Methyl Esterification Assisted MALDI FTMS Characterization of the

Casey J. Krusemark, Jonathan T. Ferguson, Craig D. Wenger, Neil L. Kelleher, and Peter J. Belshaw. Analytical Chemistry 2008 80 (3), 713-720. Abstract...
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Anal. Chem. 2007, 79, 673-681

Methyl Esterification Assisted MALDI FTMS Characterization of the Orcokinin Neuropeptide Family Mingming Ma, Kimberly K. Kutz-Naber, and Lingjun Li*

School of Pharmacy and Department of Chemistry, University of WisconsinsMadison, 777 Highland Avenue, Madison, Wisconsin 53705-2222

Methyl esterification of a peptide converts carboxylic acids, such as those present on the side chains of aspartic (D) and glutamic acid (E) as well as the free carboxyl terminus, to their corresponding methyl esters. This method has been applied to peptide and protein quantitation, de novo sequencing, and reduction of nonspecific binding in immobilized metal affinity chromatography for enrichment of phosphorylated peptides. In this study, we investigate the application of this derivatization reaction to the identification and characterization of the orcokinin neuropeptide family by screening and localizing the acidic side chains in peptides. The methyl esterification reaction drastically improves the fragmentation efficiency of modified orcokinins due to blockage of the aspartate selective cleavage pathway of the native orcokinin peptides. With the improved sustained off-resonance irradiation-collisional-induced dissociation spectra, the number and the locations of D and E residues are easily deduced. In addition, a side reaction that occurs at the carboxamide group of asparagine (N) is studied. The deamidation followed by subsequent methyl esterification reaction mechanism is proposed based on the study of an isotopelabeled standard N*FDEIDR. Reaction kinetics is studied by elevating the temperature from room temperature to 37 °C. The deamidation-methyl esterification products are greatly enhanced with elevated reaction temperature. Furthermore, we also explore the utility of this side reaction for rapid screening and characterization of Cterminally amidated neuropeptides. This derivatization reaction is applied to both in situ direct tissue neuropeptide analysis and the analysis of HPLC fractions from the separation of complex neuronal tissue extracts. Overall, this study reports a simple and effective method for profiling and localizing acidic amino acid residues (D/E), amide-containing residues (N/Q), and the Cterminal amide group in a peptide. Neuropeptides are a ubiquitous and powerful class of chemical mediators acting as neurotransmitters, neurohormones, or neuromodulators. They induce and regulate many important physi* Towhomcorrespondenceshouldbeaddressed.E-mail: [email protected]. Phone: (608)265-8491. Fax: (608)262-5345. 10.1021/ac061536r CCC: $37.00 Published on Web 11/21/2006

