecular ions, yields fragments that may be reas-sembled to provide full amino acid sequence information.That method has been called by McLafferty ‘‘top-down’’sequencing (Kelleher et al., 1999). Decompositions inthe FT-ICR cell can also be obtained by IRMPD(Hakansson et al., 2001), electron-capture dissociation(ECD; Hakansson et al., 2001), or blackbody IR radiationdissociation (BIRD; Price, Schnier, & Williams, 1996).

Other interesting studies that have been performed onintact proteins are those devoted to protein conformationinvestigations, as described in the review of Green &Lebrilla (1997). For some proteins, the conformations canbe probed by hydrogen/deuterium exchange studies, basedon the surface availability of exchangeable hydrogenatoms.

Another approach widely employed for proteinidentification and characterization is based on enzymaticdigestion of the protein, followed by MALDI-MS or LC-ESI-MS/MS analysis of the peptides obtained (Chalmers &Gaskell, 2000). An interesting application of that strategy,which illustrates the use of MALDI for protein character-ization, has been recently published by van Montfort group(van Montfort et al., 2002). That paper reports studies of in-gel digestion procedures to generate MALDI-MS peptidemaps of integral membrane proteins. It has been shown thatthe addition of the detergent octyl-b-glucopyranoside(OBG) at 0.1% concentration to the extraction solventincreases the total number of peptides detected, so as tocover at least 85% of the total protein sequence. OBGfacilitates the recovery of hydrophobic peptides when theyare SpeedVac-dried during the extraction procedure.

As emphasized from the early application of MS in theprotein field (Hamdan, Galvani, & Rigetti, 2001), post-translational modifications can be determined by shifts inthe molecular mass of an intact protein. By proteasedigestion followed by MS, it was possible to locate andcharacterize the modified sites. MALDI-MS, ESI-MS, andESI-MS/MS methods are now widely employed for therecognition and location of amino acid mutations in nativeand recombinant proteins and peptides. Recently, someN-terminal peptide derivatization procedures have beendeveloped for subsequent MALDI analysis for the se-quence determination of peptides obtained from the pro-tein by enzymatic tryptic digestion (Keough, Lacey, &Youngquist, 2000). Those sulfonation reactions convertlysine-terminated tryptic peptides into modified peptidesthat are suitable for de novo sequencing by post-source decay matrix-assisted laser desorption/ionization(MALDI) mass spectrometry. In that case, the N-terminalamino acid series is mainly observed and the peptidesequence can be obtained from the N-terminus by the de-termination of the mass difference values among the frag-ment peaks. For instance, Figure 4A–C (Keough, Lacey,& Youngquist, 2000) shows the spectrum of the VGGY-GYGAK peptide obtained with the PSD technique withand without the derivatization approach. In that case, thefragmentation of the peptides without derivatization(Fig. 4A) leads to a complicated spectrum that containsvarious fragment series (a, b, y). The main series that couldbe obtained from peptide fragmentation are summarized inScheme 1 (Cristoni et al., 2002a). It must also be empha-sized that, in this particular case, fragmentation of theunderivatized peptide is observed; however, with otherpeptides very poor fragmentation and little structuralinformation are obtained with the PSD approach withoutderivatization (Keough, Youngquist, & Lacey, 1999).Figure 4B,C show the effect of two derivatization ap-proaches. Particularly in Figure 4C, only one series wasobtained by the N-terminal sulfonation approach, and thatseries leads to the determination of the amino acidsequence. That technique is, however, very expensive anddoes not operate in the same mode for all peptides. The besttechnique to obtain a reproducible and easily interpretablepeptide fragmentation pattern still remains ESI-MS/MS(for that purpose, ion traps are widely employed).Figure 5A–C (Simpson et al., 2000) show the chromato-gram of A33 antigen protein tryptic digest, the MSspectrum of the chromatogram peak at 25.20 min, andthe MS/MS spectrum of the peptide ions at m/z 1198.4,respectively. The MS/MS spectrum was obtained with anion-trap analyzer; two fragmentation series are mainlydetected (y and b, see Scheme 1). Such a spectrum could beeasily used to perform a database search and de novodetermination of sequence in order to identify andcharacterize peptides and proteins.

FIGURE 3. Isotopic distribution obtained by using the electron-capture

dissociation (ECD) technique on the 15þ ion of cytochrome c and by

monitoring the isotopic distribution of the ions in the range 987–995.

(Reproduced from Horn, Zubarev, & McLafferty (2000) with permission

from Elsevier, copyright 2000.)


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B. Oligonucleotides

In the analysis of oligonucleotides, the presence ofabundant phosphate anion groups requires an extensiveuse of negative-ion measurements. Using DE with TOFanalyzers, satisfactory MALDI resolution, sensitivity, andmass accuracy are obtained. Fragmentation pathways thatallow the determination of the oligonucleotide sequencecan be enhanced by the selection of a MALDI matrix to

activate metastable decompositions, and by an adjustmentof the delay time to maximize the fragment-ion yield(Juhasz et al., 1996; Distler & Allison, 2001). IR MALDI(Hillenkamp, 1998) is particularly effective for the MWdetermination of large oligonucleotides. For instance, thespectra of synthetic DNA, of restriction enzyme fragmentsof plasmid DNA, and of RNA transcripts up to 2180 nuc-leotides, have been reported (Berkenkamp, Kirpekar, &Hillenkamp, 1998). The ability to handle large DNA and

FIGURE 4. PSD MALDI mass spectra obtained from VGGYGYGAK peptide. (A) Native peptide, (B)

guanidinated peptide, and (C) sulfonated homoarginine-terminated peptide. The spectra show the influence

of various chemical modifications on the peptide fragmentation patterns (chemical-assisted fragmentation).

The yi* designations indicate that the y-ions are derivatized. (Reproduced from Keough, Lacey, &

Youngquist (2000) with permission from John Wiley & Sons, Ltd., copyright 2000.)


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RNA fragments should facilitate high-throughput screen-ing applications for genotyping and clinical diagnosis.It has also been shown that the PSD technique is effectiveto investigate oligonucleotide fragmentation processes(Chan, Fung, & Li, 2002).

The ESI spectra of oligonucleotides are more difficultto obtain, due to the high number of phosphate negativegroups, which give rise to salt adducts during the ESIionization process. Consequently, it is necessary to modifythat tendency with the production of derivatives that donot allow the formation of salt adducts. An interestingapplication of that type has been recently published byDe Bellis et al. (2000). In their method, a direct-injectionelectrospray ionization mass spectrometry (ESI-MS) tech-nique was used. Aqueous 2-propanol was used as theeluent, with spermidine or triethylamine as DNA modifier.About 200 synthetic oligonucleotides with molecularmasses that ranged from 5 to 15 kDa (17–51mers) wereanalyzed with good results in terms of sensitivity, suppres-sion of sodium adduct formation, and speed of analysis.

A field of particular interest is the analysis of singlenucleotide polymorphisms (SNPs) in which extensiveprotocols that incorporate MALDI-TOF have been per-formed (Pusch et al., 2002; Ding & Cantor, 2003). Forinstance, Ding & Cantor (2003) have reported an approachfor gene expression analysis by combining competitivePCR and MALDI time-of-flight MS. A DNA standard wasdesigned with an artificial SNP in the gene of interest. Thestandard was added to the reverse transcription productbefore PCR. Subsequently, a base-extension reaction wascarried out at the SNP position, and the products werequantified by MALDI time-of-flight MS. The approach iscapable of relative and absolute quantification of gene

expression; it is extremely sensitive and highly reprodu-cible. It is so possible to obtain a high-throughput, auto-mated gene expression analysis platform, where a fewhundred genes from 20 to 500 different samples can beaccurately quantified per day. Another recent interestingapplication in that field has been published by Pusch et al.(2002). In that review, the latest developments of MALDItechnology in the gene and SNPs analysis were discussed,and the fact that MALDI-TOF mass spectrometry has thepotential to develop into a ‘‘Gold Standard’’ for high-throughput SNPs genotyping has been shown.

Other interesting papers that describe improvements ofthe ESI technique in the analysis of oligonucleotides havebeen recently published (Berggren, Westphall, & Smith,2002; Deguchi et al., 2002).

C. Oligosaccharides and Glycoconjugates

Oligosaccharides and their conjugates are difficult to anal-yze due to their many possible isomeric forms. Further-more, they contain very labile bonds, and are usuallypresent in nature as complex mixtures of closely relatedcompounds. In the case of glycoproteins, multiple modifiedsites are usually present, and each glycoprotein exhibits adifferent set of glycoforms. MALDI-TOF-MS has beeneffectively employed in the analysis of glycoproteins(Papac et al., 1998; Lapolla, Fedele, & Traldi, 2000). Thegroup of Traldi and Lapolla (Lapolla, Fedele, & Traldi,2000) has applied mass spectrometry techniques to thestudy of advanced protein glycation end-products. TheMALDI-PSD technique has been widely employed forthe study of the structure of oligosaccharides and glyco-conjugates. For instance, in the study of the Yamagakigroup (Yamagaki & Nakanishi, 2001), the native oligosac-charides of lacto-N-neotetraose (Gal beta1-4GlcNAcbeta1-3Gal beta1-4Glc; LNnT) and lacto-N-tetraose (Galbeta1-3GlcNAc beta1-3Gal beta1-4Glc; LNT) were anal-yzed with curved-field reflectron MALDI-TOF-MS.Because a curved-field reflectron TOFMS enables asimultaneous focusing of a wide mass range of metastablefragment ions, the relative ion intensities in the PSD massspectra can be evaluated. The PSD spectra of LNnT andLNT were distinguishable by their relative product ionintensities. In the case of LNT, beta-elimination occurs inthe N-acetyl glucosamine (GlcNAc) residue at the C-3position. The same process was not observed in the case ofLNnT. The 3-O elimination leads to a clear difference inPSD relative ion intensities of LNnT and LNT. In theMALDI-PSD fragmentation, the beta1-3 glycosyl linkagecleaved more easily than the beta1-4 glycosyl linkage.

An interesting approach for the characterization ofglycoproteins is based on protease digestion. That kind ofapproach is particularly relevant because, in order to studythe structure–function relationships of glycoproteins, it is

SCHEME 1. Main series that could be obtained from peptide fragmen-

tation. (Reproduced from Cristoni et al. (2002a) with permission from

John Wiley & Sons, Ltd. copyright 2002.)


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necessary to distinguish the glycoforms present on eachsite. Allmaier’s group has given a good example of thiskind of study (Bacher et al., 2001). Those authors havedescribed a general approach for the detailed characteriza-tion of sodium borohydride-reduced peptidoglycan frag-ments (syn. muropeptides), produced by muramidasedigestion of the purified sacculus isolated from Bacillussubtilis (vegetative cell form of the wild type and a dacAmutant) and Bacillus megaterium (endospore form). Thatmethod is based on MALDI-TOF and nano-electrospray

ionization (nESI) ion-trap mass spectrometry. After enzy-matic digestion and reduction of the resulting muropep-tides, the complex glycopeptide mixture was separatedand fractionated by reversed-phase liquid chromatography.Prior to mass spectrometric analysis, the muropeptidesamples were subjected to a desalting step, and an aliquotwas taken for amino acid analysis. Initial molecular-mass determination of those peptidoglycan fragments(ranging from monomeric to tetrameric muropeptides)was performed by positive- and negative-ion MALDI-MS,

FIGURE 5. ESI-MS/MS spectrum of A33 antigen protein. (A) RP-HPLC/ESI/ITMS total ion-current

profile of tryptic digestion, (B) MS spectrum of the region at 25.20 min, and (C) ESI-MS/MS spectrum of the

peptide ion at m/z 1198.4. (Reproduced from Simpson et al. (2000) with permission from Wiley-VCH,

copyright 2000.)


