Selective enrichment of glycopeptides for mass spectrometry analysis using C18 fractionation and...

$: Selective enrichment of glycopeptides for mass spectrometry analysis using C18 fractionation and titanium dioxide chromatography$
Research Article

Selective enrichment of glycopeptides formass spectrometry analysis using C18fractionation and titanium dioxidechromatography

Comprehensive glycoprotein characterization based on mass spectrometry (MS) is chal-

lenging because of low concentration of glycopeptides and suppression effect of abundant

non-glycosylated peptides in MS. Therefore, it is vital to enrich glycopeptides before MS

analysis. A new method was developed to selectively enrich glycopeptides from complex

sample by coupling C18 fractionation with titanium dioxide (TiO2) enrichment. The new

method allows to selectively enrich N-linked glycopeptides with various glycan forms and

different sequence lengths. Compared with single TiO2 method, the established method

demonstrated higher glycopeptide selectivity and higher glycosylation heterogeneity

coverage. Further application of this method to mixture of non-glycosylated protein and

glycoprotein digests at different levels reveals the feasibility of enrichment of tryptic

glycopeptides from simple proteomics samples.

Keywords: C18 fractionation / Glycopeptide / Hydrophilic interaction chromato-graphy / Mass spectrometry / TiO2 enrichmentDOI 10.1002/jssc.201100427

1 Introduction

N-Linked protein glycosylation is one of the most common

and important post-translational modifications (PTMs), with

an estimated 50% of proteins receiving glycosylation [1].

Glycoproteins are involved in many key biological processes,

such as cell adhesion, receptor activation and signal

transduction. Protein glycosylation alterations indicate

aberrant cellular changes in a number of diseases [2–5].

Comprehensive glycoprotein characterization is essential to

our understanding of the role that protein glycosylation

plays in biological processes and ultimately in the etiology of

disease. Comprehensive glycoprotein characterization

entails glycosylation site identification, glycan structure

determination, site occupancy and glycan isoform distribu-

tion. Although recent advancements in mass spectrometry

(MS) made large-scale identification of proteins feasible

[6, 7], it is still very challenging to analyze protein

glycosylation in complex samples. Low concentration and

microheterogeneity of glycopeptides create a clear challenge

to MS analysis. Moreover, non-glycosylated peptides can

exert a suppression effect on samples [8]. Therefore, it is

pivotal to remove non-glycosylated peptides and enrich

glycopeptides prior to MS analysis.

Because peptides are more compatible with liquid

chromatography (LC) separation and MS identification,

proteins are usually digested with a protease to generate a

mixture of peptides in shotgun proteomics. Several methods

have been developed to enrich glycopeptides from complex

samples at the peptide levels. This includes lectin affinity

chromatography [9–11], hydrazide chemistry [12, 13] and

hydrophilic interaction chromatography (HILIC) [14–17].

Lectin affinity chromatography is the most widely used

technique to capture glycopeptides, but different lectins

have diverse affinities toward different glycans [9, 10].

Hydrazide chemistry specifically isolates glycopeptides from

complex mixtures [12, 13] but suffers from low glycosylation

heterogeneity coverage [18]. In the last few years, HILIC in

solid-phase extraction (SPE) mode is increasingly employed

to enrich glycopeptides. Separation of glycopeptides from

non-glycosylated peptides is based mainly on hydrophilic

interaction of a large number of hydroxyl groups in the

glycans with hydrophilic matrices. Under HILIC mode,

peptides are dissolved in the high content of organic solvent

Bo Zhang1

Qianying Sheng2

Xiuling Li3

Qi Liang1

Jingyu Yan3

Xinmiao Liang3�

1Department of General Surgery,Affiliated Union Hospital ofHuazhong University of Scienceand Technology, Wuhan, HubeiProvince, P. R. China

2Engineering Research Center ofPharmaceutical ProcessChemistry, Ministry ofEducation, School of Pharmacy,East China University of Scienceand Technology, Shanghai,P. R. China

3Dalian Institute of ChemicalPhysics, Chinese Academy ofScience, Dalian, P. R. China

Received May 16, 2011Revised July 14, 2011Accepted July 14, 2011

Abbreviations: AA, amino acid; FA, formic acid; Fuc, fucose;

