Benzoyl Derivatization as a Method To Improve Retention of

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Anal. Chem. 2004, 76, 5799-5806

Benzoyl Derivatization as a Method To Improve Retention of Hydrophilic Peptides in Tryptic Peptide Mapping Samir Julka and Fred E. Regnier*

Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47907

This study exploits the increase in chromatographic retention that accrues from benzoyl derivatization of primary amines as a tool to increase sequence coverage in tryptic peptide mapping. N-hydroxysuccinamide sulfonyl benzoate quantitatively derivatizes primary amines of peptides. Introduction of the hydrophobic benzoyl moiety into peptides increased retention of peptides during reversed-phase chromatography (RPC), particularly in the case of smaller hydrophilic peptides. Short chain (1-6 amino acids) tryptic fragments of model proteins lysozyme, myoglobin, and cytochrome c derivatized with N-hydroxysuccinamide sulfonyl benzoate eluted in the linear acetonitrile gradient. Application of benzoyl derivatization was further extended to achieve complete sequence coverage of a therapeutic protein, recombinant human growth hormone, and in detection of single amino acid polymorphism. Reversed-phase chromatographic mapping of proteolytic digests has been widely used in the characterization of proteins, particularly with biopharmaceuticals where changes in the primary structure of a protein therapeutic can alter its efficacy.1,2 The problem with RPC in this application is that it does not yield complete sequence coverage of proteins, especially for proteins with high lysine/arginine content.3 Small hydrophilic peptides produced during proteolysis do not interact strongly, if at all, with the hydrophobic octadecyl silane stationary phase used in most RPC columns and are eluted together in the column void volume. Up to 5-15% of the amino acid residues in a protein can occur in these small, hydrophilic peptides.4 The concern is that an alteration in the primary structure of a protein within these peptides will be missed, and the analysis does not provide a truly representative characterization of a protein.5 Beyond biopharmaceutical applications, poor retention of hydrophilic peptides is a problem in proteomics as well. Posttranslational modifications (PTMs) such as glycosylation,6 nitra* Corresponding author. Phone: (765) 494-9390. Fax: (765) 494-0239. E-mail: [email protected]. (1) Teshima, G.; Davis. E. C. J. Chromatogr., A 1992, 625, 207-215. (2) Teh, L. C.; Murphy, L. J.; Huq, N. L.; Surus, A. S.; Friesen, H. G.; Lazarus, L.; Chapman, G. E. J. Biol. Chem. 1987, 262, 6472-6477. (3) Lei, J.; Chen, D. A.; Regnier. F. E. J. Chromatogr., A 1998, 808, 121-131. (4) Larsen, M. R.; Cordwell, S. J.; Roepstroff, P. Proteomics 2002, 2, 12771287. (5) Chin, E. T.; Papac, D. I. Anal. Biochem. 1999, 273, 179-185. (6) Otvos, L., Jr.; Urge, L.; Thurin, J. J. Chromatogr., A 1992, 599, 43-49. 10.1021/ac049688e CCC: $27.50 Published on Web 08/27/2004

© 2004 American Chemical Society

tion,7 and phosphorylation8 are often found on short peptides. Moreover, the PTM itself can increase peptide hydrophilicity. Analysis of single amino acid polymorphism among proteins from different individuals is another case where it is critical to examine the total sequence of proteins to preclude missing an alteration in primary structure. Poor sequence coverage can even be a problem in food sciences where short chain peptides are considered critical in flavor and aroma formation.9-12 Failure to recognize short peptides can lead to an erroneous evaluation of organoleptic properties. A number of strategies have been employed to improve retention of short hydrophilic peptides. One is to neutralize the charge on lysine and N-terminal amines by chromatographing them at a pH above their pKa.13 Another is to use a hydrophobic ion-pairing agent, e.g., n-alkylated chain-perfluorinated carboxylic acid analogues, in the mobile phase.14 Recently, use of hydrophilic interaction chromatography coupled to electrospray mass spectrometry was reported to specifically target small peptides.15 But these techniques are of limited utility with mixtures containing a wide variety of species ranging from small hydrophilic to large hydrophobic peptides. Another approach was to couple normal phase chromatography and RPC columns in tandem to capture the entire proteolytic tryptic digest and then elute them sequentially.16,17 Obviously, this doubles the analysis time. Yet, another popular strategy is to derivatize peptides with a nonpolar tag. 9-Fluoromethyl chloroformate (FMOC) has been used in this context to retain and resolve short peptides on RPC columns.9,18 However, side products of the derivatization reaction along with overlap of unreacted FMOC with derivatized peptides is a problem. (7) Walcher, W.; Franze, T.; Weller, M. G.; Po¨schl, U.; Huber, C. G. J. Proteome Res. 2003, 2, 534-542. (8) Neubauer, G.; Mann, M. Anal. Chem. 1998, 71, 235-242. (9) Gartenmann, K.; Kochhar, S. J. Agric. Food Chem. 1999, 47, 5068-5071. (10) Fujimaki, M.; Arai, S.; Yashamita, M.; Kato, H.; Noguchi, M. Agric. Biol. Chem. 1973, 37, 2891-2898. (11) Noguchi, M.; Arai, S.; Yamashita, M.; Kato, H.; Fujimaki, M. J. Agric. Food Chem. 1975, 23, 49-53. (12) Schlichtherle-Cerny, H.; Amado, R. J. Agric. Food Chem. 2002, 50, 15151522. (13) Dolan, J. W. LC-GC 2001, 19, 1132-1135. (14) Petritis, K.; Brussaux, S.; Guenu, S.; Elfakir, C.; Dreux, M. J. Chromatogr., A 2002, 957, 173-185. (15) Schlichtherle-Cerny, H.; Affolter, M.; Cerny. C. Anal. Chem. 2003, 75, 23492354. (16) Yoshida, T. Anal. Chem. 1997, 69, 3038-3043. (17) Yoshida, T. J. Chromatogr., A 1998, 811, 61-67. (18) Roturier, J. M.; Bars, D. L.; Gripon, J. C. J. Chromatogr., A 1995, 696, 209217.