© 2007 American Chemical Society

ological processes throughout the animal kingdom.1-4 Challenges exist in traditional techniques for neuropeptide analysis due to very low concentrations, extreme chemical diversity, and the complexity of these endogenous signaling molecules. With unique advantages such as speed, high sensitivity, chemical specificity, and the capability of de novo sequencing, mass spectrometry (MS) is playing an increasingly important role in the identification and discovery of neuropeptides in nervous systems.5-19 However, MSbased de novo sequencing brings further challenges due to the possible incompleteness of MS/MS fragmentation and the ambiguity occurring in a complex MS/MS spectrum. Therefore, a variety of derivatization reactions have been developed to overcome such difficulties and ambiguities in mass spectrometric sequencing. Derivatization techniques label the N- or C-terminus of a peptide and thus increase the information content of a fragment spectrum. To assist solving the ambiguity between the b- and y-ion series in a complex MS/MS spectrum, reductive methylation and acetylation are designed to label the N-terminus and the -amino groups of lysine residue. This not only allows the differentiation (1) Hokfelt, T.; Broberger, C.; Xu, Z. Q.; Sergeyev, V.; Ubink, R.; Diez, M. Neuropharmacology 2000, 39, 1337-1356. (2) Schwartz, M. W.; Woods, S. C.; Porte, D., Jr.; Seeley, R. J.; Baskin, D. G. Nature 2000, 404, 661-671. (3) Skiebe, P. J. Exp. Biol. 2001, 204, 2035-2048. (4) Desiderio, D. M. J. Chromatogr., B: Biomed. Sci. Appl. 1999, 731, 3-22. (5) Haskins, W. E.; Wang, Z.; Watson, C. J.; Rostand, R. R.; Witowski, S. R.; Powell, D. H.; Kennedy, R. T. Anal. Chem. 2001, 73, 5005-5014. (6) Wei, H.; Nolkrantz, K.; Parkin, M. C.; Chisolm, C. N.; O’Callaghan, J, P.; Kennedy, R. T. Anal. Chem. 2006, 78, 4342-4351. (7) Li, L.; Garden, R. W.; Sweedler, J. V. Trends Biotechnol. 2000, 18, 151160. (8) Hummon, A. B.; Amare, A.; Sweedler, J. V. Mass Spectrom. Rev. 2006, 25, 77-98. (9) Desiderio, D. M. Methods Mol. Biol. 1996, 61, 57-65. (10) Emmett, M. R.; Andren, P. E.; Caprioli, R. M. J. Neurosci. Methods 1995, 62, 141-147. (11) Skold, K.; Svensson, M.; Nilsson, A.; Zhang, X.; Nydahl, K.; Caprioli, R. M.; Svenningsson, P.; Andren, P. E. J. Proteome Res. 2006, 5, 262-269. (12) Svensson, M.; Skold, K.; Svenningsson, P.; Andren, P. E. J. Proteome Res. 2003, 2, 213-219. (13) Jimenez, C. R.; Li, K. W.; Dreisewerd, K.; Spijker, S.; Kingston, R.; Bateman, R. H.; Burlingame, A. L.; Smit, A. B.; van Minnen, J.; Geraerts, W. P. Biochemistry 1998, 37, 2070-2076. (14) Che, F. Y.; Fricker, L. D. Anal. Chem. 2002, 74, 3190-3198. (15) Fricker, L. D.; Lim, J.; Pan, H.; Che, F. Y. Mass Spectrom. Rev. 2006, 25, 327-344. (16) Baggerman, G.; Boonen, K.; Verleyen, P.; De Loof, A.; Schoofs, L. J. Mass Spectrom. 2005, 40, 250-260.