HILIC, hydrophilic interaction chromatography; HRP,horseradish peroxidase; IgG, immunoglobulin G; PTMs,post-translational modifications; RNase B, ribonuclease B;

Ser, serine; Thr, threonine; TiO2, titanium dioxide; Xyl,xylose

�Additional correspondence: Xinmiao Liang

E-mail: [email protected]

Correspondence: Dr. Xiuling Li, Dalian Institute of ChemicalPhysics, Chinese Academy of Science, 457 Zhongshan Road,Dalian 116023, P. R. ChinaE-mail: [email protected]: 186-411-84379539

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

J. Sep. Sci. 2011, 34, 2745–2750 2745

and loaded onto the hydrophilic matrix; elution of glyco-

peptides with high content of water is then achieved after

successive washing to remove non-glycosylated peptides.

Thus, various types of N-linked glycopeptides are separated

from non-glycosylated peptides and the glycosylation

heterogeneity coverage is improved. However, glycopeptide

fraction is contaminated by non-glycosylated peptides,

which contain multiple serine (Ser)/threonine (Thr) resi-

dues as well as larger molecular weight [19]. Moreover,

HILIC under general conditions is not effective for enrich-

ing less hydrophilic glycopeptides, e.g. the high-mannose

type ribonuclease B (RNase B) glycopeptides [20]. The

selectivity for glycopeptides is improved in two different

ways: the utilization of ion-pairing acids [21], such as

trifluoroacetic acid (TFA), or development of novel HILIC

matrices, such as click maltose [16] and click TE-Cys [22].

Recently, titanium dioxide (TiO2) under HILIC mode has

been utilized to enrich sialylated [23] and neutral glyco-

peptides [24]. TiO2 demonstrates stronger hydrophilic affi-

nity and higher selectivity for glycopeptides than Sepharose

and click maltose [24].

Even though TiO2 method features broad glycan speci-

ficity and high glycosylation heterogeneity coverage, it is still

difficult to achieve global glycosylation analysis because of

its medium glycopeptide selectivity. To improve the

enrichment selectivity of glycopeptides, a combination of

reverse-phase (RP) LC and HILIC SPE is developed to bind

proteinase K- and chymotrypsin-produced glycopeptides

[25]. However, these two kinds of low-specificity proteases

generate very short glycopeptides, making it difficult to

identify the peptide sequences with MS after the release of

glycans [25]. Specific trypsin is the most commonly used

protease in proteome and generates preferred peptide

length for effective fragmentation in MS/MS. To explore the

comprehensive glycoproteome, a method that can be

applied to tryptic digests of different proteins should be

developed.

In this study, we demonstrated the combination of C18

fractionation and TiO2 chromatography for selective glyco-

peptide enrichment. The efficiency of this method was

evaluated with glycopeptides containing various types of

glycans and different sequence lengths. To address the

application of this technique to the analysis of relatively

simple mixture, tryptic digests of non-glycosylated protein

and glycoprotein in different molar ratios were investigated,

and the enrichment selectivity of both acidic and neutral

glycopeptides was assessed.

2 Materials and methods

2.1 Materials

Horseradish peroxidase (HRP, 98%), human serum immu-

noglobulin G (IgG), human serum albumin (HSA), bovine

RNase B, chicken ovalbumin (re-purified by RPLC) and

ammonium bicarbonate were purchased from Sigma-

Aldrich (St. Louis, MO, USA). TiO2 was obtained from GL

Sciences (Tokyo, Japan). Formic acid (FA) was obtained

from Acros Organics (Geel, Belgium). TFA was purchased

from TEDIA (Fairfield, OH, USA). Sequencing grade-

modified trypsin was purchased from Promega (Madison,

WI, USA). Empore C18 disk was purchased from 3M (St.

Paul, MN, USA). Ammonium hydroxide was purchased

from Fluka (Buchs, Switzerland). Acetonitrile (ACN) was

purchased from Merck (Darmstadt, Germany). Water was

prepared with a Milli-Q system (Millipore, Bedford, MA,

USA).