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Figure 1. Reaction scheme for synthesis of NHSS-benzoate and subsequent derivatization with primary amines.

This problem has been addressed by solid-phase derivatization19 in a process where peptides are dried on-column, FMOC derivatized, and then redissolved for RPC analysis. This method only partially succeeded in removing interfering side products. Moreover, quantitiative drying and then redissolution of peptides has been reported to be sequence specific and can lead to sample losses as high as 30-90%.20 In another study, esterification was used to reduce charge and increase peptide retention.21 Partial derivatization at several carboxylic centers was observed to be a problem in this case.22 Recently, citraconylation of lysine residues was employed to improve sequence coverage by eliminating the tryptic cleavage at lysines, thereby increasing the overall tryptic peptide size.23 However, even in excess citraconic anhydride, all lysine residues are not modified. This increases the complexity of a tryptic digest. Moreover, short arginine-terminating tryptic peptides will still not be observed in RPC-MS analysis. In this paper, N-hydroxysuccinamide sulfonyl (NHSS)-benzoate (Figure 1) was used to quantitatively derivatize primary amines at N-termini and lysine residues of peptides. Primary amine groups of tryptic peptides have been previously quantitatively derivatized using various acylating reagents to quantify changes in protein expression.24-27 Benzoyl derivatization of amines using benzoyl chloride has been previously used for determination of biogenic amines (low molecular weight organic bases of aliphatic, aromatic, and heterocyclic structures) using HPLC28 and micellar liquid chromatography.29 However, benzoyl chloride decomposes in water and is only soluble in organic solvents. Most peptides, in contrast, are insoluble in organic solvents. Therefore, this procedure cannot be used to derivatize peptides to increase protein coverage. Thus, a water soluble benzoyl derivatizing agent is (19) Shangguan, D.; Zhao, Y.; Han, H.; Zhao, R.; Liu, G. Anal. Chem. 2001, 73, 2054-2057. (20) Stewart, I. I.; Thomson, T.; Figeys, D. Rapid Commun. Mass Spectrom. 2001, 15, 2456-2465. (21) Falick, A. M.; Maltby, D. A. Anal. Biochem. 1989, 182, 165-169. (22) Shevchenko, A.; Chernushevich, I.; Ens, W.; Standing, K. G.; Thomson, B.; Wilm, M.; Mann, M. Rapid. Commun. Mass Spectrom. 1997, 11, 10151024. (23) Kadlcˇ´ık, V.; Strohalm, M.; Kodı´cˇek, M. Biochem. Biophys. Res. Commun. 2003, 305, 1091-1093. (24) Chakroborty, A.; Regnier, F. E. J. Chromatogr., A 2002, 949, 173-184. (25) Zhang, R.; Sioma, C.; Thompson, R. A.; Xiong, L.; Regnier, F. E. Anal. Chem. 2002, 74, 3662-3669. (26) Ji, J.; Chakraborty, A.; Geng, M.; Zhang, X.; Amini, A.; Bina, M.; Regnier, F. E. J. Chromatogr., B 2000, 745, 197-210. (27) Munchbach, M.; Quadroni, M.; Miotto, G.; James, P. Anal. Chem. 2000, 72, 4047-4057. (28) Hwang, D.-F.; Chang, S.-H.; Shiua, C.-Y.; Chai, T. J. J. Chromatogr., B 1997, 693, 23-29. (29) Paleologos, E. K.; Chytiri, S. D.; Savvaidis, I. N.; Kontominas, M. G J. Chromatogr., A 2003, 1010, 217-224.