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using alpha-cyano-4-hydroxycinnamic acid as matrix. Theresults demonstrated that the linear TOF mode is themethod of choice for the fast molecular-mass determina-tion of a large number of samples (in the 0.8–10 pmolrange), providing a high mass accuracy. After that muro-peptide screening, a detailed primary structure analysisis often required, in order to resolve ambiguous data.Structural data could be obtained from peptidoglycanmonomers by PSD fragment ion analysis, but not fromdimers or higher oligomers, due to the low sensitivity.Multi-stage collision-induced dissociation (CID) experi-ments performed on a nESI-IT instrument were found to bethe best method for structural characterization, not only ofmonomeric but also of dimeric and trimeric muropeptides.Up to MS4 experiments were sometimes necessary toobtain unambiguous structural information. All MSn ex-periments were performed on singly charged precursorions, and the MS2 spectra were dominated by fragmentsderived from interglycosidic bond cleavages. The resultsthat have been obtained demonstrate the utility of nESI-ITfor the easy determination of the glycan sequence, thepeptide linkage, and the peptide sequence and branching ofpurified muropeptides (monomeric up to trimeric forms).The wealth of structural information generated by nESI-IT-MSn is unsurpassed by any other technique.

An interesting comparison of the MALDI-TOF andESI FT-ICR MS techniques for the analysis of gangliosideshas been provided by Penn et al. (1997). The ESI and theMALDI spectra were both obtained.

A detailed account of analysis of oligosaccharides andglycoconjugates by MALDI has been published by Harvey(1999).

Other glycoconjugate compounds that have beenstudied by mass spectrometry are glycolipids. An interest-ing review that discussed the mass spectrometric behaviorof those compounds has been published by van der Drift(van der Drift et al., 1998). The mass spectrometrytechniques mainly used for this class of compounds areFAB, MALDI, and ESI combined with LC.

D. Various Complexes (DNA–Protein,Antigen–Antibody, Macromolecules–Metals)

ESI allows the investigation of intermolecular interactions,and thus is highly effective for the study of complexes(Loo, 1997). In that field of analysis, mass spectrometryexhibits specificity, sensitivity, and speed. Macromolecularcomplexes are held together by specific interactions(covalent-, non-covalent, and hydrogen binding interac-tions), based on key structural and/or energetic features. Itmust be emphasized that ESI is a well-suited method toanalyze non-covalent interaction due to the fact that it is asoft ionization method and allows intact weakly boundcomplex to be detected. Small structural changes can

dramatically affect the nature of the complexes. Great careshould be taken to ensure that the complexes are studiedunder biologically significant conditions, and in this con-text the specificity of ESI-MS approach is particularlyvaluable. Speed and sensitivity are the most obviousadvantages of using ESI-MS methods to study macro-molecular complexes. In some cases, pmol-fmol sensitivitywas achieved (Jedrzejewski & Lehmann, 1997). In aninteresting study of macromolecular non-covalent com-plexes, the analysis of the calcium-dependent interaction ofcalmodulin with two amphipathic peptides, calmodulin-dependent protein kinase II and mellitin, was studied bymicro-ESI MS techniques (Veenstra et al., 1998).

It must be emphasized that the number of subunits thatform a unique and biologically relevant complex is animportant parameter that can be determined by the ESI-MStechnique. That parameter is easily obtained from themolecular mass of the complex, which is directly measuredby mass spectrometry. Very large MW complexes can bestudied with that approach. Many enzymes are composedof identical and non-identical subunits that associatetogether to give rise to the active species. It is often neces-sary to study the chemical interactions that maintain thequaternary structures in order to understand the structure-function relationship of the complexes.

The study of the interactions of DNAwith drugs, metalions, and proteins is also important—as shown by Becket al. (2001). In that case, the ESI technique again gives thebest results, due to the gentle nature of the ionizationphenomenon. In that area, the ESI-MS and MS/MS tech-niques have proven to be valuable complements to otherstructural methods (computational modeling, X-ray crys-tallography, and nuclear magnetic resonance spectro-scopy), and offer an advantage in terms of speed,specificity, and sensitivity.

III. COMMON PROBLEMS AND DEVELOPMENTSIN THE ANALYSIS OF BIOORGANICMACROMOLECULES BY MASS SPECTROMETRY

A. Ionization Source Problems and Developments

As discussed above, the ESI and MALDI techniques havesignificantly improved the analysis of bioorganic macro-molecules. On the other hand, they suffer from somelimitations that mainly concern sensitivity, the effect ofthe solution buffer and salts, the limited throughput, andthe ion-charging mechanism.

1. Sensitivity

In the analysis of bioorganic macromolecules, it is oftennecessary to analyze a very small quantity of analytes.


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A typical example is the detection of a protein that has beenexpressed in a tissue or in a selected organism at very lowconcentration. Sensitivity depends on instrumental designand operating parameters. A relevant problem with the ESIsource originates from the presence of solvent and buffermolecules, which, if partially introduced into the massspectrometer, lead to an increase of chemical noise. Inorder to alleviate that problem, the orthogonal ionizationESI source has been recently developed. Figure 6A showsa schematic drawing of an axial injection ESI source,and Figure 6B an orthogonal ionization source. In theorthogonal-injection ionization source, the ESI needle is

orthogonal to the mass-spectrometer entrance, so that alarge amount of neutral molecules is not admitted into themass-analyzer region. A flow of nitrogen is also used toreduce the entrance of neutral molecules into the massanalyzer. Another method to improve the ion-sourceefficiency is to reduce the sample flow. That technique[named micro- (micro flow) or nano- (nano flow) ESI]allows a large improvement in sensitivity. For instance,Quenzer et al. (2001) have shown that nano-LC-ESI-FTICR can achieve attomole detection limits in theanalysis of peptides. For that purpose, three peptides in amixture that totaled 500 amol (10 mL, 50 amol/mL) were

FIGURE 6. Schemes of two different ESI ionization sources: (A) axial injection ESI ionization source and

(B) orthogonal-injection ESI ionization source.


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separated and detected to demonstrate the detection froma mixture at low endogenous biological concentration.Furthermore, Emmett et al. (1998) have shown that micro-ESI permits low-level sample analysis by constant infusionover a wide range of solvent conditions in the positive- andthe negative-ion modes. The system is flexible and allowsrapid conversion to allow routine LC/MS analysis on low-level mixtures presented in biological media. Anotherinteresting study by Bahr et al. (1997) showed that nano-ESI provides good results in term of sensitivity in theanalysis of oligosaccharides. For a series of compounds,ranging from trisaccharides to larger polymers withmolecular masses up to 6 kDa and 10 pmol total sampleload, very intense singly or multiply cationized moleculeions were detected on an ion-trap mass spectrometer. In adilution series, it is exemplified that, at the 10�8 M level,molecule-ion signals can be clearly registered with a S/Nratio of about 7. Only 100 amol of sample had beenconsumed in that experiment. Investigation of an oligo-saccharide-peptide mixture revealed that the oligosacchar-ide signals were suppressed in conventional ESI, whereasin nano-ESI the analytes were clearly detected.

Sensitivity is an important parameter that affects MS/MS determinations because, to isolate and fragment theions, it is necessary to reach a good signal intensity forthe precursor ions. For that reason, new ESI geometriesand new approaches have been developed (Hsieh, 1998;Hutchens & Yip, 2001; Guzzetta, Thakur, & Mylchreest,2002). For example, Guzzetta, Thakur, & Mylchreest(2002) have recently developed a method based on the useof a 60 mm internal diameter stainless-steel needle. It wasadapted to the orthogonal ESI probe (micro-ESI) of acommercial ion-trap mass spectrometer, and was usedfor protein identification experiments based on capillaryliquid chromatography/tandem mass spectrometry (LC-MS/MS). That modification allows the use of a nitrogensheath gas, which helps the nebulization process at LCflow rates that exceed 500 nL/min. Phosphorylated pep-tides in a beta-casein tryptic digest were also successfullyidentified. In addition, it was found that a mild sanding ofthe metal needle tip led to a significant improvement ofthe system.

The MALDI method has high sensitivity, especiallyif coupled with a TOF mass analyzer. However, newimprovements are continuously being developed to im-prove the sensitivity. For example, the Chen group (Chen &Tsai, 2000) has examined the effect of surface activity insurface-assisted laser desorption/ionization (SALDI) massspectrometry. That technique is based on the phenomenon,observed in the FAB ionization source, that chargedanalytes are significantly affected by oppositely chargedsurfactant additives in the matrix (Ligon & Dorn, 1984;Huang et al., 1994). It has been proposed that the chargedsurfactant concentrates the oppositely charged analyte near

the surface to produce larger signals for the analyte(Huang et al., 1994). Surfactants such as SDS present in thesample solution usually are removed to prevent the degra-dation of sensitivity, resolution, and mass accuracy inMALDI mass spectra. However, surfactants applied in thesimultaneous analysis of hydrophobic and hydrophiliccomponents of mixtures have recently been reported(Breaux et al., 2000). In SALDI analysis, a sample solutionis mixed with a matrix that is composed of a suspension ofmicrometer-sized carbon powder in a mixture of glycerol,sucrose, and methanol. The carbon/glycerol suspensionand the analyte mixture, containing the surfactant, areirradiated with a pulsed laser, and the produced ions areanalyzed by a TOF. It must be stressed that a liquid is one ofthe essential compositions in the SALDI matrix. Moleculeswith surface activity may cover the surface of the liquidmatrix, as demonstrated in FAB and liquid SIMS. Thus, thesurface activity of analytes may play a role in the SALDIanalysis. Breaux et al. (2000) have used several surfac-tants, including p-toluenesulfonic acid (PTSA), sodiumdodecyl sulfate, and alkyltrimethylammonium bromide, inthe SALDI matrix to demonstrate the surface-activityeffect. The experimental results demonstrate that analytesthat have a good surface activity can be detected with highsensitivity. Adding suitable amounts of surfactants to theSALDI matrix can dramatically enhance the determinationof low quantities of analytes that lack surface activity.

Other systems that have been used to increase thesensitivity of MALDI instruments are based on bioreac-tive MALDI probe tips (Nelson, 1997). To avoid pos-sible sample loss and autolytic interferences in the massspectrum, mass spectrometer targets can be covalentlyderivatized with enzymes used for the protein character-ization. The analyte is deposited on the enzymaticallyactive, or bioreactive, activated surface, followed by appli-cation of a suitable MALDI matrix and laser irradiation.Limited transfer- and handling-steps eliminate samplelosses, and surface-tethered enzymes are prohibited frominterfering with analytical signals in the mass spectra(autolytic fragments are avoided). In addition, the probesare rapid and easy to use.

