Approaches towards the quantitative analysis of peptides and proteins by reversed-phase...

Journal of Chromatography A, 891 (2000) 235–242www.elsevier.com/ locate /chroma

Approaches towards the quantitative analysis of peptides andproteins by reversed-phase high-performance liquid chromatography

in the absence of a pure reference sample1*Frank Moffatt , Paul Senkans, Dean Ricketts

Zeneca Agrochemicals, Jealotts Hill Research Station, Bracknell, Berks RG42 6ET, UK

Received 7 March 2000; received in revised form 25 May 2000; accepted 25 May 2000

Abstract

A reversed-phase HPLC protocol for the quantitative analysis of peptides and proteins is presented. It is applicable topurified samples and potentially to crude biological extracts. The key feature is that an analytically pure reference sample ofthe analyte is not required because the extinction coefficient for the UV absorbance at 280 nm can be accurately estimatedfrom the amino acid sequence. The concentration of a protein can therefore be calculated from the peak area relative to aninternal standard. Sources of error and limitations of the method are systematically considered. Tryptophan containingpeptides gave closer agreement to expected values than those with only tyrosine. It was found that analogous, previouslyused methods could not be directly applied to lower wavelengths. 2000 Elsevier Science B.V. All rights reserved.

Keywords: Peptides; Proteins

1. Introduction reference sample. Nowadays, molecular biologyprovides protein sequences from DNA sequences so

It is beyond the scope of this introduction to the necessary information for such calculations isreview the vast number ways in which proteins can readily available.be quantified [2] except to note that the majority The determination of protein concentration byrequire a calibration using an analytically pure spectrophotometric means is widely practiced in

1%reference sample of the analyte, which may often not biochemical laboratories. Absorptivity, A values280

be available in the first instance. Our aim was to [1] absorbance at 280 nm, 1% (w/v) solution, pathdevise a convenient and reliable protocol for the length of 1 cm are relied upon for the analysis of

21quantitative analysis that would be applicable to proteins at concentrations of 20–3000 mg ml . Theimpure proteins without the need for a purified limiting concentrations for the avoidance of interfer-

ence from common biochemical media are known.Specific corrections can be made for nucleic acids

*Corresponding author. Tel.: 144-134-441-4689; fax: 144- [1,2]. The method [2] is direct and rapid but a major134-441-3677.

disadvantage is that the analyte must be sufficientlyE-mail addresses: [email protected] (F. Mof-pure.fatt), [email protected] (D. Ricketts).

1Co-corresponding author. Wetlaufer [3] described how the extinction coeffi-

0021-9673/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0021-9673( 00 )00620-8

236 F. Moffatt et al. / J. Chromatogr. A 891 (2000) 235 –242

cient of proteins may be regarded as the sum of the (from measurement at 280 nm) as described for 205nm by Scopes [16]. The limitations of the Scopesindividual contributions of the amino acids. It hasmethod and of the ratio methods listed above werealso been pointed out [4] that measurement ofoutlined by van Irsel et al. [17]. Silvestre et al. [18]absorbance is straightforward, but conversely theanalyzed casein hydrolysates by size-exclusion LCdetermination of concentration (based upon thewith detection at the wavelengths 300, 280 and 230extinction coefficient) is not. Nevertheless, Edelhochnm. This allowed for substraction of the individual[5] calculated the extinction coefficients of proteinscontributions of tryptophan and tyrosine to theat 280 nm from the tryptophan and tyrosine content.absorbance (peak area) at 230 nm. The correctedGill and Von Hippel [6] proposed slightly revisedfraction (of peak) area at 230 nm was shown to bevalues and also included the contribution of cystine.proportional to the amount of amino acids present soMach et al. [7] produced average values for thethat the proportion in molar terms of peptides as aextinction coefficients of tryptophan and tyrosine infunction of size could be determined. These ap-proteins themselves but the most comprehensiveproaches were not pursued because if measurementsevaluation is the Pace et al. [8] analysis of 116can be performed at 280 nm this is itself sufficientproteins which gave an average deviation of 63.8%for the determination of protein concentration.from the Edelhoch method. Thus, for any protein the