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required. The presence of a sulfonic acid group in NHSS-benzoate ensures complete solubility of the benzoyl acylating agent in water. It will be shown that the addition of the benzoyl moiety to peptides increases their retention in RPC, particularly in the case of small, hydrophilic peptides, thereby increasing overall protein coverage. The utility of benzoyl derivatization in characterization of therapeutic proteins and in detection of single amino acid polymorphism is also demonstrated. EXPERIMENTAL SECTION Materials and Reagents. Amino acid standard solution (AA-S-18), cytochrome c (horse), cytochrome c (bovine), lysozyme (chicken), benzoic acid, HPLC grade acetonitrile (AcN), dicyclohexylcarbodiimide (DCC), dithiothreitol (DTT), N-hydroxysuccinamide sulfonate (NHSS), iodoacetic acid (IAA), urea, N-[2hydroxyethyl]piperazine-N′-[2-ethane sulfonic acid] (HEPES), dimethylformamide (DMF), ethyl acetate, and calcium chloride were purchased from Sigma-Aldrich (St. Louis, MO). Research grade (i.e., not intended for human use) rhGH was a generous gift from Eli Lily & Co (Indianapolis, IN). Trifluoroacetic acid (Sequanal Grade) was obtained from Pierce (Rockford, IL). Sequencing grade, modified trypsin was purchased from Promega (Madison, WI). The C18 RPC column (4.6 mm × 250 mm) was obtained from Vydac (Hesperia, CA). The low-pressure microsplitter valve was from Upchurch Scientific (Oak Harbor, WA). Double-deionized water (ddI H2O) was produced by a Milli-Q gradient A10 system from Millipore (Bedford, MA). Proteolysis of Cytochrome c (Equine and Bovine), Myoglobin (Equine), Lysozyme (Chicken), and Recombinant Human Growth Hormone. An amount of 1 mg of recombinant hGH and lysozyme (chicken) were separately reduced in 1 mL of 0.2 M HEPES buffer (pH 8.5) containing 8 M urea and 10 mM DTT. After a 2 h incubation at 50 °C, iodoacetic acid was added to a final concentration of 20 mM and incubated in darkness on ice for an additional 2 h. After dilution with 0.2 M HEPES buffer to a final urea concentration of 0.8 M, modified sequencing grade trypsin was added to the sample at a 50:1 protein/trypsin mass ratio and the solution was incubated for 24 h at 37 °C. Digestion was stopped by addition of trypsin inhibitor, type I-S. Trypsin digestion of 1 mg each of cytochrome c (equine, bovine) and myoglobin (equine) was directly carried out with 0.1 M ammonium bicarbonate buffer at pH 8.0 by addition of trypsin as aforementioned. Synthesis of N-hydroxysuccinamide Sulfonyl (NHSS)benzoate. N-hydroxysuccinamide sulfonyl (NHSS)-benzoate was synthesized according to the protocol by Staros30 with slight modifications. An amount of 2 mol of Na+NHSS, 2.0 mol of benzoic