2. Problems with Buffers

ESI and MALDI methods both show a clear decrease insensitivity if buffers, salts, or detergents are present in thesample solutions. ESI, in particular, shows a low toler-ance to the presence of those compounds. Generally,volatile salts lead to a lower signal decrease than non-volatile salts. Detergents, in particular, strongly decreaseESI signals. The effect of various detergents on signaldecrease is reported in Table 1. In order to minimize thosesignal suppression effects, LC techniques are often coupledto the ESI source (Niessen, 1999) to separate contaminants


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from the analytes before ESI-MS analysis. The chromato-graphic separation allows sample purification/enrichmentfrom most common laboratory buffers and endogenoussalts, and provides some additional useful parameters suchas retention time. On the other hand, it must be emphasizedthat the LC step significantly increases analysis time anddecreases throughput. As an alternative to LC purification,several laboratories have used on-line dialysis prior to massspectral analysis (Huber & Buchmeiser, 1998; Xu et al.,1998). However, that procedure is also time-consuming. Adetailed explanation of the decrease in signal due to thesalts effect has been given by Constantopoulos, Jackson, &Enke (1999). Using electrospray ionization mass spectro-metry (ESI-MS), the effect of salt concentration on analyteresponse was measured, and was compared with thatpredicted by Enke’s equilibrium partitioning model (Enke,1997). That model predicts that analyte response is pro-portional to concentration, and that the response factor willdecrease with an increased electrolyte concentration. The

concentration of excess charge generated by the ESIprocess increases significantly at 10�3 M ionic concentra-tion, but the response factor decreases at that concentration.Changes in the shape of the spray cause a loss of iontransmission efficiency, and that effect may be the cause ofthe decrease in response.

The MALDI technique seems to be less affected by thepresence of salts than ESI. That fact is very importantbecause MALDI analyses co-crystallized samples, andthus cannot be coupled with an on-line LC separation. Inthe off-line approach, the molecules are separated by LCand are collected. The fractions are co-crystallized with thematrix molecules, and are analyzed by MALDI. However,even if that source is less affected by the presence of saltsand buffers than ESI, those compounds do lead to somesignal decrease. The detergents often used to extractproteins from various organisms also lead to a severe signaldecrease. Various papers describe the effects due to salts,buffers, and detergents (Shaler et al., 1996; Amini et al.,2000; Bajuk, Gluch, & Michalak, 2001). In particular,Bajuk, Gluch, & Michalak (2001) have measured the effectof contaminants such as sodium sulfate (Na2SO4), coppersulfate (CuSO4), potassium ferrocyanide (K4Fe(CN)6), andtriammonium citrate ((NH4)3C6H5O7) on the positive-ionMALDI-TOF spectra of insulin. 2,5-Dihydroxybenzoicacid was used as matrix, and the concentrations of dif-ferent salts that reduce the insulin signal to zero were alsodetermined. Amini et al. (2000) described the effect ofvarious buffers, surfactants, and organic additives that arecommonly found in capillary zone electrophoresis andmicellar electrokinetic chromatography separation on theMW determination of peptides by MALDI mass spec-trometry. The signal-to-noise ratio (S/N) generally de-creased with increasing buffer concentration, withoutaffecting mass accuracy. Good spectra were obtained withan ammonium acetate buffer up to a concentration of500 mM without impacting on the ionization of peptides.Ionization of organic additives—such as anionic sur-factants, non-ionic surfactants, and cyclodextrins—wasbuffer-dependent. Some problems arose when the mole-cular mass of the additive was in the same range as thepeptide mass. Brij-35, Tween-80, and cyclodextrins allproduced abundant molecular species in the presence ofsodium- or potassium-containing buffers, but not withammonium acetate. Cationization of those neutral specieswith sodium or potassium ions appears to be a highlyfavored process. In contrast, the ammonium ion appearsto be a poor cationizing agent for those compounds.Ionization of neutral surfactants was suppressed in ammo-nium acetate without impacting the spectra of peptides.Ammonium acetate buffers that contain 30 mM sodiumdodecyl phosphate also lead to spectra with abundantmolecular species and without interference from the sur-factant. Suppression of peptide ionization in MALDI was

TABLE 1. Effect of the detergents on the ESI signal decrease

þþ, weak signal intensity decrease; þþþ, moderately signal

intensity decrease; þþþþ, significant signal intensity decrease;

þþþþþ, strong signal intensity decrease.


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found when methanol, tetrabutyl amine, or poly(vinylalcohol) were used with ammonium acetate, sodium phos-phate, and N-(2-hydroxyethyl)piperazine-N-(2-ethansul-fonic acid).

Finally, the study performed by Shaler et al. (1996) onthe quality of MALDI spectra is particularly interesting andshowed the effect of impurities in single-stranded DNAoligomers. That study has been performed with 3-hydroxypicolinic acid as matrix and a 355-nm pulsedlaser. By mixing the DNA oligomers with different con-centrations of impurities and recording mass spectra, limitswere set on the tolerable level of a given impurity in asample. The tolerance limits for sodium chloride, potas-sium chloride, sodium acetate, sodium fluoride, sodiumdodecyl sulfate (SDS), and manganese(II) chloride weredetermined in this study.

3. Multicharge Effect

The production of multi-charged ions is a typical aspect ofthe ESI method, and provides a means of accurate andprecise measurement of molecular masses of macromole-cules; however, it also gives rise to some problems. Theexcessive overlap of signals in ESI spectra, in fact, presentssome limitations in the analysis of mixtures. Isotopespacings in m/z units are inversely proportional to thecharge of ions, and the spacing of multi-charged ions onthe m/z scale decreases with increasing charge. The re-solution of mixture components with modest differencesin their mass/charge ratios can became impossible, due tothe m/z scale compression. Algorithms that allow the de-convolution of overlapping charge states distribution—thereby facilitating the identification of mixture compo-nents—have been proposed (Mann, Meng, & Fenn, 1989;Zhang & Marshall, 1998). However, as spectral complexityand chemical noise levels increase, those algorithms arelikely to produce artifactual peaks and might also missanalyte peaks with a low signal intensity. That situationimproves when high-resolution mass spectrometers areemployed. High resolution makes the deconvolution ofcomplex multi-charged ion spectra possible. Furthermore,it must be emphasized that signal distribution over severalmolecular charge states necessarily results in a decrease ofthe absolute signal intensity for each individual ion species,and thus also of the S/N ratio.

The MALDI method does not usually give rise to themulti-charge effect, and mainly produces singly chargedions. The analysis of complex mixtures of bioorganicmacromolecules by that method is thus more effective.However, that positive aspect is balanced by a negative one,reflecting the fact that, to perform MS/MS studies onpeptides, the best ions to fragment for structural investiga-tion are the doubly charged ions. Cramer & Corless (2001)have performed a comparative MS/MS study on singly and

doubly charged ESI- and MALDI-generated peptideprecursor ions. The spectra from those experiments havebeen evaluated-paying particular attention to the dataquality for subsequent processing to yield protein/peptidesequence identification. It has been shown that, once pep-tide ions are formed by ESI or MALDI, their charge stateand the collision energy are the most important parametersthat determine the quality of the CID spectra of a givenpeptide. MALDI of medium-to-high MW peptides ob-tained by protein tryptic digestion, are predominantlyformed as singly charged species and ESI ions as doublycharged, the difference in the quality of MS/MS spectraobtained by the two different ionization techniques has anobvious effect on data processing, database searching usingion fragmentation data, and de novo sequencing.

B. Mass Analyzer Problems and Developments

The mass analyzer is another important part of the massspectrometer to be considered in evaluating instrumentalperformance. Companies strive to improve the ion-opticalsystem in order to enhance the number of ions that reach theanalyzer to improve sensitivity (Cha, Blades, & Douglas,2000). The most-used mass analyzers are ion trap (IT),triple quadrupole (Q1Q2Q3), time of flight (TOF), andfourier transform ion cyclotron (FT-ICR). Each massanalyzer presents some limitations in regard to lineardynamic range, tandem mass spectrometry, sensitivity,resolution, and mass accuracy.

1. Linear Dynamic Range

Linear dynamic range strongly depends on the massanalyzer. The IT is much affected by a dynamic rangelimitation; the FTICR analyzer also suffers from thesame problem. Q1Q2Q3 gives rise to high performance interms of linear dynamic range. For instance, Xu, Veals, &Korfmacher (2003) have described a new triple-quadrupolemass spectrometry system with enhanced capability interms of linear dynamic range and resolution in the analysisof biological compounds. However, Q1Q2Q3 gives rise tothe best performances when it is used to analyze moleculeswith medium-to-low molecular mass (100–1000 Da). Forexample, many applications exist for the analysis ofmetabolites (Yang et al., 2002; Josefsson et al., 2003).

TOF analyzer also usually exhibits a high lineardynamic range if coupled with separating LC techniques.For example, Clauwaert et al. (1999) have described theinvestigation of the potential of a quadrupole orthogonalacceleration time-of-flight mass spectrometer equippedwith an atmospheric pressure ionization interface for quan-titative measurements of molecules separated by reversedphase liquid chromatography. The results revealed excel-lent sensitivity and linear dynamic range.


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2. Tandem Mass Spectrometry

The TOF and Q1Q2Q3 instruments are typically used inMS2 (Premstaller & Huber, 2001; Yergey et al., 2002) butnot in MSn experiments, whereas ITand FTICR are usuallyemployed for that purpose (Kruppa et al., 2002; Wu et al.,2002). However, it must be emphasized that Q1Q2Q3

instruments can produce medium-energy fragmentation(more structural information in the MS2 spectra), whereasIT gives rise to low-energy fragmentation (it is oftennecessary to perform MSn analysis to obtain detailed struc-tural information). Premstaller & Huber (2001) haverecently investigated the sequence coverage by fragmentions that result from collision-induced dissociation ina Q1Q2Q3 and an IT mass spectrometer of 10–20-meroligonucleotides. Whereas (a-B) and w ion series were themost abundant on both instruments, additional ion-series ofsequence relevance were preferably formed in the Q1Q2Q3.Thus, a total number of 83 fragment ions were used todeduce the complete sequence of a 10-mer oligonucleotideof mixed sequence from a tandem mass spectrum record-ed on the Q1Q2Q3. The complete sequence was alsoencoded in the 28 fragments that were obtained from the IT.However, it must be emphasized that spectrum complexityincreases considerably at the cost of signal-to-noise ratioupon fragmentation of a 20-mer oligonucleotide in theQ1Q2Q3, whereas spectrum interpretation with longeroligonucleotides was significantly more straightforwardin spectra recorded on the IT. The extent of fragmenta-tion had to be optimized by an appropriate setting ofcollision energy and the choice of the precursor-ion charge-state in order to obtain full sequence coverage by frag-ments for de novo sequencing. Moreover, full sequenceinformation was also dependent on the base sequencebecause of the low tendency of backbone cleavage atthymidines. However, tandem mass spectrometry on theIT yielded information that was successfully utilized todeduce with high confidence the complete sequence of20-mer oligonucleotides.

Especially the TOF instrument exhibits problems dueto the low fragmentation efficiency of the PSD approachused to fragment the ions. However, it must be emphasiz-ed that the fragmentation efficiency strongly depends onthe kind of molecule that is analyzed (Dikler, Kelly, &Russell, 1997; Peng et al., 2003). For instance, Peng et al.(2003) showed that C(alpha)-formylglycine-containingpeptides give rise to a unique and prominent fragmention by using MALDI PSD technique, whereas Dikler,Kelly, & Russell (1997) clearly showed that modificationof arginine residues in bradykinin, [1–5]-bradykinin, sple-nopentin, and two synthetic pentapeptides with acety-lacetone (pentane-2,4-dione) significantly increased therelative abundance of the sequence-specific fragment ionsproduced by PSD technique.