2.2 Trypsin digestion of glycoproteins

Glycoprotein standard was separately dissolved in denatur-

ing buffer containing 8 M urea and 50 mM ammonium

bicarbonate and incubated for 3 h. The resulting protein

solution was reduced with 50 mM dithiothreitol for 2 h at

371C. Then, 50 mM iodoacetamide was used for alkylation,

and the mixture was incubated in the dark for 30 min at

room temperature. The solution was diluted to tenfold with

50 mM ammonium bicarbonate and digested with trypsin at

an enzyme/protein ratio of 1:30 w/w. Digestion was stopped

with FA to a final concentration of 0.5%.

2.3 Enrichment of glycopeptides

For C18 fractionation, two small pieces of Empore C18 disk

were packed into the GELoader tips. The microcolumns

were conditioned and equilibrated with 20 mL of 50% ACN/

0.1% FA and 0.1% FA, respectively. Then tryptic digests in

20 mL of 0.1% FA were loaded into the microcolumns.

RNase B glycopeptides were obtained with pooling flow-

through fraction and 20 mL of 0.1% FA wash fraction from

C18 microcolumns. IgG and HRP glycopeptides were eluted

with 20 mL of 10% ACN/0.1% FA and 20% ACN/0.1% FA,

respectively, after rinsing with 20 mL of 0.1% FA. Ovalbumin

glycopeptides were eluted with 20 mL of 25% ACN/0.1% FA

after washing with 0.1% FA. The collected peptides were

dried with speed vacuum centrifuge (Labconco, Kansas City,

MO, USA) and redissolved with 20 mL of 50% ACN/0.1% FA

for further TiO2 enrichment.

For TiO2 enrichment, the protocol was carried out as

reported [24] with minor modifications. Briefly, 1 mg of

TiO2 microspheres was packed into GELoader tips. Glyco-

peptide fractions from C18 were loaded into the TiO2 tips.

After rinsing with 50% ACN/0.1% FA, RNase B glycopep-

tides were eluted with 0.1% FA. HRP glycopeptides were

released from the TiO2 with 10% ACN/0.1% FA after

stepwise washing with 50 mM NH4HCO3 and 50% ACN/

0.1% FA. IgG glycopeptides were detached from the TiO2

with 5% TFA after successive washing with 50% ACN/0.1%

FA and 0.1% FA. Ovalbumin glycopeptides were released

from the TiO2 with 5% ammonium hydroxide after washing

twice with 0.25% ammonium hydroxide.

J. Sep. Sci. 2011, 34, 2745–27502746 B. Zhang et al.


2.4 MS analysis

Nano electrospray ionization (ESI)-MS and MS/MS analysis

under positive ion mode were carried out on a quadrupole

time-of-flight (Q-TOF) tandem mass spectrometer (Waters,

Manchester, UK). Peptides dissolved in 50% ACN/0.1% FA

were pumped into nano ESI source with an X’TremeSimple

nano-LC system (Micro-Tech Scientific, Vista, CA, USA).

Full-scan MS data and MS/MS data were acquired in the

mass range 500–2000 and 100–2000, respectively. The

collision energy in MS/MS analysis was varied from 30 to

45 eV according to the size and charge of the peptides.

Peptides producing marker oxonium ions such as m/z 163

(Hex1), m/z 204 (HexNAc1), m/z 366 (Hex-HexNAc1) and

m/z 292 (NeuNAc1) were considered as glycopeptides.

3 Results and discussion

3.1 General strategy

HILIC isolates glycopeptides based primarily on hydrogen

bonding between the hydroxyl groups in the matrices and

glycopeptides. Enrichment is thus universal for glycopep-

tides with various types of N-linked glycans. Therefore, high

coverage of glycosylation heterogeneity is achieved with this

method. However, glycopeptide fractions are contaminated

by non-glycosylated peptides with hydrophilic property or

large molecular weight, and such contamination decreases

the enrichment selectivity. Because both the composition of

peptides [26] and glycans [16] affect the retention of

glycopeptides on HILIC matrixes, HILIC under general

conditions is not effective for the enrichment of all

glycopeptides. In order to increase in glycopeptide profile

and improve the selectivity of glycopeptides, it is necessary

to develop appropriate conditions for glycopeptides with

different peptide sizes and glycan types. In this study, we

established a new method for glycopeptide enrichment with

the combination of C18 fractionation and TiO2 enrichment.