acid, and 2.2 mol of DCC was dissolved in 5.0 mL of dry DMF, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was cooled to 3-4 °C and then stirred for another 4 h. The precipitated dicyclohexylurea was removed by filtration and washed with a minimum volume of dry DMF. The product was then precipitated from solution by addition of excess ethyl acetate. The precipitate was dried and stored in a desiccator. Derivatization of Peptides. A 50-fold molar excess of NHSSbenzoate was added individually to tryptic peptides at pH 8.0. The reaction was allowed to proceed for 4 h at room temperature. After derivatization, the pH was increased to 11 by addition of 0.5 M hydroxylamine and then decreased back to 8.0 by dilute acetic acid for reversed-phase chromatography. Reversed-Phase Chromatography of Derivatized Peptides. Benzoyl-coded peptide mixtures were separated by linear gradient elution from a Vydac C18 column (4.6 mm × 250 mm) on an Integral Micro-Analytical Workstation (Applied Biosystems, Framingham, MA). The C18 column was equilibrated using 100% mobile phase A (0.01% TFA in ddI H2O) at a flow rate of 1 mL/ min for two column volumes (cv ) 4.155 mL). The peptide mixtures (2 nmol) were injected and eluted at a flow rate of 1 mL/min in a linear gradient ranging over 70 min from 100% mobile phase A to 70% mobile phase B (95% AcN/0.01% TFA in ddI H2O). At the end of this period, a second linear gradient was applied in 10 min from 70% B to 100% B at the same flow rate. The gradient was then held at 100% mobile phase B for an additional 10 min. Throughout the analysis, an on-line UV detector set at 214 nm was used to monitor separation of the peptide mixtures. ESI-MS Analysis. Tryptic peptides were simultaneously monitored by ESI-MS by directing 5-8% of the flow from the column using a low-pressure postcolumn microsplitter to the QSTAR workstation (Applied Biosystems, Framingham, MA) equipped with an ESI source. All spectra were obtained in the positive-ion TOF mode at a sampling rate of 1 spectrum/s. During LC-MS data acquisition, mass scans from m/z 100 to 2000 were recorded. The peptides were identified using extracted ion chromatograms (XIC) based on monoisotopic mass of known peptide sequences. MS/MS Analysis. MS/MS analysis of model peptides dissolved in CH3OH/H2O/formic acid (50%/49%/1%) were done by flow injection at 3 to ∼5 µL/min. Identification of the peptide sequence was achieved by denovo sequencing. The tandem MS of synthetic peptides and their corresponding benzoyl-coded peptide was studied and compared. RESULTS AND DISCUSSIONS Hydrophilic analytes that do not interact with the hydrophobic stationary phase of an RPC column are typically eluted in the column void volume. These peptides are very difficult to identify because the void volume is a complex mixture comprising excess derivatizing agent, reagents used in trypsin digestion (L-cysteine, iodoacetic acid, HEPES, urea, dithiothreitol (DTT), trypsin inhibitors, short autolytic tryptic fragments, mobile phase additives used in the RPC column, and often additives from preceding chromatographic steps such as strong-cation exchange chromatography (SCX) or immobilized metal affinity chromatography (IMAC)). (30) Staros, J. V. Biochemistry 1982, 21, 3950-3955.

Table 1. Retention Times and Observed Monoisotopic Molecular Masses for Derivatized Single Amino Acids

acid

observed monoisotopic masses of benzoyl-derivatized amino acids (Da)

retention time (min)

G A P V T L/I D E M H F R Y S K CC

180.04 194.09 220.10 222.12 224.00 236.11 238.01 252.10 254.09 260.10 270.10 279.15 286.11 314.12 355.17 449.13

20.8 25.5 31.2 36.7 21.9 42.8/44.0 20.9 24.2 37.2 23.0 45.3 25.7 34.3 37.2 41.8 43.6

L-amino

Due to the complexity of the nonretained (void volume) peak, it is undesirable to introduce it into an electrospray mass spectrometer. Unfortunately, this void volume also contains short, hydrophilic peptides. But even when the void volume peak is introduced into the ion source, peptide ionization can be masked by coeluting reagents.31 The best solution to this problem would be to increase the retention of all peptides beyond the void volume peak. It is in this context that derivatization of peptides at their N-termini and at the -amino groups of lysine residues with a benzoyl group was examined. During preliminary experiments, succinimidyl benzoate32 was used to derivatize primary amines. However, inadequate water solubility of this reagent led to poor derivatization, even in water/ dioxane (1:3) at 50 °C for 7-8 h (data not shown). This problem was circumvented by using a sulfonic acid derivative of N-hydroxy succinamide (NHSS) in the preparation of the benzoyl acylating agent. HPLC of the Standard Amino Acid Mixture. A standard mixture of 17 amino acids was derivatized with NHSS-benzoate at pH 8.0 using the derivatization protocol described in the methods. Esterification of amino acids L-tyrosine and L-serine was preserved for better retention of the benzoylated amino acids. Benzoyl derivatization resulted in elution of all derivatized amino acids in a linear acetonitrile gradient (Table 1). A relatively small signal was observed for L-aspartic acid, L-glutamic acid, and L-serine (Figure 2). This was attributed to poor ionization due to an inherent negative charge present on these amino acids and due to double derivatization of L-serine. On the basis of the above results, it can be concluded that even single amino acid residues generated during trypsin digestion will be retained in the linear acetonitrile gradient. Effect of Benzoyl Derivatization on Retention of Peptides. The efficacy of benzoyl derivatization of peptides to increase retention times and sequence coverage was examined using tryptic digests of equine cytochrome c, lysozyme (chicken), and myo(31) Brancia, F. l.; Openshaw, M. E.; Kumashiro, S. Rapid Commun. Mass Spectrom. 2002, 16, 2255-2259. (32) Colton, I. J.; Anderson, J. R.; Gao, J.; Chapman, R. G.; Isaacs, L.; Whitesides, G. M. J. Am. Chem. Soc. 1997, 119, 12701-12709.