3. Sensitivity

In the case of the ITanalyzer, the main factors that affect thesensitivity are the trapping efficiency (Yoshinari, 2000;Vaden, Ardhal, & Lynn, 2001) and ion-ejection efficiency(He & Lubman, 1997). Yoshinari (2000) applied an in-teresting theoretical approach to increase the trappingefficiency. In that study, a numerical simulation methodwas developed for the analysis of ions injected into an ion-trap mass spectrometer. That method was applied to clarifythe effects of the following parameters on trappingefficiency: (1) initial phase of the radio frequency (RF)drive voltage, (2) ion injection energy, and (3) RF peakvoltage while injecting ions. Some conclusions wereobtained by theoretical approaches. A relationship amongthe operating parameters that gives the maximum trappingefficiency was derived, and an advanced injection methodwas proposed in which the RF peak voltage is decreasedduring the ion injection. The ability of that method to solvethe problem of unequal sensitivities for different ionspecies has been described, using a numerical simulation.He & Lubman (1997) have also developed an interestingtheoretical study on ion-trap ejection efficiency. A PC-based simulation was described to study ion injection,cooling, and extraction processes for multiple ions in a trap/reflectron time-of-flight (IT/reTOF) system. The results ofthose simulations made it possible to describe the relation-ship between trapping efficiency for external injection ofions into the trap and the RF phase. The effects of initialkinetic energy and ramp-up rate on the dynamic trapping ofexternally produced ions was also described, as well assingle-pulsing and bipolar-pulsing schemes for ejectingions from the trap. The simulations show that bipolarpulsing can markedly improve the performance of theinstrument.

The Q1Q2Q3 mass analyzer exhibits a sensitivity thatis higher than that of IT systems when the spectra areacquired in the multiple-reaction monitoring (MRM)mode. In fact, it must be emphasized that, for the full-scan spectra acquisition mode, the IT is definitely moresensitive than a linear mass filter, but the opposite is true forMRM mode also if the new linear ion trap seems to reachsimilar performances (Hager & Yves Le Blanc, 2003).

Another factor that can limit the sensitivity is related tothe micro-channel plate (MCP) impact detector (Franket al., 1999). The main problem of that detector is that, in allthe arrangements unless post-acceleration, its ion trans-mission is, to some extent, mass-dependent: the sensitivitydecreases with an increase in the molecular mass of theanalyzed compounds. The efficiency of the MCP impactdetector, in fact, drops considerably at high molecular massdue to the fact that it relies on secondary electron emission,which becomes increasingly inefficient as molecular massincreases and impact velocity decreases.


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FT ICR is by far the most-sensitive device currentlyavailable; it can detect ions at attomole levels (Soloukiet al., 1995; Hannis & Muddiman, 2001; Quenzer et al.,2001). However, its cost remains prohibitive for manyresearch laboratories. It must also be emphasized that alimit in the sensitivity of FTICR is caused by broadbandimage current detection, which typically requires approxi-mately 100 charges to generate a measurable signal at agiven m/z ratio (Quenzer et al., 2001). For that reason, theFTICR mass analyzer has a problem with acquiring datafast enough if interfaced directly to high-resolution capil-lary chromatography. In order to solve that problem,ions can be accumulated in a linear octopole ion trapbefore injection into an FTICR mass analyzer (Wilcox,Hendrickson, & Marshall, 2002). Such an instrumentalconfiguration has been shown to provide an improvedsensitivity, scan rate, and duty cycle relative to accumu-lated trapping in the ICR cell.

As shown by Chernushevich (2000), normal orthogo-nal TOF instruments benefit from a simultaneous recordingof all masses, and their sensitivity is higher than that ofa triple quadrupole when a full scan is recorded. Thissensitivity is not the case when only a single type of ionmust be monitored as in the precursor-ion scanning orMRM mode. This limitation is because ion losses in a TOFare higher than in a quadrupole. A significant part of thoselosses is due to the low duty—cycle of an orthogonal TOFinstrument typically—5–30%.

4. Resolution

The IT is the mass analyzer that is most affected byresolution problems—mainly due to space-charge effectsthat occur in the ion-trap analyzer. The resolution of thisinstrument in normal operation is typically 1 unit mass ifthe spectrum is obtained in the full-scan mode. For thatreason, when LC-MS/MS is used to characterize elutingsubstances (e.g., metabolites, peptides), it is often neces-sary to perform a zoom-scan spectrum (high-resolutionspectrum in a small m/z range around the peak of interest)before performing the MS/MS experiment (Gatlin et al.,2000; Ramanathan et al., 2002). An interesting approachthat can be used in the analysis of complex biologicalmixture is called the data-dependent scan. In that case, theprecursor ion is automatically chosen by the instrument forMS/MS analysis. For instance, in the data-dependence scantriple-play, the full-scan spectrum is acquired, a zoom scanis performed on the peak with the highest abundance, andfinally an MS/MS spectrum is obtained on the same peak.Another interesting approach could include dynamicexclusion, which allows one to select parameters to pre-vent, for a pre-selected length of time, an ion fromtriggering a subsequent data-dependent scan after it hasalready triggered a data-dependent scan. That procedure

strongly increases the number of ions that are automaticallyselected and analyzed. Ramanathan et al. (2002) perform-ed a rapid identification of metabolites, using the data-dependent scan function of an ion-trap mass spectrometerand semi-automated metabolite-identification software.Gatlin et al. (2000) applied data dependent scan methodsto study amino acid sequence variations that resulted fromsingle-nucleotide polymorphisms (SNPs). In that case,using the zoom-scan step, it was possible to obtain thecharge state of the molecule by analyzing its isotopicdistribution, before fragmentation. Nowadays, some com-mercial IT instruments are able to acquire a high-resolutionspectrum in the full-scan mode.

It must be emphasized that, if the spectra are acquiredin the full-scan mode, the resolution increases in this order:IT<Q1Q2Q3<TOF< FTICR. The advantages of the FT-ICR spectrometer have been explored by several groups(Marshall & Guan, 1996; Kelleher et al., 1997; Williams,1998). In particular, an interesting study of McLaffertyand co-workers (Kelleher et al., 1997) reported unit-mass resolution (i.e., separation of individual isotopicvariants) during the analysis of a 112 kDa protein byelectrospray-FT-ICR.

5. Mass Accuracy

The ion-trap instrument provides the worst mass accuracyof all of the commonly used analyzers. It has typically anuncertainly of ca. �0.2–0.4 Da. Due to the space-chargeeffect in the small space of the ion-trap analyzer. Marinaet al. (1999) attempted to increase the sensitivity and themass accuracy of an ion-trap instrument. The standardfront-end of the electrospray probe was replaced with amicromanipulator, which, with the aid of a magnifyingdevice, allowed the use of a variety of miniaturizedspraying interfaces. The low sample consumption andextended analysis time of that device improved the resultsin terms of sensitivity and mass accuracy. However,nowadays many mass spectrometer manufactures aretrying to find a new solution in order to increase the massaccuracy of the ion trap.

The mass accuracy of TOF instruments, on the otherhand, is very high, which makes them suitable tocharacterize proteins and peptides.

The FT ICR mass analyzer has a high mass accuracy,in some cases, <1 ppm (Williams, 1998). It must beemphasized that, as for the resolution parameters, themass accuracy increases following this order: IT<Q1Q2Q3<TOF< FTICR. For instance, Kosaka, Takazawa,& Nakamura (2000) developed a new method for theidentification and C-terminal characterization of proteinsseparated by two-dimensional polyacrylamide gel electro-phoresis (2D-PAGE), using the high-resolution and -massaccuracy of the FT-ICR technique. Proteins were in-gel


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digested, using a buffer solution that contained 50% 18O-labeled water, and mixtures of 18O/16O-labeled peptideswere analyzed by nanoelectrospray Fourier transform ioncyclotron resonance mass spectrometry (FT-ICR MS).That method was evaluated, using horse skeletal musclemyoglobin as the model protein in a SDS gel.

IV. NEW PROMISING TECHNOLOGIES

In this section, some of the most interesting new techniquesthat have been recently developed for the study of bio-organic macromolecules will be reviewed.

A. Interfaces and Ionization Sources

1. Improvements in the Coupling of Liquid-PhaseSeparation Systems to Mass Spectrometry

The latest improvements in interfacing liquid-phase sep-aration systems are continuous-flow matrix-assisted laserdesorption/ionization, micro- and nano-liquid chromato-graphy/MS, capillary electrophoresis/MS, and on-chipseparation technologies/MS.

In recent years, new methods have been developed inorder to couple on-line LC and MALDI instruments (Gelpı,2002). The technology used for that purpose is namedcontinuous-flow MALDI (cf-MALDI). The main advan-tage of coupling LC to MALDI is the introduction of adesalting step. Li and co-workers (Nagra & Li, 1995)improved their continuous-flow probe in order to maximizethe detection sensitivity. To maximize the ratio between thearea of sample desorption (laser spot size) and the total areaover which the sample diffuses on the probe, the flow-probesample area was reduced, and the frit was eliminated. Thematrix was added post-column. The main improvementwas the independence of LC and liquid matrix flows, sothat the two systems can be independently optimizedfor separation speed and ion detection sensitivity. Thatconventional LC/on-line MALDI system was also adaptedfor micro-LC/MALDI by inserting a pre-column solventsplitting tee to allow LC flow rates of 1–10 mL min�1 withpost-column matrix addition set at 3-5 mL min�1.

Two problems are associated with the cf-MALDIapproach. The first is the need for non-crystalline matricesand the relative lack of suitable liquid matrices forirradiation by an UV laser. At present, only 3-NBA and2-nitrophenyl octyl ether can be used for this particularapplication. The second problem of this technique is due tothe fact that none of these devices has the level ofstandardization of the electrospray systems.

Other interesting approaches that have steadily devel-oped are based on the liquid-junction interfacing of the

separation column outlet, and the inlet of the capillarysprayer, using micro- and nano-ESI (Gelpı, 2002). Theminiaturized ESI techniques have also been essential forthe successful interfacing of capillary electrophoresis andmass spectrometry (Ding & Vouros, 1999). Those emer-ging new technologies may become dominant forces in thenear future because they perfectly couple the concept oflimited sample amounts with the requirements for highseparation efficiency and detection sensitivity.