It is common in HPLC to quantify proteins byextinction coefficient at 280 nm, may be calculatedconstructing a standard curve. Zhu et al. [19] found[8]:that the sensitivity by UV absorbance at 215 nm was

e 5 (n 5500) 1 (n 1490) 1 (n 125) (1) about ten times that at 280 nm for bovine serumpro280 W Y S-S

albumin. A recent, typical example, is the analysis ofwhere n is the number of each group per protein tear proteins [20]. The key concept behind ourmolecule, and the subscripts W, Y and S-S denote investigation, namely protein analysis in the absencetryptophan, tyrosine and cystine, respectively. of an analytically pure reference sample of the

Analogous formulae may be constructed for lower analyte, was exemplified by the work of Eberlainwavelengths (adapted from Buck et al. [9]): [21], in which the response of LC–UV detection at

215 and 277 nm was calculated from spectroscopice 5 (n 2 1 1 n 1 n ) 2846 1 n 7200pro214 AA N Q F data. However, the method is rather cumbersome for

1 n 6309 1 n 22 735 1 n 5755 (2) routine use.H W Y

LC detection, using the native fluorescence oftryptophan or fluorescent tags can provide highere 5 (n 2 1 1 n 1 n ) 2400 1 n 8600pro205 AA N Q Fsensitivity than UV absorbance. Unfortunately, the

1 n 5200 1 n 20 400 1 n 6080 (3)H W Y quantum yield for the fluorescence of tryptophan canvary from 0.00 to 0.35 through quenching by up towhere the subscript AA represents all amino acids,four different mechanisms [22]. Therefore, the fluo-and N, Q, F and H denote the usual single letterrescence response of a protein cannot be readilyamino acid codes.predicted, for which reason UV-detection was chosenAlternative assays for proteins using UV absor-for this study.bance differences that may compensate for the

The approach described in this paper for thepresence of non-proteinacious components, havedetermination of the concentration of a protein (c )Probeen reviewed [10]. The wavelengths selected byis based upon the calculated extinction coefficientvarious workers include A –A [11,12], A –215 225 224(e ) and peak areas by HPLC:proA [13], A –A [14], and A –A [15].233 230 260 235 280

These approaches were not followed in this in- c 5 c (e /e )(A /A ) (4)pro cal cal280 pro280 pro280 cal280vestigation because the use of a high resolutionseparation technique achieves purification that in where A is the peak area, the subscripts pro and calprinciple renders such compensation redundant. refer to the protein and the calibrant, respectively, for

A further possibility is use of low wavelengths absorbance at 280 nm, e is calculated using Eq.pro

with corrections for tryptophan and tyrosine content (1).

F. Moffatt et al. / J. Chromatogr. A 891 (2000) 235 –242 237

Table 1Comparison of experimentally determined purity with the supplier’s specification for a range of tryptophan containing peptides and proteins

Compound Observed, Expected, Observed,% peptide % peptide % of expected

Lys–Trp–Lys 76 79 96a-Lactalbumin 91 85 107D-Lys–Tyr–D-Trp–D-Trp–Phe 84 79 106pGlu–Lys–Trp–Ala–Pro 90 |95 95Lysozyme 99 94 105b-Ala–Trp–Met–Asp–Phe 86 91 95Trp–Met–Asp–Phe 88 91 97pGlu–Ser–Leu–Arg–Trp 88 89 99Average (RSD, %) 100 (5.4)

This expression is derived by equating the path supplied by Sigma–Aldrich (Poole, UK) included:length of the UV detection cell in terms of the ethylenediamine tetracetic acid disodium salt,Beer–Lambert parameters for the protein and guanidine hydrochloride, guanidine thiocyanate,tryptophan, and recognising that in HPLC the ab- lysozyme (chick), ribonuclease A (bovine pancreas),sorbance must be integrated over time (i.e. peak trifluoroacetic acid (TFA) ($99%), D-tryptophan,area). The principle of using absorbance in this other proteins and peptides listed in Tables 1 and 2.manner is thus well supported by the prior work a-Dithioreitol was obtained from Pierce and War-cited. Surprisingly, we have found no applications of riner (Chester, UK). The plant defensin, Rs-AFP1this principle in LC or CE. This study illustrates how [23] was obtained from an in-house source (S.the method may be applied in practice. Particular Attenborough, Zeneca Agrochemicals, Jealotts Hillcomplications are addressed, with emphasis upon Research Station, Bracknell, UK).experimental design.