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Figure 2. Overlaid extracted ion chromatograms of NHSS-benzoate-derivatized amino acids.

globin (equine heart) by comparing the retention time of tryptic peptides with and without benzoyl derivatization. On derivatizing peptides using NHSS-benzoate, complete sequence coverage was achieved for cytochrome c, myoglobin, and lysozyme. The coverage lost for underivatized tryptic peptides was most evident with equine cytochrome c. Only 79% sequence coverage (number of amino acids observed/number of amino acids in the known protein sequence) was achieved without derivatization, i.e., tryptic peptides comprising 21% of equine cytochrome c elute in the void volume during the sample loading step. An overall increase in retention time in the range of 6.8-42.1 min was observed in comparison to underivatized peptides. The effect of benzoyl derivatization on retention to the reversed phase was observed to be least for larger peptides (6.8-13.7 min). This fact can be valuable in mass spectrometry as will be described below. All cytochrome c derivatized tryptic peptides eluted before 52.65% AcN during gradient elution. With the myoglobin tryptic digest, an overall 91.5% of the underivatized tryptic peptides eluted in linear acetonitrile gradient. The amino acids and peptides K, HK, YK, FDK were only retained after benzoyl derivatization (Figure 3). With lysozyme, 90.7% sequence coverage was achieved without NHSS derivatization with amino acids and peptides L, R, K, CK, NR, GCR, GY absent in the LC-MS analysis. These amino acids and hydrophilic peptides were observed only after derivatization with NHSS-benzoate (Table 2). Trypsin has also been associated with chymotryptic activity as was observed during lysozyme digestion. This often results in formation of even shorter fragments, thereby increasing the losses during reversed-phase chromatography. On the basis of the data in parts a, b, and c of Table 2, for all the three model proteins the following conclusions can be made. First, benzoyl derivatization increases the retention of all peptides and as a consequence increases sequence coverage by retaining more small peptides. Second, derivatization increases the retention of small peptides more than that of large peptides and causes the size distribution in chromatographic fractions to be larger. This has the advantage of increasing the use of separation space in mass spectra. Third, changes in retention time with derivatization are highly sequence specific. For example, lysine-containing tryptic peptides will be derivatized twice and their increase in retention will be greater than that of arginine-containing peptides that are derivatized once. 5802 Analytical Chemistry, Vol. 76, No. 19, October 1, 2004

Figure 3. Overlaid extracted ion chromatograms of previously unretained amino acids and peptides during reversed-phase chromatography for (a) cytochrome c, (b) myoglobin, and (c) lysozyme.

The increase in peptide retention after benzoyl derivatization, especially for short hydrophilic peptides, can be attributed to two phenomena: charge neutralization and addition of the hydrophobic benzoyl moiety. An additional advantage of stronger retention is that elution occurs in a relatively more organic portion of the mobile phase gradient. This facilitates peptide detection in both the MALDI and ESI forms of ionization.33-35 Electrospray ionization of peptides is less efficient when the mobile phase contains less than 10% acetonitrile.33 Enhancement of detection in MALDI after derivatization is apparently due to an increase in peptide hydrophobicity.34 The mass increase upon derivatization (+104.05 amu for arginine-terminated peptides, +208.1 amu for lysineterminated peptides) is also helpful. This aids in bringing short peptides into a mass range amenable to both MALDI-MS and ESIMS. Derivatization of tyrosine and serine residues can be preserved by eliminating the use of hydroxylamine. Quantitative acylation of primary amine groups have been carried out previously down (33) Srebalus-Barnes, C. A.; Hilderbrand, A. E.; Valentine, S. J.; Clemmer, D. E.; Anal. Chem. 2002, 74, 26-36. (34) Krause, E.; Wenschuh, H.; Jungblut, P. R. Anal.Chem. 1999, 71, 41604165. (35) Mason, D. E.; Liebler, D. C. J. Proteome Res. 2003, 2, 265-272.