The enrichment strategy is summarized in Scheme 1. Four

glycoprotein standards attached with different types of

glycans were separately digested with trypsin. Glycopeptides

with varied sequence lengths and different glycan forms

were fractionated with C18 SPE microcolumns. Because of

their hydrophobicity, non-glycosylated peptides with large

molecular weight tend to be well retained on C18

microcolumns. Compared with their counterpart non-

glycosylated peptides, glycopeptides are more hydrophilic

because of a large number of hydroxyl groups in the glycans.

Therefore, glycopeptides and hydrophilic non-glycosylated

peptides are not or weakly retained on C18 and are found in

the flow through or fractions eluted with low content of

organic solvent, resulting in the removal of majority of non-

glycosylated peptides. The glycopeptide fraction from C18 is

further enriched with TiO2 under SPE mode. Compared

with N-linked glycopeptides containing a common core

pentasaccharide, the remaining non-glycosylated peptides

are less hydrophilic and are removed in the loading or

washing step. Finally, the glycopeptides are eluted from

TiO2 with elution buffer. Glycopeptides are selectively

captured with the above-mentioned procedures and

characterized with Q-TOF MS and MS/MS. For compar-

ison, same amounts of different tryptic peptides were

enriched with single TiO2 microcolumns. Three technical

replicates were performed for each sample with different

methods.

3.2 Selective enrichment of glycopeptides

To demonstrate the efficiency for glycopeptide enrichment,

three well-characterized glycoproteins, bovine RNase B, HRP

and ovalbumin, were selected as model samples. RNase B is a

small glycoprotein containing one N-glycosylation site

attached with high-mannose type glycans. Tryptic peptide

of the single glycosylation site of RNase B consists of four

amino acid (AA) residues. In theory, more than 10 peptides

are produced after trypsin digestion. Signals of glycopeptides

were very low in the RNase B digests because of the

suppression effect of high-abundance non-glycosylated

peptides (Fig. 1A). Majority of non-glycosylated peptides

were removed after enrichment with TiO2, notably enhancing

the signals of glycopeptides (Fig. 1B). However, some non-

glycosylated peptides, such as peptides at m/z 789.31 (31)/

1183.46 (21), 840.38 (31) and 1113.03 (21), co-eluted with

glycopeptides. Sequences of these contaminating peptides are

QHMDSSTSAASSSNYCNQMMK, CKPVNTFVHESLADV-

QAVCSQK and HIIVACEGNPYVPVHFDASV, respectively,

which either contain several Ser/Thr residues or bear

relatively large molecular weight. This result is consistent

C18 SPE

TiO2 SPE

Trypsin digestion

Small peptides

ESI-MS and MS/MS

Glycoproteins

Glycopeptides and

non-glycosylated peptides

Glycopeptides and hydrophilic

non-glycosylated peptides

Large peptides

Glycopeptides

Scheme 1. Strategy for selective glycopeptide enrichment.

J. Sep. Sci. 2011, 34, 2745–2750 Sample Preparation 2747


with the previous study that peptides with multiple Ser/Thr

residues as well as with larger molecular weight tend to co-

elute with glycopeptides under HILIC mode [19]. The

interfering non-glycosylated peptides were thoroughly elimi-

nated through sequential C18 fractionation and TiO2

enrichment. Only signals of glycopeptides were found in

the mass spectrum (Fig. 1C), demonstrating higher glyco-

peptide selectivity of the established method. The higher

selectivity of glycopeptides originates from the fact that non-

glycosylated peptides with large molecular weight retain well

on C18 and are therefore separated from glycopeptides. Small

or less hydrophilic non-glycosylated peptides in the glycopep-

tide fractions of C18 are detached from TiO2 in the

subsequent loading and washing steps. Intensities of

glycopeptides enriched with these two methods were also

investigated. In theory, the intensities of glycopeptides should

be improved because the suppression effect is decreased with

the sequential C18 and TiO2 method. However, no remark-

able difference of glycopeptide intensities was observed with

or without additional enrichment (Table 1), which might

have resulted from sample loss during additional enrich-

ment. All in all the combinations of C18 fractionation and

TiO2 enrichment efficiently improved the selectivity of

glycopeptides.