2.2. Sample preparation2. Experimental

The protein or peptide (1–5 mg) was dissolved inaqueous guanidine thiocyanate (2.4 M, 1–5 ml).2.1. Chemicals and solventsSome hydrophobic peptides required additional ace-tonitrile for dissolution. In general, peptides of .20Deionised water was obtained from a Milli-Qamino acids were found to be soluble in thewater purification system [Millipore, Watford, UK].guanidine thiocyanate alone. Peptides were diluted toAll solvents were of HPLC grade (Romil, Cam-the following concentrations: 1–5 amino acids (10 orbridge, UK), and were passed through a 0.22 mm

2150 mg ml ), 6–50 amino acids (100 or 250nylon membrane filter [Millipore, UK] prior to use21 21as the mobile phase for HPLC analysis. Substances mg ml ), .50 amino acids (1 mg ml ). Solutions

Table 2Comparison of experimentally determined purity with the supplier’s specification for a range of tyrosine containing peptides and proteins

Compound Found, Expected, Found, %% peptide % peptide of expected

Thr–Tyr–Ser 77 92 84Gly–Gly–Thr–Arg 68 80 85Thr–Gly–Gly–Phe–Met 73 83 88des-Asp angiotensin I human 71 82 87Average (RSD) 86 (2.1)


21of D-tryptophan (10 or 100 mg ml ) in aqueous21guanidine thiocyanate (2.4 mol l ) were prepared

weekly and were stored in a refrigerator when not inuse. Samples were analyzed by HPLC within 24 h.

2.3. HPLC conditions

An HP1100 Series HPLC instrument (Hewlett-Packard, Bracknell, UK) was used, consisting of adegasser, binary pump, autosampler, column tem-perature control compartment and a UV detector.Detection was by UV absorbance at a wavelength of280 nm and a bandwidth of 2 nm. Reversed-phase

Fig. 1. Quantitation of a peptide by RP-HPLC–UV – typical280HPLC was performed on a Jupiter column (15 cm3 chromatogram.4.6 mm I.D., C bonded to silica particles of18

diameter 5 mm and pore size 30 nm) (Phenomenex,Cheshire, UK). Analyses were performed at a col- 5630, under acid conditions [24]). Reversed-phaseumn temperature of 408C and a flow-rate of 1.0 HPLC analyses were performed using a solvent

21ml min. . The following gradient elution profiles gradient owing to the steep adsorption isothermswere used: Solvent A, 0.1% (v/v) TFA in water, associated with proteins in this mode of LC [25].Solvent B, 0.085% (v/v) TFA in acetonitrile. The TFA was added to the mobile phase in order toscouting conditions, for substances of unknown obtain good chromatography [26]. Many proteinselution properties were 5–80% B over 30 min. and peptides are isolated as salts of the basic aminoGradients were adjusted to provide resolution of acid residues [27] histidine (pK 6–7), arginine (pKa a

tryptophan from the analyte and generally started at 12) and lysine (pK 10.4–11.1). Commercial sourcesa

5 or 10% B followed by an increase in the range of were used that provided peptide, counter-ion, salt1–3% B per minute. The sample injection volume and solvent composition data. The calibrant was keptwas 10 ml in all cases. The calibrant, D-tryptophan in a separate vial to the sample but equal volumes

21(10 ml injection volume, 10 or 100 mg ml in 2.4 M were injected in each run via an injection program.aqueous guanidine thiocyanate), was injected with All analytes were well resolved from tryptophan bythe sample via an injection program. reversed-phase HPLC (e.g. Figs. 1 and 2). The

observed purity of the test analytes (from Eq. (4)),2.4. Reduction of proteins with a-dithiothreitol

The protein (ca. 1 mg) was dissolved in ‘‘GET’’buffer (1 ml, 6 M guanidine hydrochloride, 8 mMEDTA, 0.5 M Tris, pH 8.6). a-Dithiotreitol (40 molequiv., 65 mM, in ‘‘GET’’ buffer) was added at roomtemperature. The resulting mixture was analyzed byHPLC within 0.5–1.5 h.