Table 2. Comparison of Retention Profilea

peptide sequence

observed monoisotopic peptide mass after benzoyl derivatization (Da)

observed retention time of underivatized peptides (min)

observed retention time of benzoyl-derivatized peptides (min)

(a) Cytochrome c (Equine) Amino Acids/Peptides before and after Derivatization with NHSS-benzoate K 355.16 GK 412.25 GGK 469.19 NK 469.15 HK 492.22 TER 509.29 ATNE 538.20 Ac-GDVEK 755.32 27.8 GITWK 812.35 26.4 IFVQK 842.41 21.2 YIPGTK 886.38 18.5 MIFAGIK 987.44 35.7 EDLIAY* 827.36 37.2 LK* 468.23 29.6 heme-CAQCHTVEK 921.32(2+) 28.2 TGPNLHGLFGR 636.80(2+) 36.3 TGQAPGFTYTDANK 839.85(2+) 26.3 EETLMEYLENPK 852.39(2+) 43.4

40.9 38.2 33.5 36.9 32.7 20.3 20.5 43.1 51.5 55.7 48.8 62.4 45.9 54.8 35.0 42.6 40.1 55.4

(b) Lysozyme (Chicken) Amino Acids/Peptides before and after Derivatization with NHSS-benzoate L 236.14 K 355.21 R 279.15 C* K 516.18 NR 393.19 GC* R 497.18 VFGR 582.32 21.4 TPGSR 621.2 14.2 C* ELAAAMK 1102.4 27.5 HGLDNYR 978.47 21.3 WWC* NDGR 1098.38 32.6 GTDVQAWIR 575.28(2+) 24.2 GY( 343.15 SLGNW( 680.32 31.0 VC* AAK( 757.40 23.7 FESNFNTQATNR 766.83 (2+) 26.2 IVSDGNGMNAWVAWR 890.89 (2+) 34.5 NTDGSTDY 502.72 (2+) 26.4 GILQINSR 976.34 20.2 * * * NLC NIPC SALLSS DITASVNC AK 1361.48(2+) 38.4

45.7 43.3 23.0 44.5 22.1 25.5 42.8 27.7 54.7 33.8 48.7 33.7 27.4 47.8 39.3 43.1 47.4 41.7 32.2 40.2

(c) Myoglobin (Equine) Amino Acids/Peptides before and after Derivatization with NHSS-benzoate K 355.21 HK 492.34 FK 502.22 11.5 YK 518.22 HLK 605.32 12.1 FDK 617.26 IPIK 678.24 22.8 NDIAAK 839.13 19.4 ELGFQG 754.08 30.4 ASEDLK 870.12 20.0 TEAEMK 916.21 19.6 ALELFR 852.44 36.7 LFTGHPETLEK 740.35(2+) 29.7 HGTVVLTALGGILK(Na+) 803.75(2+) 43.7 HPGDFGADAQGAMTK 855.56(2+) 28.2 VEADIAGHGQEVLIR 855.69(2+) 36.6 GLSDGEW( 867.15 34.3 QQVLNVWGK( 640.12(2+) 39.5 GHHEAELKPLAQSHATK(2Na+) 1105.22(2+) 25.4 YLEFISDAIIHVLHSK 1046.89(2+) 40.6

43.6 40.5 24.0 47.2 44.5 41.7 57.3 44.5 44.5 44.8 44.3 55.2 41.5 52.2 46.0 52.2 39.5 48.5 56.2 47.2

a ( indicates chymotryptic fragments, Ac- indicates acylated peptide, - indicates the nonretained amino acids and peptides, 2+ indicates the observed molecular mass for the doublet, and *indicates the cysteine peptides alkylated with iodoacetic acid.

to femtmolar concentrations.36 Figure 4 depicts derivatization of 500 fmol of C3a peptide with NHSS-benzoate. Derivatization of the N-termini of peptides with benzoyl moiety does not compromise fragmentation patterns as depicted in parts a and b of Figure

5. The database search with readily available N-terminus modifications in MASCOT using isotopic analogues of benzoate further aids sequencing by identification of “b” ions. A minor limitation of derivatization of N-termini of peptides with hydrophobic Analytical Chemistry, Vol. 76, No. 19, October 1, 2004

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Figure 4. Averaged full scan ESI-MS spectrum of 500 fmol of model peptide C3a (464.7 amu, doublet) quantitatively derivatized with benzoyl-NHSS. The inset is the expanded spectrum indicating the absence of any underivatized peptide at 412.7 amu (doublet).