To validate the enrichment selectivity of glycopeptides

with various N-linked glycans and different sequence

lengths, HRP was digested with trypsin and subjected to

similar experiments. HRP has nine potential glycosylation

sites and eight of them were modified with complex type of

glycans [27]. Sequence length of tryptic HRP glycopeptides

ranges from 6 to 24 AA, which is much larger than that of

RNase B glycopeptides. Figure 2 shows the mass spectra of

desalted HRP digest and glycopeptide fractions enriched

with TiO2 and sequential C18 and TiO2, respectively. Only

seven glycopeptide signals were observed in the HRP digest

resulting from the suppression effect of abundant non-

glycosylated peptides (Fig. 2A). After treatment with only

TiO2, most high-abundance non-glycosylated peptides were

separated from glycopeptides and 13 glycopeptide signals

were characterized (Fig. 2B). After the sequential treatment

with C18 and TiO2, the interfering non-glycosylated

peptides were efficiently removed from the glycopeptide

fraction and totally 20 glycopeptide ions were detected.

Among them, 13 glycopeptide signals are reported and 7

glycopeptide signals were confirmed with MS/MS

(Supporting Information Fig. 1). Confirmation of glycan

structures from two glycopeptides with MS/MS is shown in

Fig. 3. Tryptic peptide at m/z 1179.66 (21) is attached with

Man3GlcNAc2Xyl1Fuc1 (Mannose (Man), N-acetyl-

glucosamine (GlcNAc), xylose (Xyl) and fucose (Fuc))

glycan. At the low mass range, the presence of sugar

oxonium ions at m/z 163.10, 204.12 and 366.21 indicated

that the precursor was indeed glycopeptide. At the high

mass range, sequential loss of Fuc, Xyl and Man residues

from glycopeptide was observed until the fragment ion for

peptide and its GlcNAc bearing species was detected at m/z695.90 (21) and 1390.79 (11) (Fig. 3A). The other peptide at

m/z 1237.78 (31) presents same indicator ions, such as

163.10, 204.12 and 366.21 at low mass range and sequential

loss of Fuc, Xyl and Man residues at high mass range

(Fig. 3B). However, elucidation of these glycopeptide

sequences was impaired using collision-induced dissocia-

tion (CID), resulting from its preferential fragmentation of

the glycosidic bonds.

To confirm the efficacy of the established method for

the enrichment of larger glycopeptides, tryptic digest of

ovalbumin was enriched with this two-step method. Oval-

bumin has one glycosylation site modified with high-

mannose and hybrid-type glycans. Trypsin digestion of

ovalbumin results in the production of glycopeptides

containing 32 AA (YN292LTSVLMAMGITDVFSSSA

NLSGISSAESLK). As many as 11 glycopeptides were iden-

tified after enrichment with sequential C18 fractionation

and TiO2 enrichment compared with 2 glycopeptides in the

C18 desalted digest (Supporting Information Fig. 2). These

results suggested that the established method can be applied

to glycopeptides with various N-linked glycans and different

sequence lengths.

3.3 Application to simple mixture

To validate the selectivity of the established method for

glycopeptide enrichment, a mixture of non-glycosylated

protein HSA and glycoprotein IgG digests with molar ratio

1:1 and 10:1 was used to mimic simple proteomics sample.

Figure 1. Mass spectra of tryptic peptides from RNase B. (A)Total digest desalted with C18; (B) glycopeptide fraction fromTiO2 and (C) glycopeptide fraction from sequential C18 and TiO2.Glycopeptides are labeled with their structures.



Human IgG is an antibody molecule and constitutes 75% of

serum Igs in humans. IgG has one glycosylation site and is

modified with both sialylated and neutral N-glycans.

Commercial IgG is composed of, IgG1 and IgG2, two

variants. The sequences of tryptic IgG glycopeptides contain

nine AA and they are EEQYN180STYR and EEQFN176STFR

for two variants, respectively. HSA is the most abundant

protein in human serum. More than 170 peptides with up to

one missed cleavage site will be generated in the tryptic

solution theoretically. No signals of glycopeptides were

identified in the mass spectrum of C18-desalted HSA and

IgG digests with molar ratio 1:1 (Supporting Information

Fig. 3A). The presence of large amount of non-glycosylated

peptides seriously suppressed the signals of glycopeptides.