2. AP-MALDI

An interesting approach that has been recently introducedis the construction of a MALDI source that operates atatmospheric pressure (Laiko, Baldwin, & Burlingame,2000; Laiko, Moyer, & Cotter, 2000; Laiko et al., 2002).The transfer of the ions from the atmospheric pressureionization region to the high vacuum is pneumaticallyassisted (PA) by a stream of nitrogen—hence, the acronymPA-AP MALDI. That source is readily interchangeablewith the ESI source of an orthogonal acceleration time-of-flight (oaTOF) mass spectrometer. The performance of thation source on the oaTOF mass spectrometer has beencompared by Laiko, Baldwin, & Burlingame (2000) withthat of conventional vacuum MALDI-TOF for the analysisof peptides. PA-AP MALDI can detect low fmol amountsof peptides in mixtures with good signal-to-noise ratio andwith less discrimination for the detection of individualpeptides in a protein digest. Peptide ions produced by thismethod generally exhibit no metastable fragmentation,whereas an oligosaccharide ionized by PA-AP MALDIshows several structurally diagnostic fragment ions. TheAP MALDI source can be coupled with all of the massanalyzers that are suitable for atmospheric-pressure ioni-zation sources. Laiko, Moyer, & Cotter (2000) havedescribed the coupling of this ionization source with acommercial ion-trap mass spectrometer. The detectionlimit of the new AP MALDI/IT is 10–50 fmol of analytedeposited on the target surface for a four-componentmixture of peptides with 800–1700 Da MW. Thepossibility of peptide structural analysis by MS/MS andMS3 experiments for AP MALDI-generated ions was de-monstrated. Using the ion trap, it was in fact possible tofragment the ions with an efficiency higher than thatachieved with the PSD approach. Some strategies have alsobeen recently described to increase the sensitivity of thisionization technique. For example, Laiko et al. (2002) havereported atmospheric pressure laser desorption/ionizationon porous silicon (AP-DIOS). That new method fea-tures reasonably good sensitivity (subpicomole range forstandard peptide mixtures), simple preparation of sample,uniformity of target spots, and the absence of matrix peaksin the spectra. The AP-DIOS spectrum of 250 fmol ofunseparated tryptic digest of bovine serum albumin (BSA)


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was compared with that of AP-MALDI, and showed thatAP-DIOS offers a significantly better coverage in the massrange 200–1000 Da. The AP-MALDI technique, however,gives better results in the 1000–2000 Da range. Thecombined data from those two techniques yield nearlydouble the number of matched peaks in the analyses ofBSA digests as compared with separate AP-DIOS orAP-MALDI analysis. Figure 7A,B (Laiko et al., 2002)shows the AP DIOS and AP-MALDI mass spectra of thetryptic digest of BSA acquired in the m/z 1000–2000range; the AP-MALDI technique seems to work betterin the characterization of peptides with high molecularmasses, and detected a large fraction of the predicted tryp-tic peptides of BSA. Figure 8A,B (Laiko et al., 2002) showthe AP-DIOS and AP-MALDI BSA digest spectra acquiredin the m/z 200–1000 range, and indicate that the bestresults were obtained with the AP-DIOS technology.

In summary, the AP-DIOS and AP-MALDI ionizationsources show a good sensitivity, and make it possible toperform effective MS/MS experiments on the ions pro-duced. Moreover, they are less affected by the presence ofsalts and buffers than ESI, making possible a higheranalytical throughput. The main problem for the structuralanalysis of peptides is that the doubly charged ions (the beston which to perform MS/MS experiments) are not mainlyproduced by the AP-DIOS and AP-MALDI sources.

3. Deprotonant Agents

As mentioned above (Section III), one of the problems thataffects the ESI source in the direct analysis of complexmixtures is the overlap of the signals due to the formationof multi-charged species. McLuckey and Stephenson(Stephenson & McLuckey, 1996, 1998; McLuckey &Stephenson, 1998) have studied various systems, devotedto ion-charge reduction, using ion/ion chemistry in the gasphase. They have found that ion/ion proton transferreactions are effective means to facilitate the resolutionof ions different in mass and charge, but similar in mass-to-charge ratios. In their 1996 paper, examples are shown inwhich a minor protein contaminant in a ribonuclease Bsolution, not detectable in the conventional ESI spectrum,becomes clearly apparent after ion/ion proton transfer. Afurther experiment, involving a mixture of bovine serumalbumin and bovine transferrin, was also performed inorder to detect high MW contaminants. That study alsoillustrated some important issues in the use of thequadrupole ion trap as a reaction vessel and mass analyzerfor high mass-to-charge ratio ions. The results indicatedthat the use of IT-operating parameters—specificallytailored for storage, ejection, detection, and mass-to-charge analysis of high mass-to-charge ratio ions—canhave important applications in the analysis of relativelyhigh-mass proteins or other high-mass biopolymers. IT

physical parameters may be changed in order to analyzemolecules with high mass and low charge. In anotherapplication described by McLuckey & Stephenson (1998),data are shown for the þ4 and þ3 precursor ions derivedfrom the electrospray of melittin, and the þ12 to þ4 pre-cursor ions of bovine ubiquitin, whereby product ions wereformed in a conventional quadrupole ion-trap tandemmass spectrometry experiment. Data are also shown forproduct-ion mixtures derived from the interface-induceddissociation of multiply charged ions derived from bovineubiquitin, tuna cytochrome c, bovine cytochrome c, andequine cytochrome c. It was shown that the use of ion/ionchemistry to simplify product-ion spectra derived frommultiply charged precursor ions significantly extends thesize range of macromolecules for which the quadrupole iontrap can obtain structural information.

A similar approach has been tried by Cristoni (2002) toreduce the charge state of bioorganic macromolecules.A new approach, based on the use of triethylamine asdeprotonating agent, was performed to reduce the charge ofprotein multi-charged ions and to decompress the masssignals. That approach makes it possible to extend theprotein mass-deconvolution range, and to increase thesignal/noise ratio due to the low chemical noise present inthe high m/z region. It was also possible to analyze com-plex protein mixtures that are difficult to analyze in therange m/z 100–2000 due to the extensive overlap of therelated mass signals and the high chemical noise of thatregion. The same approach was used in the analysis of asolution that contained three standard proteins (horsecytochrome c, horse myoglobin, and bovine albumin) inorder to verify the ability of the proposed method toanalyze protein mixtures. Different deprotonating molec-ules were tried (Table 2; Cristoni, 2002), leading to thechoice of TEA as deprotonating reagent due to its highproton affinity (Proton Affinity: 981.8 kJ/mol, data obtain-ed from NIST chemistry WebBook: http://webbook.nist.gov/chemistry/) and low cost. TEA molecules may reactwith protein molecules in solution through a nucleophilicattack at the protonated protein amino groups (Scheme 2i;Cristoni, 2002). The acid-base reactions of TEA with acidgroups may also take place, reducing the net charge of theprotein (Scheme 2ii; Cristoni, 2002). Moreover, the basiccondition in the solution makes easier the deprotonationof �NH3

þ protein groups and the formation of proteincarboanions �COO�. It must also be considered that thepartially or fully desolvated gas-phase protein ions mayundergo charge-exchange reactions, including the transferof protons to water molecules, inside an ion trap.Furthermore, the ion-molecule reactions may also occurinside the ion trap, between the protein multi-charged ionsand TEA neutral molecules (the same as in Scheme 2i and2ii). In order to reduce the chemical noise due to the TEAions, an injection waveform was used to eliminate TEA


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FIGURE 7. AP MALDI mass spectrum of the tryptic digest of BSA. High-mass region (1000–2000 Th) of

BSA digest spectra recorded by (A) AP-DIOS and (B) AP-MALDI techniques (250 fmol loaded in each

case). (Reproduced from Laiko et al. (2002) with permission from John Wiley & Sons, Ltd., copyright 2002.)


386

FIGURE 8. Low-mass region (200–1000 Th) of BSA digest spectra recorded by (A) AP-DIOS and (B) AP-

MALDI techniques (250 fmol loaded in each case). (Reproduced from Laiko et al. (2002) with permission

from John Wiley & Sons, Ltd., copyright 2002.)


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ions at the entrance of the ion trap during the injectiontrapping time. Figure 9A–C (Cristoni, 2002) shows thespectra of horse cytochrome c, horse myoglobin, andbovine albumin, respectively, that were acquired withoutTEA and adding 0.025% (v/v) of trifluoroacetic acid (TFA)to the sample solutions. In the case of horse cytochrome cand horse myoglobin, the protein multi-charged ions weredetected in the m/z 100–2000 range. It must be emphasiz-ed that, in the spectrum of bovine albumin, the proteinsignals give rise to a very complicated spectrum in them/z 1000–4000 range. In Figure 9A–C, the deconvolutionspectra obtained for each protein are also shown; in thecase of bovine albumin, the deconvolution algorithmcould not calculate the correct mass of the protein due tothe complexity of the mass spectrum. In Figure 10A–C(Cristoni, 2002), the spectra obtained with TEA are shown.In the case of horse myoglobin and horse cytochrome c,

the multi-charged ions were detected in the m/z 1000–4000 range. In that region, the chemical noise is lower thanin the m/z 100–2000 region and, consequently, the signal/noise ratio increases. As shown in Figure 10C, in the caseof bovine albumin, no positive multi-charged ions wereobserved on adding TEA to the sample solution. Multi-charged ions of that protein were observed in the negative-mode in the m/z 4000–10,000 range (Fig. 11; Cristoni,2002).

A mixture of the three standard proteins was analyzedin order to verify the power of the method in the anal-ysis of protein mixtures. In Figure 12A,B (Cristoni,2002), the spectra of a protein mixture that containedcytochrome c, horse myoglobin, and bovine albumin, ac-quired without and with using TEA, are shown. Figure 12Ashows the spectrum of that solution acquired in them/z 100–2000 range without using TEA and adding0.025% (v/v) TFA to the analyzed solution; the spectrumobtained is very complex, partly due to the high chemicalnoise. The C-series corresponds to the multi-charged ionsof horse cytochrome c, whereas the M-series correspondsto multi-charged ions of horse myoglobin. Figure 12Bshows the spectrum obtained with TEA; the multi-chargedsignals due to horse cytochrome c (C-series) and horsemyoglobin (M-series) are well-resolved in the m/z 1000–4000 range, and bovine albumin multi-charged signalswere detected with the negative-acquisition mode in them/z 4000–10,000 range (data not shown).

This example clearly shows that ion/ion or ion/molecule chemistry in an ion trap may facilitate theanalysis of complex bioorganic macromolecule mixtures.

4. Sonic Spray Ionization

Sonic spray ionization (SSI) was developed in 1994 byHirabayashi (Hirabayashi, Sakairi, & Koizumi, 1994,

TABLE 2. Table of deprotonant molecules used in order to reduce

the charge of the multi-charge ions. The proton affinity of the

compounds is also shown.

(Reproduced from Cristoni et al. (2002) with permission from

Internet Journal of Chemistry, copyright 2002.)

SCHEME 2. Proton transfer reactions that can take place among protein and TEA molecules. (Reproduced

from Cristoni (2002) with permission from Internet Journal of Chemistry, copyright 2002.)


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1995) as a technique to generate molecular ions under avery wide range of solvent systems and flow rates. Arinobuet al. (2002) recently published an article that showedthe high sensitivity of that ionization method. In SSI,vaporization is performed so that the surface layer of thesolution, in the region of charge separation, is stripped by afast gas flow with the formation of electrically chargedairborne droplets. The diameters of those electricallycharged droplets shrink by vaporization of solvent molec-ules from the surface, and protonated molecules are formedin the gas-phase. Various studies have shown the high

sensitivity of this ionization technique in the analysisof bioorganic macromolecules (Huang et al., 2001;Hirabayashi & Hirabayashi, 2002; Huang & Hirabayashi,2002). As an example, Huang et al. (2001) and Huang &Hirabayashi (2002) have analyzed a 20-base oligonucleo-tide. The effects of contaminants and the parameters thataffect the ion production (e.g., a high voltage applied to theionization source, and the sample solution-flow rate) wereinvestigated. Signal intensity and mass spectrum patternswere found to be dependent on the matrix and operatingparameters. One of the reasons for such behavior is the

FIGURE 9. Spectra of (A) horse cytochrome c, (B) horse myoglobin, and (C) bovine albumin, acquired

without using TEA and adding 0.025% (v/v) of TFA to sample solutions (Reproduced from Cristoni (2002)

with permission from Internet Journal of Chemistry, copyright 2002.)


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presence of various ion-states for the oligonucleotide underinvestigation. Changes in the matrix or in the operatingparameters may shift the balance among the ion-chargestates. The addition of Tris or (hydroxymethyl)amino-methane enhanced the signal intensity of the oligonu-cleotide, promoting the formation of oligonucleotide ionswith a higher number of charges, whereas adding aceticacid favored the formation of ions with a lower numberof charges.