reagents, including FMOC, is the slight decrease in ionization efficiency during MALDI and to a lesser extent in ESI forms of ionization.37 Sequence grade trypsin is specific for post lysine/arginine amide bonds. Proteolysis of peptides with a sequence having a proline residue on the C-terminal side of a lysine and arginine residue is substantially reduced.38 Taking into consideration only these potential missed cleavages, complete sequence coverage of proteins can be ascertained using benzoyl derivatization for proteins susceptible to trypsin proteolysis. An exception will be certain membrane proteins which lack solubility, thereby requiring initial cleavage with cyanogen bromide.39 Another exception will be the peptides which have poor ionization efficiency in the

positive-ion mode, e.g., peptides with inherent negative charges due to the presence of several aspartatic acid/glutamic acid residues. Therapeutic Protein Analysis. Both in vivo and in vitro modifications of therapeutic proteins can alter their biological activity.2,40 It is for this reason that primary structure analysis is such an important part of QC/QA and regulation in the biopharmaceutical industry. Genetic drift, misincorporation of amino acids, limited proteolysis, methionine oxidation, and deamidation are but a few of the phenomena that can lead to therapeutic proteins of potentially reduced biological activity and immunogenicity. During the past decade federal regulatory agencies internationally have come to depend heavily on peptide mapping by RPC and RPC/MS to confirm primary structure and regulate the production of biopharmaceuticals.41 The sequence coverage issue caused by poor retention of small, hydrophilic peptides is still a nagging problem with this otherwise good method. This problem can be illustrated with recombinant human growth hormone (rhGH). On the basis of previous tryptic mapping studies involving recombinant human growth hormone, amino acid and peptides K, AHR, EQK, EETQQK have not been observed during RPC-MS analysis.3 The peptides are not retained in the reversed-phase columns and elute in 100% mobile phase A. These peptides constitute 7.4% of the sequence. The objective of the work described below was to study the effect of benzoyl derivatization on retention of these short tryptic fragments. After derivatization with NHSS-benzoate, it was observed that all derivatized peptides were retained during the initial loading and equilibration steps. The amino acid and short peptides

Figure 5. MS/MS spectra of (a) underivatized C3a peptide and (b) benzoyl-derivatized C3a peptide. 5804

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Table 3. Comparison of Retention Profile for Human Growth Hormone Amino Acids/Peptides before and after Derivatization with NHSS-benzoatea

peptide fragment

peptide sequence

observed monoisotopic peptide mass after benzoyl derivatization (Da)

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 T17 T18-T19 T20 T21

FPTIPLSR LFDNAMLR AHR LHQLAFDTYQEFEEAYIPK EQK YSFLQNPQTSLC*FSESIPTPSNR EETQQK SNLELLR ISLLLIQSWLEPVQFLR SVFANSLVYGASDSNVYDLLK DLEEGIQTLMGR LEDGSPR TGQIFK QTYSK FDTNSHNDDALLK NYGLLYC*FR K DMDKVETFLR IVQC*R SVEGSC*GF

1034.56 1083.53 487.24 1275.50(2+) 612.27 1389.61(2+) 970.41 948.51 1080.11(2+) 1235.58(2+) 1465.69 877.40 901.45 834.37 849.38(2+) 1310.57 355.16 731.319(2+) 780.37 947.34

observed retention time of underivatized peptides (min)

observed retention time of benzoyl-derivatized peptides (min)

37.3 39.1 42.9 42.3 34.5 56.7 46.5 42.9 20.6 27.2 7.6 30.9 42.5 36.7 30.9 20.8

52.4 53.8 24.9 54.5 38.5 49.1 37.1 44.2 70.4 57.8 52.8 37.1 48.6 39.5 48.8 49.7 39.5 55.7 54.8 39.6

a An * indicates the cysteine peptides alkylated with iodoacetic acid, - indicates the nonretained amino acids and peptides, and 2+ indicates the observed molecular mass for the doublet.

Table 4. Short Peptides and Amino Acids Retained after Benzoyl Derivatization and the Corresponding Increase in Sequence Coverage

protein

underivatized amino acids and peptides not retained in RPC

cytochrome c

K, GK, GGK, HK, TER, ATNE

myoglobin

K, HK, YK, FDK

lysozyme

L, R, K, CK, NR, GCR, GY

recombinant human growth hormone

K, AHR, EQK, EEQTTK

which were previously lost in the void volume eluted in the linear trifluoroacetic acid/acetonitrile gradient leading to complete sequence coverage (Figure 6). As was the case with model proteins, benzoyl derivatization had a very large impact on the retention of small, hydrophilic peptides; the effect on larger hydrophobic peptides, T4 (MW 2342.14 Da) and T6 (MW 2616.13 Da) was relatively minor. (Table 3). The previously unretained amino acid and peptides eluted separately during the linear gradient as mentioned in the parenthesis: K (29.5 AcN%), AHR (14.9 AcN%), EQK (28.5 AcN%), EETQQK (27.1 AcN%) (Table 4). (36) Thompson, A.; Schaefer, J.; Kuhn, K.; Kiene, S.; Schwarz, J.; Schmidt, G.; Johnstone, R.; Neumann, T.; Hamon, C. Anal. Chem. 2003, 75, 1895-1904. (37) Julka, S.; Regnier, F. J. Proteome Res. 2004, 3, 350-363. (38) Thiede, B.; Lamer, S.; Mattow, J.; Siejak, J.; Dimmler, C.; Rudel, T.; Jungblut, P. Rapid Commun. Mass Spectrom. 2000, 14, 496-502. (39) Blonder, J.; Goshe, M. B.; Moore, R. J.; Pasa-Tolic, L.; Masselon, C. D.; Lipton, M. S.; Smith, R. D. J. Proteome Res. 2002, 1, 351-360.