After enrichment with sequential C18 and TiO2, signals of

glycopeptides were notably improved and 13 glycopeptide

ions were easily detected (Supporting Information Fig. 3B).

For the case of mixture of HSA and IgG digests with molar

ratio 10:1, no glycopeptide was found in the mass spectrum

of C18-desalted sample (Fig. 4A). After enrichment with the

established method, 12 glycopeptide signals were observed

(Fig. 4B). These data reveal the selectivity of glycopeptides

with this method.

Figure 2. Mass spectra of tryptic peptides from HRP. (A) Totaldigest desalted with C18; (B) glycopeptide fraction from TiO2

and (C) glycopeptide fraction from sequential C18 and TiO2. %,Reported glycopeptides; ~, confirmed glycopeptides with MS/MS spectrum; other peaks are non-glycosylated peptides.

Table 1. Intensities of RNase B glycopeptides enriched with only TiO2 and sequential C18 and TiO2

Peptides Monosaccharide compositiona)

of RNase B glcopeptides

Observed N-glycopeptides

with charge states

Glycopeptide intensity

with TiO2 � (103) b)

Glycopeptide intensity

with C18 and TiO2 � (103) b)

34NLTK37 Man3GlcNAc2 684.3186 (21) 2.0370.16 1.8270.1734NLTK37 Man4GlcNAc2 765.3433 (21) 5.3970.95 4.8570.7234NLTK37 Man5GlcNAc2 846.368 (21) 16.472.5 16.472.934NLTK37 Man6GlcNAc2 927.4015 (21) 9.0671.69 9.4071.6134NLTK37 Man7GlcNAc2 1008.4272 (21) 2.2770.52 2.3370.5434NLTK37 Man8GlcNAc2 1089.4642 (21) 3.1970.47 3.2070.5434NLTK37 Man9GlcNAc2 1170.4951 (21) 0.9370.17 0.9070.16

a) Key for monosaccharide composition: Man, mannose; GlcNAc, N-acetylglucosamine.

b) Results from triplicate experiments.

Figure 3. MS/MS spectra confirmation of HRP glycopeptides. (A)MS/MS spectrum of glycopeptides with m/z 1179.66 (21). (B)MS/MS spectrum of glycopeptides with m/z 1237.89 (31).Glycopeptide fragments are labeled with their structures. P,Peptide backbone; & , N-acetylglucosamine; �, mannose orgalactose; m, fucose; %, xylose.

J. Sep. Sci. 2011, 34, 2745–2750 Sample Preparation 2749


4 Concluding remarks

A selective method for N-linked glycopeptide enrichment

was established through the combination of C18 fractiona-

tion and TiO2 enrichment. Glycopeptides with different

sequence lengths and various glycan types were chosen as

model samples. Compared with single TiO2 enrichment,

sequential C18 fractionation and TiO2 enrichment demon-

strated higher selectivity and higher glycosylation hetero-

geneity coverage for tryptic glycopeptides. Furthermore, this

method was applied to relatively complex sample and

showed superior selectivity. Further studies are now in

progress for selective enrichment and characterization of

glycopeptides in biologically relevant samples.

This work was supported by ‘Project of National ScienceFoundation of China (30801513 and 20975100)’.

The authors have declared no conflict of interest.

5 References

[1] Apweiler, R., Hermjakob, H., Sharon, N., Biochim.Biophys. Acta 1999, 1473, 4–8.

[2] Parekh, R. B., Dwek, R. A., Sutton, B. J., Fernandes, D. L.,Leung, A., Stanworth, D., Rademacher, T. W., Mizuochi,T., Taniguchi, T., Matsuta, K., Nature 1985, 316, 452–457.

[3] Campbell, B. J., Yu, L. G., Rhodes, J. M., GlycoconjugateJ. 2001, 18, 851–858.

[4] Valenzuela, H. F., Pace, K. E., Cabrera, P. V., White, R.,Porvari, K., Kaija, H., Vihko, P., Baum, L. G., Cancer Res.2007, 67, 6155–6162.

[5] Ohtsubo, K., Takamatsu, S., Minowa, M. T., Yoshida, A.,Takeuchi, M., Marth, J. D., Cell 2005, 123, 1307–1321.