3. Results and discussion

To measure the concentration of a protein accord-ing to Eq. (4), a calibrant is required. Tryptophanwas selected because it is readily available in pure Fig. 2. Quantitation of a protein by RP-HPLC–UV – typical280

form and has a known extinction coefficient (e 5 chromatogram.cal


were compared with the expected values (supplier’s choice of this salt follows the empirical rules ofdata). Excellent agreement was found, the overall Hofmeister [27] for the stabilization by anions andaverage agreement for tryptophan containing samples cations of proteins in solution. It is advisable towas 100% (relative standard deviation (RSD) 5.1%) assess the risk of losses through adsorption, for(Table 1). example, by dipping pipettes and LC tubing into a

One possible source of inaccuracy is the attenua- dilute solution of the analyte and by transfer of thetion of light in the solution phase through light protein solution between two and three vials. At-scattering owing to turbidity, which becomes more tenuation of LC signal intensity after dipping orsignificant as the size of the proteins increases – the transfer is an unambiguous indicator of loss. Conse-Tyndall effect [28]. This obeys a Beer–Lambert type quently, the consistent use of one source of glass orof relationship: polypropylene, pipettes and vials, and one type of

LC tubing are recommended. Additives for proteinln I /I 5 t l (5)0 stabilization may only be effective up to the point

that the sample reaches the head of the HPLCwhere t is the turbidity of the solution, l is the pathcolumn. Losses from the column onwards can belength.diagnosed via a linearity check. Deviation fromThe full expression for the turbidity includes thelinearity, or sudden loss of signal, shows that stick-concentration, relative molecular mass and the wave-ing is significant. A difference in the proportions oflength of light. Turbidity can be used for theanalyte and calibrant lost within the LC systemquantitation of proteins [29] but in this paper thewould produce systematic error. It has been a regularconcern is to assess the impact upon ‘‘apparent’’ UVobservation, that the first one to two injections canabsorbance values. Mach et al. [30,31] have pro-give spurious results. This is attributable to theposed that turbidity can be accounted for by measur-blocking of irreversible binding sites. Sufficienting absorbance readings at 320 and 350 nm, on thereplication is therefore also recommended.assumption that there is no interfering absorbance at

In order to adapt the A method to separation280these wavelengths, and adjusting the 280 nm ab-techniques such as LC or CE, it is necessary tosorbance accordingly:establish the effect of mobile phase composition

(m11) log A 2m log A320 350 upon the chromophores. The absorbance of tyrosineA 5 10 (6)lundergoes significant shifts [27] at higher pH (asso-

where m 5 64.32 2 25.67, log l51.5 at 280 nm, l is ciated with the pK of the phenolic group). Organica

the wavelength of light. solvents can also alter the pK of both acid and basica

We found that peptides /proteins of molecular groups by as much several pH units in aqueousmass 5000–6000 exhibited light scattering of around acetonitrile mobile phases [36,37]. The tryptophan1% by HPLC. The impact of light scattering depends chromophore has been found to be unaffected by theupon the size of protein, but need only be considered presence of urea or guanidine in aqueous solution,for realization of the highest accuracy in the case of but had a 9% higher extinction coefficient in 1-large or aggregated proteins. propanol compared with water [8] but others have

Sample handling is an important practical issue claimed that denaturing agents may cause hypso-because there is a high potential for proteins to chromic and hypochromic effects [38]. In any eventadhere to LC vials and the LC system. The stabiliza- spectral differences may arise though a combinationtion of proteins in solution, to avoid non-specific of intra- and intermolecular effects upon ionisablebinding or sticking to surfaces, is a classical problem groups that influence the chromophore, as discussedaddressed both in the biochemical literature [32,33] elsewhere [39]. It has often been found that reversed-and in the context of capillary electrophoresis phase HPLC denatures proteins [40], giving rise to[34,35]. The ‘‘sticking problem’’ is the outcome of greater exposure of hydrophobic amino acids whichdiverse mechanisms but we have found that in many include tryptophan and tyrosine, to the solventcases dissolving samples in guanidine thiocyanate medium. This would reduce any potential influences

21(2.4 mol l ) provides reliable stabilization. The of the local structural environment within folded


proteins, upon the chromophore. By working under acontrolled pH remote to the pK of the amino acidsa

this source of variation could be eliminated. A UVspectrophotometer was used to determine the effectof a range of HPLC mobile phases containing water(TFA, 0.1%) and acetonitrile (TFA, 0.085%) uponproteins, peptides and D-tryptophan. These testsshowed that the proportions of these mobile phasesdid not affect the responses at 280 and 205 nmrelative to that in water.