The SSI source has been shown to be highly effectiveeven in the protein field. Hirabayashi & Hirabayashi (2002)have used capillary isoelectric focusing (CIEF) separationcombined with mass spectrometry for protein analysis byusing a SSI interface. With that approach, they were able todetect 160 fmol of myoglobin and cytochrome c.

The main advantages of that ionization source arerelated to its sensitivity and to the fact that it operateswithout using needle potentials. In those conditions, even if

FIGURE 10. Spectra of (A) horse cytochrome c, (B) horse myoglobin, and (C) bovine albumin, obtained

using TEA. (Reproduced from Cristoni (2002) with permission from Internet Journal of Chemistry,

copyright 2002.)


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salts and buffers are present in the analyzed mixture, theydo not lead to discharge phenomena between the needle ofthe ionization source and the instrument. However, thematrix composition strongly influences the yield of theproduced ions (e.g., with the formation of salt, sodium, andpotassium adducts), and, consequently must be carefullycontrolled.

5. APCI without Corona Discharge

In previous investigations (Cristoni et al., 2002a,b), it hasbeen shown that the vaporization of sample solutions inatmospheric pressure conditions, without the presence ofany corona discharge, leads anyway to the production of ahigh number of ions. Those experiments were perform-ed by a commercially available APCI source, and thatapproach was called ‘‘no-discharge APCI.’’ It must beemphasized that, in these conditions and in the case ofproteins, the formation of doubly charged species isprivileged and, considering their usefulness in furtherMS/MS studies (Cramer & Corless, 2001), the observedphenomenon was considered of high interest.

Figure 13A,B (Cristoni et al., 2002b) shows the spectraof horse cytochrome c and horse myoglobin, obtained withthat technique; in the case of horse cytochrome c, [MþH]þ,[Mþ2H]2þ, [Mþ3H]3þ, and [Mþ4H]4þ ions are readilydetectable, whereas in the case of horse myoglobin, the[MþH]þ ion was not detected and only [Mþ2H]2þ and[Mþ3H]3þ ions were observed with good Signal/Noiseratios. The counts per second value of the most abundantsignal was 2� 106 for horse cytochrome c and 5� 105 forhorse myoglobin. The width of the peaks is mainly due to

the low resolution of the mass analyzer used (Ion Trap) inthe m/z 3000–12,000 range, combined with the effect ofisotopic distributions. In Figure 13A,B the zoom scans of[Mþ2H]2þ of horse cytochrome c and [Mþ2H]2þ of horsemyoglobin clearly show the broadening effect. When thecorona discharge was activated, the signals completelydisappeared. This result possibly suggests that the coronadischarge leads to an extensive decomposition of theprotein molecules or, alternatively, that the protein ionicspecies, observed without corona discharge, must neces-sarily already be present in the solution (i.e., they do notoriginate from gas-phase reactions activated by the coronadischarge). A mixture of the two standard proteins was alsoanalyzed in order to verify the power of the proposedmethod in the analysis of protein mixtures. The spectrawere acquired without and with TEA addition. TEA isused, as described in the ‘‘Deprotonant Agents,’’ in order toreduce the protein charge state. Figure 14A,B (Cristoniet al., 2002b) show the spectra obtained; the C-seriescorresponds to the m/z ions of horse cytochrome c, and theM-series correspond to the ions of horse myoglobin.Figure 14A shows the spectrum of the protein mixtureacquired in the positive-ion mode in the m/z 3000–12,000range without using TEA. In that spectrum, the peaks thatcorresponds to the ions of horse cytochrome c were moni-tored at higher Signal/Noise ratio (counts per second valueof the most abundant signal of horse cytochrome c was5� 106) than for the pure (single-protein) solution (countsper second value of the most abundant signals of horsecytochrome c was 2� 106), whereas in the case of horsemyoglobin only the [Mþ2H]2þ ion was detected. Thatbehavior is probably due to proton-transfer reactions

FIGURE 11. Multicharge ions of bovine albumin acquired in the negative-ion acquisition mode in the range

4000–10,000 Th. (Reproduced from Cristoni (2002) with permission from Internet Journal of Chemistry,

copyright 2002.)


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between the two proteins that take place in solution and inthe gas phase. Figure 14B (Cristoni et al., 2002b) shows thespectrum obtained with the proposed approach and adding1%(v/v) TEA to the protein mixture solution; the multi-charged signals due to horse cytochrome c and horsemyoglobin are both clearly detected when acquiring thespectrum in the negative-acquisition mode.

In order to verify the ability of the proposed methodbased on the use of APCI conditions (but withoutdischarge) for peptide analysis, three standard peptides

(bombesin, trityrosine, and YGG) were analyzed. Themethod was also applied in the analysis of peptides ob-tained by enzymatic digestion of horse cytochrome c andhorse myoglobin. The spectra of a mixture of the threestandard peptides obtained by ESI and no-discharge APCIare shown in Figure 15A,B, respectively, (Cristoni et al.,2002b); in the case of ESI spectra [MþH]þ, [MþNa]þ,[Mþ2H]2þ, and [MþNaþH]2þ ions of bombesin weredetected. For trityrosine, only [MþH]þ and [MþNa]þ ionswere generated, whereas in the case of YGG, only the

FIGURE 12. ESI spectra of a mixture of cytochrome c, horse myoglobin, and bovine albumin acquired

(A) without using TEA and (B) using TEA. (Reproduced from Cristoni (2002) with permission from Internet

Journal of Chemistry copyright 2002.)


392

[MþH]þ ion is present. Good signal intensity was achieved(counts per seconds of the most abundant signals was1� 106). In the spectrum obtained by no-discharge APCI(Fig. 15B), the same behavior was observed, but with lowerchemical noise.

Some experiments were performed by HPLC-ESI-MS/MS and HPLC-APCI-MS/MS to analyze the pep-tides obtained by tryptic digestion of two standardproteins (horse cytochrome c and horse myoglobin). TheSEQUEST program (Eng, McCormack, & Yates, 1994)was used to characterize the protein, using a protein fastadatabase (nr.fasta). To confirm the results obtained withthe SEQUEST software, a de novo sequence analysis wasperformed, using the Lutefisk program (Taylor & Johnson,1997; Johnson & Taylor, 2002), and the results obtained bydatabase search were fully confirmed.

In summary, this new approach makes it possible todecompress the multi-charged signal and provides goodsensitivity because the signals are moved to a region ofthe spectrum in which the chemical noise is low. Moreover,the formation of doubly charged ions of high MW peptides(m/z 2000–6000) is privileged. This fact makes it possibleto perform MS/MS experiment on the peptide doublycharged ions the best ions to fragment in order to obtainpeptide identification and characterization (Cramer &Corless, 2001).

B. Mass Analyzers

In this section, some interesting new analyzers, which leadto significant improvements of the instruments’ perfor-mance, are described. In particular, three new inventions

FIGURE 13. Mass spectra of (A) horse cytochrome c and (B) horse myoglobin, both acquired in the

positive-ion acquisition mode, without corona discharge. (Reproduced from Cristoni et al. (2002b) with

permission from John Wiley & Sons, Ltd., copyright 2000.)


393

are described: (1) the linear quadrupole ion trap; (2) TOFinstruments with a new cryogenic detector; and (3) thecombination of various mass analyzers.

1. Linear Quadrupole Ion Trap

The linear quadruple ion trap has introduced an improve-ment in terms of sensitivity, resolution, and mass accuracyrelative to the usually employed 3D-Ion Trap (Hager, 2002;Schwartz, Senko, & Syka, 2002). In the linear quadrupoleion trap, ion ejection is shown to occur by coupling radialand axial motion in the exit fringing fields of the linear iontrap. This coupling is efficient, and can result in extractionof as much as 20% of the trapped ions. It must be

emphasized that the trapping efficiency of the linear iontrap is high. Thus, this efficiency is an improvement on theusually employed 3D ion traps that have a low trappingefficiency primarily due to their small volume. The ITsmall volume also results in a limited dynamic range,because there is a maximum charge density beyond whichresponse becomes non-linear and the quality of massspectra deteriorates. For this reason, some kind of ion-density control system, for example, Automatic GainControl, is usually employed in 3D ion traps. A very highefficiency in ion trapping and ejection can yield highsensitivity mass spectral responses that have been reach-ed by the linear quadrupole ion trap. The experimentalapparatus performed by Hager (2002) is based on the ion

FIGURE 14. Spectra of a protein mixture of horse cytochrome c and horse myoglobin: (A) spectrum

acquired in the positive-ion acquisition mode and without adding TEA to the solution and (B) spectrum

acquired in the negative-ion acquisition mode in the presence of 1% (v/v) TEA. Corona discharge was off.

(Reproduced from Cristoni et al. (2002b) with permission from John Wiley & Sons, Ltd., copyright 2002.)


394

path of a triple-quadrupole mass spectrometer, allowingeither the Q2 collision cell or the final mass analysisquadrupole (Q3) to be used as a linear ion trap. Distortions(induced by space-charge effects) of the mass-resolvedfeatures occur at approximately the same ion density asreported for conventional three-dimensional ion trapswhen the Q2 region operating at 4� 10�4 Torr pressureis used for trapping the ions. Those distortions are,however, much reduced when the ions are analyzed atlow pressure (3� 10�5 Torr) in the Q3 region and the Q3system operates in linear ion-trap mode. That behavior ispossibly due to the proposed axial ejection mechanism,which leads to ion ejection only for ions of considerableradial amplitude. An interesting application of this newtechnology to the analysis of proteins has been recentlypublished (Mao, Ding, & Douglas, 2002). In that paper, thehydrogen/deuterium (H/D) exchange of gas-phase ions ofholo- and apo-myoglobin was studied by confining the ionsin a linear quadrupole ion trap with D2O or CD3OD at apressure of several mTorr. The exchange takes place on a

time scale of seconds. It is thus possible to study the ionsfolding or unfolding to new conformations.

The linear ion-trap analyzers are also highly effectivein producing MS/MS and MSn spectra for structuralcharacterization of macromolecules (Schwartz, Senko, &Syka, 2002). It is also possible to use the MRM as well asprecursor- and neutral-loss scanning with the enhancedcapability of the linear ion trap (Hager & Yves Le Blanc,2003).

Nowadays, the cost of those instruments is quite highwith respect to the usually employed 3D ion trap, but itprobably will decrease in the near future with the advent ofnew advanced commercial instruments based on the use oflinear ion trap combined with the FTICR or other highaccurate and precise analyzers.

2. TOF Analyzers with New Detectors

As discussed above in the section ‘‘Common Pro-blems and Developments in the Analysis of Bioorganic

FIGURE 15. Direct-infusion spectra of a mixture of bombesin, trityrosine, and YGG obtained by (A) ESI

and (B) APCI (no discharge) ionization methods. (Reproduced from Cristoni et al. (2002a) with permission

from John Wiley & Sons, Ltd., copyright 2002.)


395

Macromolecules by Mass Spectrometry,’’ in TOF analy-zers the sensitivity decreases on increasing the molecularmass of the analyzed ion. An interesting solution toovercome this weak point has been proposed by Frank et al.(1999) and is based on the use of cryogenic detector. Thisimpact detector measures low-energy solid-state excita-tions created by a particle impact. Cryogenic detectorsare expected to have near 100% efficiency even for verylarge, slow-moving molecules, in contrast to MCPs, whoseefficiency drops considerably at large mass. They have thepotential to contribute to extending the mass range thatis accessible by TOF-MS, and to help to improve thedetection limit. In addition, the energy resolution providedby cryogenic detectors can be used for charge discrimina-tion and to study fragmentations and internal energies ofbioorganic macromolecules.