[6] Sweet, S. M. M., Bailey, C. M., Cunningham, D. L.,Heath, J. K., Cooper, H. J., Mol. Cell. Proteomics 2009, 8,904–912.

[7] de Godoy, L. M. F., Olsen, J. V., Cox, J., Nielsen, M. L.,Hubner, N. C., Frohlich, F., Walther, T. C., Mann, M.,Nature 2008, 455, 1251–1254.

[8] Dell, A., Morris, H. R., Science 2001, 291, 2351–2356.

[9] Ueda, K., Fukase, Y., Katagiri, T., Ishikawa, N., Irie, S.,Sato, T.-A., Ito, H., Nakayama, H., Miyagi, Y., Tsuchiya,E., Kohno, N., Shiwa, M., Nakamura, Y., Daigo, Y.,Proteomics 2009, 9, 2182–2192.

[10] Drake, R. R., Schwegler, E. E., Malik, G., Diaz, J., Block,T., Mehta, A., Semmes, O. J., Mol. Cell. Proteomics2006, 5, 1957–1967.

[11] Zielinska, D. F., Gnad, F., Wisniewski, J. R., Mann, M.,Cell 2010, 141, 897–907.

[12] Zhang, H., Li, X.-J., Martin, D. B., Aebersold, R., Nat.Biotechnol. 2003, 21, 660–666.

[13] Blake, T. A., Williams, T. L., Pirkle, J. L., Barr, J. R., Anal.Chem. 2009, 81, 3109–3118.

[14] Wada, Y., Tajiri, M., Yoshida, S., Anal. Chem. 2004, 76,6560–6565.

[15] Snovida, S. I., Bodnar, E. D., Viner, R., Saba, J.,Perreault, H., Carbohydr. Res. 2010, 345, 792–801.

[16] Yu, L., Li, X., Guo, Z., Zhang, X., Liang, X., Chem. Eur. J.2009, 15, 12618–12626.

[17] Takegawa, Y., Ito, H., Keira, T., Deguchi, K., Nakagawa,H., Nishimura, S., J. Sep. Sci. 2008, 31, 1585–1593.

[18] Wohlgemuth, J., Karas, M., Eichhorn, T., Hendriks, R.,Andrecht, S., Anal. Biochem. 2009, 395, 178–188.

[19] Wada, Y., Tajiri, M., Yoshida, S., Anal. Chem. 2004, 76,6560–6565.

[20] Hagglund, P., Bunkenborg, J., Elortza, F., Jensen, O. N.,Roepstorff, P., J. Proteome Res. 2004, 3, 556–566.

[21] Ding, W., Nothaft, H., Szymanski, C. M., Kelly, J., Mol.Cell. Proteomics 2009, 8, 2170–2185.

[22] Shen, A., Guo, Z., Yu, L., Cao, L., Liang, X., Chem.Commun. 2011, 47, 4550–4552.

[23] Larsen, M. R., Jensen, S. S., Jakobsen, L. A., Heegaard,N. H., Mol. Cell. Proteomics 2007, 6, 1778–1787.

[24] Yan, J., Li, X., Yu, L., Jin, Y., Zhang, X., Xue, X., Ke, Y.,Liang, X., Chem. Commun. 2010, 46, 5488–5490.

[25] Kondo, A., Thaysen-Andersen, M., Hjerno, K., Jensen,O. N., J. Sep. Sci. 2010, 33, 891–902.

[26] Gilar, M., Jaworski, A., J. Chromatogr. A, in press. DOI:10.1016/j.chroma.2011.04.005.

[27] Wuhrer, M., Balog, C. I. A., Koeleman, C. A. M., Deelder,A. M., Hokke, C. H., Biochim. Biophys. Acta 2005, 1723,229–239.

Figure 4. Mass spectra of tryptic peptides from HSA and IgGwith molar ratio 10:1. (A) Total digest desalted with C18; (B)glycopeptide fraction enriched with sequential C18 and TiO2.Glycopeptides are labeled with asterisk.



Selective enrichment of glycopeptides for mass spectrometry analysis using C18 fractionation and...

Documents

Transcript of Selective enrichment of glycopeptides for mass spectrometry analysis using C18 fractionation and...