Measurements at 280 nm impose a limitationbecause tryptophan has the lowest average natural

Fig. 3. The effect of reduction of cystine linkages upon theabundance of any amino acid. In some cases the chromatography of selected proteins. Conditions: 5–60% acetoni-absorbance will depend upon tyrosine alone. Com- trile (0.085% TFA) water (0.1% TFA) in over 55 min.mercially available peptides containing tyrosine butnot tryptophan consistently gave a value that wasabout 14% lower than the expected value (Table 2).Further study is required to establish whether this is reported herein were confirmed by electrospray massdue to inaccuracy in the specification of the purity of spectrometry. The reduced forms all eluted at longerthe sample or another source. retention times by reversed-phase LC (Fig. 3).

Attempts to apply Eqs. (2) and (3) using 205 and Comparison of the ratios of peak areas at selected214 nm data proved unreliable (Table 3). The wavelengths shows significant differences in theexplanation for this was considered. The UV ab- absorbance of the reduced and non-reduced forms ofsorbance of peptides has been shown [41] to be three proteins (Fig. 4). These changes cannot simplyproportional to the number of peptide bonds at 200 be accounted for by the loss of the cystine linkages.nm. However, in the case of proteins, absorbance has It must therefore be advantageous to minimizebeen seen to be highly dependent upon the in- effects upon the chromophore that are not purely duetramolecular environment with alpha helices showing to amino acid composition in the development ofattenuation of up to 40% of the absorbance found in lower wavelength methods. Reduction of cystinesextended forms [42]. Evidence of such influences is and denaturation would contribute to this. A furtherprovided by the observation that enzymatic digests of refinement of this strategy would be to convert theglobular proteins produce hypochromic and hypso-chromic shifts by as much as 10–15% which wasattributed to the removal of conformational contribu-tions [38]. To study this possibility, three proteinswere reduced. The completeness of the reductions

Table 3Comparative of values for the quantification of peptides at severalwavelengths

aCompound 214 nm 205 nm

b-Ala–Trp–Met–Asp–Phe 62.8 94.2pGLu–Lys–Trp–Ala–Pro 68.7 103.8Insulin B 44.0 118.9Angiotensin I human 68.8 27.5Thr–Tyr–Ser 73.6 44.5

Fig. 4. Percentage changes in absorbance ratios upon reduction ofa Values are percentages relative to the 280 nm value. selected proteins.


reduced protein to smaller fragments by enzymatic 4. Conclusionsdigestion, however this would introduce an extrasource of inaccuracy. (1) Quantitation of peptides and proteins in solu-

The A method can be used on a ‘‘rule of tion by RP-HPLC with detection by UV absorbance280

thumb’’ basis for the quantification of proteins of at 280 nm relative to a calibrant is both highlyunspecified amino acid composition [33] (a 1 convenient and accurate.

21mg ml solution is estimated to have an absorbance (2) The protocol is limited to tyrosine andof 1 in a 1 cm path length cuvette). A similar tryptophan containing substances and may be lessapproximation can be made to the LC method accurate for non-tryptophan containing analytes.described in this paper. Taking the occurrence in (3) An analytically pure reference sample of theproteins [43] of tryptophan, tyrosine and cysteine as analyte is not required.1.3 and 3.2, 1.7 molar percentages, respectively, the (4) Good experimental practices include consid-relative response of the average protein would be eration of errors due to non-specific binding ofabout 0.04 relative to tryptophan in HPLC (by peak proteins and the significance of light scattering in thearea at 280 nm, after correcting abundance to a mass case of larger proteins.basis). However, the associated errors would be large (5) Changes to the methodology that would re-because of the dependency upon two amino acids of quire validation include the use of solvents otherrelatively low and variable abundance and does not than water–acetonitrile, and non-acidic pH values.therefore play to the main strengths of the method. (6) Use of absorbance at wavelengths below 280

In summing up, the protocol outlined in this paper nm is complex and would require considerably moreenables the determination of the concentration of a validation than that provided in the current literature.protein in solution, using only microlitre volumes, atconcentrations in the microgram–milligram permillilitre range using standard 4.6 mm I.D. RP

Acknowledgementscolumns running at a mobile phase flow-rate of 121ml min . This is sufficient for many bio-efficacy

Useful background investigations were performedtests and analysis of matrix samples.by S. King, T. Ost (as part of the B.Sc. degree fromQuantitation by mass of a solid protein by thethe University of Edinburgh, UK), and R. Dunk (asmethod described herein, is more sample demandingpart of the M.Chem. degree from the University ofbeing limited by weighing accuracy. In our ex-Southampton, UK).perience, with ordinary four or five figure balances,

1–10 mg of solid protein is required. For proteincharacterization, where high accuracy may be re-

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