Conventional MCD detectors rely in their detectionmechanism on secondary-electron formation. In contrast,cryogenic detectors are energy-sensitive and energy-resolving (‘‘calorimetric’’) detectors. When applied toTOF-MS, cryogenic detectors measure the low-energysolid-state excitations, called phonons, that are created by aparticle impact. Phonons are quantized crystal-latticevibrations, and can be measured, in the simplest case, asheat created by the impact. The energy of the phonons istypically less than a few meV, which is much smaller thanthe energy (in the electron volt range) needed to producesecondary electrons or electronic excitation in conven-tional ionization detectors. Those detectors respond to low-energy crystal-lattice excitations, and this response is thereason why cryogenic detectors are more sensitive toweakly ionizing, slow-moving particles.

There are not, at present, many applications published,but this technology represents a possible improvement ifapplied to the TOF analyzer. Much work must be done tomake the use of cryogenic detectors more user-friendly.In fact, the main advantage of the MCP detector is thatthey work at room temperature and do not need a coolingcontrol system.

3. Multiple Mass Analyzers

Various combinations and arrangements of mass analyzershave been described in order to improve instrumentalperformance (Chen et al., 1999; Medzihradszky et al.,2000; Collings et al., 2001; Ni & Chan, 2001; Kurahashiet al., 2002; Wattenberg et al., 2002; Yergey et al., 2002).The multiple mass analyzers mainly produced are: IT/TOF,quadrupole/TOF, and TOF/TOF. For instance, a powerfulsystem that combines a linear ion trap with TOF has beendescribed by Collings et al. (2001). The high trappingefficiency of the linear ion trap was combined with thehigh-resolution and mass accuracy of TOF. Chen et al.(1999) analyzed protein spots from two-dimensional (2-D)

gel electrophoresis of a human erythroleukemia cell lineby analysis of the in-gel tryptic digests, using capillaryliquid chromatography (LC) separation coupled withelectrospray ionization mass spectrometry (ESI-MS). Thiswork was performed on an electrospray/ion trap storage/reflectron time-of-flight mass spectrometer system (ESI-IT-reTOFMS). Using that approach, a substantial fractionof the protein sequence could be covered and identified. Ithas also been shown that a considerably improved coverageof the protein sequence can be obtained with this methodrelative to matrix-assisted laser desorption/ionization massspectrometry (MALDI-MS).

A further improvement in the analysis of bioorganicmacromolecules has been recently obtained by thequadrupole/TOF combination (Standing et al., 2001). Thatinstrument can use ESI and MALDI sources. Wattenberget al. (2002) evaluated the usefulness of MALDI Q/TOFdata for protein identification. The comparison of MS dataof protein digests obtained by a conventional MALDI TOFinstrument with the MS data from the MALDI Q/TOFreveals peptide patterns with similar intensity ratios. TheMALDI Q/TOF MS/MS data of 24 out of 26 proteolyticpeptides produced by trypsin or Asp-N digestions weresuccessfully used for protein identification via database-searching, thus indicating the general usefulness of the datafor protein identification. De novo sequencing, using amixture of 16O/18O water during digestion, was explored,and de novo sequences for a number of peptides wereobtained. Moreover, Kurahashi et al. (2002) used an ESI-tandem quadrupole/orthogonal-acceleration time-of-flight(Q/TOF) mass spectrometer combined with a nano-HPLCsystem to determine the glycosylation site and theglycan structure in glycoprotein TIME-EA4 (EA4) fromBombyx diapause eggs. EA4 was digested with trypsin andChymotrypsin to identify the glycosylated peptide. LC-MS/MS analysis of that peptide fragment revealed, at thesame time, the sequence of the attached oligosaccharideand the glycosylation site. The combination of a nano-HPLC system and a highly sensitive Q/TOF massspectrometer has been demonstrated to be quite effectivefor the analysis of glycoproteins from natural sources ofrelatively low purity and limited availability. Furthermore,Ni & Chan (2001) have used the Q/TOF system to analyzeoligonucleotides. The combination of ESI and quad-rupole time-of-flight (Q/TOF) mass spectrometry repre-sents a powerful tool to verify/determine oligonucleotidesequences. An ESI-Q/TOF instrument provides better sen-sitivity and much higher resolution compared with eitherESI-triple quadrupole or ESI-ion trap devices. With itshigh-resolution capability, the quadrupole time-of-flightinstrument can provide an isotope pattern to support thecharge-state assignment. It must also be emphasized that,as shown by Bateman et al. (2002), tandem quadrupoletime-of-flight (Q-TOF) mass spectrometry gives rise to


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very good results if used in the precursor-ion or neutral-lossscanning mode.

Another interesting configuration to perform fragmen-tation studies is the TOF/TOF system. Yergey et al. (2002)have shown that the recently developed MALDI TOF/TOFinstrument yields relatively complex but interpretablefragmentation spectra. When coupled with a straight-forward sequence extension algorithm, it is possible todevelop from the spectra complete de novo peptide se-quences. That approach has been applied to a set ofpeptides derived from the tryptic digestion of electrophor-etically separated sea urchin egg membrane proteins. Theresults were in essential agreement with those obtained byconventional database-searching approaches. The TOF/TOF was able to detect errors in published sequences, andcan obtain sequences of peptides that differ in mass by 1Dalton (Da). Another application of this technique topeptide characterization was described by Medzihradszkyet al. (2000). In that application, a new matrix-assisted laserdesorption/ionization (MALDI) time-of-flight/time-of-flight (TOF/TOF) high-resolution tandem mass spectro-meter was described for sequencing peptides. The maindisadvantage of that technique is related to the fact thatthe MALDI ion source used with the TOF/TOF mainlyproduces singly charged ions that give rise to morecomplex fragmentation spectra than those obtained byfragmenting the doubly charged ions. On the other hand,it provides high sensitivity, resolution (resolution withreflector >12,000; resolution obtained working in MS/MSmode >2000), and mass accuracy (mass accuracy withreflector and internal standard �� 5 ppm; mass accuracyin MS/MS mode �� 50 ppm), acquiring the spectra withMS and MS/MS approaches.

Another interesting mass analyzer coupling is that oflinear ion trap with FTICR. It must be emphasized thatexternal ion accumulation in a two-dimensional (2D)multi-pole trap has been shown to increase the sensitivity,dynamic range, and duty cycle of the Fourier transform ioncyclotron resonance (FTICR) mass spectrometer (Belovet al., 2001; Harkewicz et al., 2002). For instance, in aninteresting paper Harkewicz et al. (2002) have shown thedevelopment of a data-dependent external m/z selectionand accumulation. The ions are data-dependently selectedon the fly in a linear quadrupole ion guide, and accumulatedin a second linear RF-only quadrupole trap that immedi-ately follows. A major benefit of ion pre-selection priorto external accumulation is the enhancement of ionpopulations for low-level species. That development isexpected to expand the dynamic range and sensitivity ofFTICR for applications, including analysis of complexpolypeptide mixtures.

Other configurations are theoretically possible, but themost promising technologies have been described in thissection. It must also be emphasized that, nowadays, the cost

of these combined instruments is higher than those ofinstruments with a single mass analyzer, but it is possiblethat their fast diffusion, thanks to their high capability fromthe point of view of sensitivity, mass resolution, massaccuracy, precision, MS/MS, parent-ion and neutral-lossscan, will lead to a decrease in their cost.

V. SOFTWARE FOR DATA TREATMENT

The software that is used in the mass spectrometricanalyses of bioorganic macromolecules is mainly requiredto predict, acquire, and analyze data. Predictive softwarecan give several kinds of information about knownbioorganic macromolecules. For example, knowing thechemical composition of the macromolecule, it is possibleto predict the isotopic profile. Some programs that providethose results are: ‘‘Isotopic Profiler’’ (http://www.chemsw.com/13063.htm) and ‘‘Isotope Pattern Calculators’’ (http://homepages.ihug.co.nz/�les/). Those algorithms are usefulif a high-mass resolution analyzer (for instance, an FTICR)is used. In that case, a useful method to characterizebioorganic macromolecules is to interpret their isotopicclusters. For example, the McLafferty’s group (Horn,Zubarev, & McLafferty, 2000) developed a fully automatedcomputer algorithm to analyze mass spectra of peptidesand proteins. That method uses a subtractive peak-findingroutine to locate possible isotopic clusters in the spectrum.Those clusters are subjected to a primary charge determi-nation. Finally, the method of least-squares fitting isapplied to the theoretically derived isotopic abundancedistribution for the m/z determination of the most-abundantisotopic peak, and the statistical reliability of that de-termination. The program is generally applicable toclasses of large molecules, including DNA and polymers.Other predictive software can predict the envelope ofESI-generated protein ions. For example, the ESI calcu-lator software (http://www.geocities.com/CapeCanaveral/6513/esiframe.html) can perform that calculation. Theeffects of metal-ion adducts are also fully considered. Anumber of predictive tools is available for proteins andpeptides. For example, the software JPAT (http://www.pix-elgate.net/mjones/index.htm) is a Java API (ApplicationProgram Interface) designed to calculate the results of theprotease digestion of proteins and the MS/MS fragmenta-tion of peptides. JPAT also contains GUI components todisplay the results. The software MassXpert (http://frl.lptc.u-bordeaux.fr/website-frl/massxpert/massxpert-main.html) can predict and analyze the mass spectrometric dataobtained for proteins and peptides. That software is alsoable to predict the fragmentation pathway of peptidesobtained by the enzymatic digestion of the proteins.

Software PAWS (http://prowl.rockefeller.edu/software/contents.htm) is another interesting predictive algorithm.


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That tool can predict which peptide fragments can beproduced by enzymatic digestion with various enzymes. Itis also able to predict the amino acid residues exposed to thesolvent, and it can provide other structural predictions.

Software for spectral acquisition is typically soldby mass spectrometer manufacturers; many alternativescan be taken into account (http://www.spectroscopynow.com/).

Many algorithms have been developed for the massspectrometric data analysis of bioorganic macromolec-ules. An important tool is represented by the mass-deconvolution program. In practice, multi-charged ionsare typically produced by the ESI source, in the m/z 100–2000 range. In order to obtain the molecular mass of theanalyte, it is necessary to use a deconvolution program. Thecalculation of mass by processing the ESI multi-chargedspectrum is quite easy—in principle. By considering twoadjacent multi-charged peaks (differing by one proton), it ispossible to write the follow equations:

m=z1ð Þ ¼ M þ nHð Þ=n ð1Þm=z2ð Þ ¼ M þ ðn þ 1ÞHð Þ=ðn þ 1Þ ð2Þ

(m/z1) and (m/z2) are the m/z ratios of the two adjacentmulti-charged peaks, M is the mass of the (neutral)bioorganic macromolecule, and n is an integer. Thus,solving Equations (1) and (2) gives a simple method tocalculate the value of M and n. This simple algorithmworks well if only a single macromolecule is present insolution; otherwise, a more complex and resolving algo-rithm must be used.

Other software tools can predict the sequence ofproteins and peptides by a database search (Eng,McCormack, & Yates, 1994; Pevzner, Dancik, & Tang,2000) and de novo sequence (Taylor & Johnson, 1997;Johnson & Taylor, 2002; IsoPro software: http://members.aol.com/msmssoft/) by analyzing the tandem mass spectra(MS/MS) of the peptide obtained by enzymatic digestionof the protein molecules. An interesting paper of Fenyo(2000) describes the various tools usually employed toanalyze the proteome. Furthermore, some interesting bio-informatic solutions for the analysis of proteins andpeptides have been recently reported by Field, Fenyo, &Beavis (2002) and Li et al. (2002).