observed monoisotopic masses of retained amino acids and peptides after benzoyl derivatization (Da)

increase in sequence coverage accrued (%)

355.15, 412.17, 492.20, 509.22, 538.20 355.31, 492.34, 518.22, 617.26 236.14, 279.15 355.17, 516.18, 393.19, 497.18, 343.15 355.16, 487.24, 612.27, 970.41

21.2 8.5 9.3

7.4

Detection of Amino Acid Polymorphism. Amino acid polymorphism can appear anywhere in the primary structure of a protein, thus necessitating the need for complete sequence coverage. This problem is well recognized in the detection of single amino acid polymorphism (SAAP) in wild-type populations where poor RPC retention of small, hydrophilic peptides has been cited as the reason that tryptic peptide analysis of proteins can fail to detect some SAAP variants.42 The current solution to this problem is to conduct the analysis several times using proteolytic enzymes with different cleavage patterns.43,44 But this is both time (40) Gomord, V.; Faye, L. Curr. Opin. Plant Biol. 2004, 7, 171-181. (41) Dormandy, S. J.; Lei, J.; Regnier, F. E. J. Chromatogr., A 1999, 864, 237245. (42) Liu, P.; Regnier, F. E. Anal. Chem. 2003, 75, 4956-4963. (43) Gatlin, C. L.; Eng, J. K.; Cross, S. T.; Detter, J. C.; Yates, J. R., III. Anal. Chem. 2000, 72, 757-763. (44) Choudhary, G.; Wu, S.-L.; Shieh, P.; Hancock, W. S. J. Proteome Res. 2003, 2, 59-67.

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presence of SAAPs between two individuals was identified by searching for singlets varying no more than +139 mass units, i.e., the mass difference between glycine and tryptophan in a pool of doublets.42 Clearly benzoyl derivatization has the potential to allow detection of most amino acid polymorphism in proteins except a leucine/isoleucine substitution. Another possible exception would be an amino acid substitution in a glycopeptide.

Figure 6. Overlaid extracted ion chromatograms of previously unretained amino acid and peptides during reversed-phase chromatography of recombinant human growth hormone.

intensive and redundant in the case of at least 80% of protein sequence. The objective in the work described below was to assess whether derivatization of peptides with NHSS-benzoate would overcome this problem. The model protein, cytochrome c was chosen because its natural amino acid variants are readily available. Equine and bovine cytochrome c differ in their sequence at positions 60 and 89. Tryptic digestion of these proteins produces peptide variants TER (MW 405.20 Da) and GER (MW 361.18 Da) from equine and bovine cytochrome c, respectively, that carry an SAAP on position 89. Without derivatization both of these peptides were observed to elute in the column void volume. After benzoyl derivatization, TER eluted at ∼11% acetonitrile while GER eluted at 16% acetonitrile in a linear trifluoroacetic acid/acetonitrile gradient. The isotopic analogues of NHSS-benzoate can also be synthesized using the same protocol from commercially available 13C6 and d5-benzoic acid. With the use of isotopic analogues, the

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CONCLUSIONS It is concluded that, due to complete water solubility of N-hydroxysuccinamide sulfonyl-benzoate, tryptic peptides can be derivatized in one single step and that benzoylation of most tryptic peptides will lead to retention during reversed-phase chromatography. Benzoyl derivatization is especially useful for proteins with high lysine-to-arginine ratio. For such proteins, proteolytic digestion can be carried out with the proteolytic enzyme lys-C resulting in derivatization of all peptides with two benzoyl moieties. In conclusion, derivatization of short peptides with a hydrophobic moiety will be particularly useful in therapeutic protein analysis, detection of single amino acid polymorphism, and detection of posttranslational modifications occurring in short hydrophilic peptides. ACKNOWLEDGMENT The authors gratefully acknowledge support from the NIH Grant 5R01 GM 59996-04. The authors thank Eli Lilly for the generous gift of recombinant human growth hormone.

Received for review February 26, 2004. Accepted July 9, 2004. AC049688E