Another important aspect of the data analysis is high-throughput and automatization. Nowadays, a great volumeof data is produced and stored, and the time needed toanalyze these data must be reduced in order to increase theproductivity. For instance, an interesting application ofKurvinen et al. (2002) describes a new algorithm for theautomatic interpretation of mass spectra of glycerolipids.The algorithm utilizes a user-specified list of para-meters needed to process the spectra. The compounds areidentified according to range of measured m/z values, after

which the spectra are automatically corrected by thecontent of naturally occurring isotopes, and ion intensitiesof identified compounds by a response-correlation factor.If quantitative analysis (using internal standards) is per-formed, then all the identified compounds in the sample canbe automatically quantified.

A high-throughput and automation are both requiredfor the determination of SNPs (Pusch et al., 2001). Analysisof SNPs is, in fact, a rapidly growing field of research thatprovides insights into the most common type of muta-tions. The resulting information has a strong impact inthe fields of pharamacogenomics, drug development, for-ensic medicine, and the determination of specific diseasemarkers. The software is suitable for highly automatedMALDI-TOF-MS SNP genotyping.

Some software can be used in the determination of theoptimal analytical conditions for mass spectrometer asproposed by Whalen et al. (2000). In that application, anautomated flow-injection analysis mass spectrometry sys-tem, named AutoScan, was developed to allow the rapiddetermination of the optimal conditions for the mass (MS)and tandem mass spectrometry (MS/MS) analysis of newchemical compounds. Analytes are injected, four at a time,into an injection manifold, and conventional mass spectraare acquired in both polarities (þ/�). The software de-termines the optimal polarity for all analytes, and uses theresults to build the injection sequence for product-ionscanning. Samples are automatically re-injected underMS/MS conditions, and product ion scans that loopamong different collision energies are collected for eachanalyte. The resulting data are processed automatically,and the optimal MS/MS transitions for each analyte areselected.

It must be emphasized, however, that software de-signed for high-throughput and automation purposes canlead to false positive results. Thus, it is necessary to controlthe results by statistical analysis (Eriksson, Chait, & Fenyo,2000; Pevzner et al., 2001).

It must also be stressed that the entire data-elaborationprocedure (prediction, acquisition, and analysis of massspectrometric data) has been simplified by various pro-grams. Particularly in the case of acquisition, nowadays,many programs simplify the use of the instrument by anautomatic setting of instrumental parameters (e.g., auto-matic tune on a peak of interest, and automatic calibrationusing a standard mixture). Another characteristic of someprograms is that they do not allow one to modify someadvanced parameters of the mass spectrometers to avoidany operator-caused damage of the instrument. However, ifon one hand that strategy simplifies and makes surer the useof instruments, then the versatility of the use for an expertoperator is strongly reduced.

Other information, on the applications of bioinfor-matics to the analysis of bioorganic macromolecules by


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mass spectrometry, can be found in the spectroscopynowweb site (http://www.spectroscopynow.com/).

VI. CONCLUSIONS ANDFUTURE DEVELOPMENTS

MS studies of bioorganic macromolecules have recentlyundergone rapid development. Many bioorganic macro-molecules have been analyzed with mass spectrometrytechniques. Some macromolecules of particular interestare: (1) proteins and peptides; (2) oligonucleotides; (3)oligosaccharides and glycoconjugates; and (4) variouscomplexes (DNA-protein, antibody receptor, macromole-cules-metals). The two most useful techniques in this fieldare MALDI and ESI. The MALDI source produces mainlylow-charge (typically monocharge) ions, whereas the ESIsource produces multi-charged ions of macromolecules.

New instrumental approaches have been developed inorder to increase sensitivity, mass accuracy, and resolution.Those results can be obtained by improving the ionizationsource and the mass analyzer. Moreover, it is necessary tosolve the problem of the ESI source with buffers, salts, anddetergents. Nowadays, it is, in fact, necessary to use LC,CE, and other separation techniques coupled with ESI-MSinstruments in order to separate the sample from salts,buffers, and detergents before mass analysis. Furthermore,the development of new mass analyzers that can monitorhigh m/z ranges (for instance, the new generation of iontraps or the TOF analyzer) could be of interest to analyzeions with high mass and low charge. In fact, in the analysisof mixtures of bioorganic macromolecules, the excessiveoverlap of the signals in ESI spectra makes this tech-nique not highly effective. The isotope-spacing of m/z unitsare inversely proportional to the charge of the ions, and thedistance to the adjacent multi-charge ions on the m/z scaledecreases with increasing charge. Thus, the resolution ofmixture components with small differences in the mass/charge ratio could be impossible due to the mass chargescale compression. Another interesting objective is toobtain abundant doubly charged ions of peptides with ahigh molecular mass (2000–6000 Da). Those ions give riseto MS/MS spectra that are much simpler to elaborate bydatabase and de novo sequence approaches. That fact canimprove the characterization of high MW peptides thatare obtained by enzymatic digestion.

Some interesting methods and new ionization sourceshave been recently described in order to improve theionization source performances. For instance, two inter-esting approaches have been recently used to reduce thecharge of the multi-charged ions: (1) deprotonating agents,and (2) APCI without corona discharge. The use ofdeprotonating agents is based on ion/ion and ion/moleculeproton-transfer reactions. Such mechanisms are an effec-

tive way to facilitate the resolution of ions in electrospraymass spectrometry by reducing the charge state of themulti-charged ions of the bioorganic macromolecules.The no-discharge APCI is based on the vaporization ofsample solutions in atmospheric pressure conditions, with-out the presence of any corona discharge, leading anywayto the production of a rather high number of ions. In thoseconditions, in the case of proteins, the formation ofdoubly charged species is privileged and, considering theirusefulness in further MS/MS studies, the observedphenomenon was considered of high interest. Those newapproaches make it possible to decompress the multi-charge signals and to obtain good sensitivity because thesignals are moved into a region of the spectrum in whichchemical noise is low. Moreover, it must be emphasizedthat the no-discharge APCI ionization technique seemsto be only slightly affected by the presence of salts andbuffers in solution.

An interesting improvement has been obtained bycoupling LC and MALDI techniques. Such technologiesare usually named continuous-flow MALDI (cf-MALDI).The main advantage of directly coupling LC to a MALDIsource is the introduction of a desalting step beforeexecuting the MALDI analysis. This technique is, in ouropinion, very promising and some groups are workingto improve its sensitivity. However, two problems areassociated with cf-MALDI. The first is the limitation tonon-crystalline matrices, and the lack of suitable liquidmatrices for irradiation at UV laser wavelengths. Anotherlimit is the low standardization level of this method com-pared with ESI that is commonly employed for LC/MS.

Other interesting approaches that have been developedare the liquid-junction interfacing of the separation columnoutlet and the inlet of the capillary sprayer, using micro-and nano-ESI, and the miniaturized ESI techniques.

Two new ionization interfaces recently producedare AP-MALDI and SSI. A MALDI source that works atatmospheric pressure has the advantage that it can beconnected to a mass analyzer, which produces high-qualityfragmentation spectra (e.g., an ion trap). In fact, the PSDtechnique is not as efficient as the MS/MS approach usedwith an ion trap or a triple quadrupole. The main problem ofthat approach is that the MALDI source mainly producessingly charged ions that give rise to MS/MS spectra that aremore difficult to interpret than those obtained by frag-menting the doubly charged ions. However, that ionizationsource has high-sensitivity and makes it possible toperform high-throughput analyses. Furthermore, it is lessaffected than ESI by the presence of salts and buffers.

A promising ionization technique recently developedto analyze bioorganic macromolecules is SSI. That sourceis similar to the ESI source, but it does not use a highpotential to ionize the sample molecules. In SSI, vaporiza-tion is done so that the surface layer of the solution, in the


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region of charge separation, is stripped by a fast gas flow,and electrically charged airborne droplets are created. Thediameter of those electrically charged droplets shrink byvaporization of solvent molecules from the surface, andprotonated molecules are formed in the gas-phase. Themain advantages of that ionization source are related to itssensitivity, and to the fact that it does not use high poten-tials. Also, in this mode, if salts and buffers are present inthe analyzed mixture, they do not lead to a discharge effectbetween the needle of the ionization source and the instru-ment. On the other hand, the solution matrix compositiondoes strongly influence the kind of ions that are produced.

In the field of the analyzers, some progress has alsobeen made. For example, a linear quadrupole ion trap hasbeen developed to increase the sensitivity, resolution, andthe mass accuracy of ion-trap instruments. In the linearquadrupole ion trap, ion ejection is shown to occur throughthe coupling of radial and axial motion in the exit fringingfields of the linear ion trap. The coupling is efficient, andcan result in a high trapping efficiency and in an ejectionefficiency of 20% of the trapped ions. This increase is animprovement with respect to the usually employed 3D iontraps that have low trapping efficiency primarily due totheir small volumes.

Also interesting is the development of new cryogenicdetectors for TOF mass spectrometry. Those detectorsrespond to low-energy crystal-lattice excitation, and makethem more sensitive to weakly ionizing, slow-movingparticles. For the moment, not many applications havebeen produced, but this technology represents a possibleimprovement applied to the TOF analyzer. The MCPremains, for now, more user-friendly due to the fact thatthey work at room temperature.

Many instruments make use of multiple mass analy-zers. IT/TOF, quadrupole/TOF, and TOF/TOF multipleanalyzers are now more used due to their advantages interms of sensitivity, resolution, and mass accuracy.

Finally, it must be emphasized that significant pro-gresses have been achieved in the development of softwareto predict, acquire, and interpret the data. New softwarealso improves the throughput of data analysis; howeverparticular attention must be used in order to detect falsepositive results.

In the future, the development of new solutions toincrease the sensitivity, mass accuracy, and mass resolutionof the mass analyzers will be possible. Those parametersmust be constantly improved particularly for the analysis ofbioorganic macromolecules. New possible developmentsinclude new analyzers and new multiple mass analyzers.New ion sources, normal and miniaturized, and lensgeometries are required in order to increase the numberof ion carried to the mass analyzer, and to increase thesensitivity. A promising solution to reduce the negativeeffect of the salts and buffers present in the biological

solutions is the no-discharge APCI. Further developmentof that source should be carried on in order to increase theion production efficiency. Furthermore, new chemicalapproaches should be studied in order to reduce the chargeof multi-charge ions. Those chemical approaches, in fact,combined with the existing ionization sources and withmass analyzers with a high acquisition range, could beuseful to reduce the complexity of the spectra of biologicalmixtures. Furthermore, it must also be emphasized thatenhanced capability in term of MRM as well as precursor-,MS/MS-, neutral-loss scanning and linear dynamic rangeare present in the new generation of mass spectrometer(e.g., combined instrument and linear ion trap). With thefast development of the mass analyzer technology, thosecharacteristics will have probably a fast improvement. Forinstance, the linear ion trap combined with FTICRinstruments will permit one to obtain the high-accuracymass of the MS/MS fragments as well as of the precursorion. Finally, it must be emphasized that ion pre-selection isexpected to expand the linear dynamic range, duty cycle,and sensitivity of FTICR for applications that include theanalysis of complex macromolecules mixtures.

ACKNOWLEDGMENTS

The authors thank Dr. Remo Cristoni, Mrs. Maria Florio,and Mrs. Karim Amaya Mendoza for their support.

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