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between b- and y-type fragment ions but also can be used to confirm the number of lysine residues in a peptide. Also, these derivatization reactions show utility for quantitative analysis because of the readily available isotope-labeled reagents.20-24 Methyl esterification converts carboxylic acids on the side chains of aspartic (D) and glutamic acids (E) as well as the carboxyl terminus to methyl esters with a 14-Da mass increment.25 It is a well-established derivatization technique used in gas chromatographic analysis of fatty acids by converting them into fatty acid methyl esters with lower boiling points.26,27 This simple derivatization method has been demonstrated for peptide and protein quantitation as well as de novo sequence derivation.25 More recently, it was used to reduce nonspecific binding in the enrichment of phosphopeptides via immobilized metal affinity chromatography.28,29 Here, we explore the utility of methyl esterification for the determination of the number and locations of acidic residues such as D/E as well as amide-containing amino acids Asn (N)/Gln (Q) in a peptide by comparing the native peptides and the derivatized products. Because the orcokinin neuropeptide family is enriched with D and E as well as contains N-terminus N, this peptide family provides an excellent model system to demonstrate the application of this derivatization reaction. Orcokinins comprise a family of neuropeptides that has been identified in a large number of decapod crustaceans and possess conserved sequences and myotropic activity.30-33 Orcokinin was originally purified from the nervous system of the crayfish Orconectes limosus,34 and its structural analogues have been subsequently isolated from numerous other crustacean species.32,33,35-38 Recently, members of the orcokinin family were (17) Husson, S. J.; Clynen, E.; Baggerman, G.; De Loof, A.; Schoofs, L. Biochem. Biophys. Res. Commun. 2005, 335, 76-86. (18) Cruz-Bermudez, N. D.; Fu, Q.; Kutz-Naber, K. K.; Christie, A. E.; Li, L.; Marder, E. J. Neurochem. 2006, 97, 784-799. (19) Fu, Q.; Kutz, K. K.; Schmidt, J. J.; Hsu, Y. W.; Messinger, D. I.; Cain, S. D.; de la Iglesia, H. O.; Christie, A. E.; Li, L. J. Comp. Neurol. 2005, 493, 607626. (20) Hsu, J. L.; Huang, S. Y.; Chow, N. H.; Chen, S. H. Anal. Chem. 2003, 75, 6843-6852. (21) Hsu, J. L.; Huang, S. Y.; Shiea, J. T.; Huang, W. Y.; Chen, S. H. J. Proteome Res. 2005, 4, 101-108. (22) Ji, C.; Li, L. J. Proteome Res. 2005, 4, 734-742. (23) Fu, Q.; Li, L. Anal. Chem. 2005, 77, 7783-7795. (24) Chei, F. Y.; Eipper, B. A.; Mains, R. E.; Fricker, L. D. Cell. Mol. Biol. (Noisyle-grand) 2003, 49, 713-722. (25) Goodlett, D. R.; Keller, A.; Watts, J. D.; Newitt, R.; Yi, E. C.; Purvine, S.; Eng, J. K.; von Haller, P.; Aebersold, R.; Kolker, E. Rapid Commun. Mass Spectrom. 2001, 15, 1214-1221. (26) Barnes, P. C.; Holaday, C. E.; J. Chromatogr. Sci. 1972, 10, 181. (27) Hornstein, I.; Alford, J. A.; Elliott, L. E.; Crowe, P. F. Anal. Chem. 1960, 32, 540-542. (28) Ficarro, S. B.; McCleland, M. L.; Stukenberg, P. T.; Burke, D. J.; Ross, M. M.; Shabanowitz, J.; Hunt, D. F.; White, F. M. Nat. Biotechnol. 2002, 20, 301-305. (29) Haydon, C. E.; Eyers, P. A.; Aveline-Wolf, L. D.; Resing, K. A.; Maller, J. L.; Ahn, N. G. Mol. Cell. Proteomics 2003, 2, 1055-1067. (30) Bungart, D.; Dircksen, H.; Keller, R. Peptides 1994, 15, 393-400. (31) Dircksen, H.; Burdzik, S.; Sauter, A.; Keller, R. J. Exp. Biol. 2000, 203 (Pt. 18), 2807-2818. (32) Li, L.; Pulver, S. R.; Kelley, W. P.; Thirumalai, V.; Sweedler, J. V.; Marder, E. J. Comp. Neurol. 2002, 444, 227-244. (33) Skiebe, P.; Dreger, M.; Meseke, M.; Evers, J. F.; Hucho, F. J. Comp. Neurol. 2002, 444, 245-259. (34) Stangier, J.; Hilbich, C.; Burdzik, S.; Keller, R. Peptides 1992, 13, 859864. (35) Bungart, D.; Hilbich, C.; Dircksen, H.; Keller, R. Peptides 1995, 16, 6772.

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also identified in several insect species including cockroach Blattella germanica,39,40 Leucophaea maderae,38 and locust Schistocerca gregaria.41 In addition, several orcokinin neuropeptide genes were identified in Caenorhabditis elegans42 and in Drosophila melanogaster.43 These studies demonstrate the widespread distribution of this peptide family and suggest that the orcokinins may have numerous roles in the regulation of complex behaviors. Physiological studies of orcokinins show that they elicit potent myotropic effects in the hind gut of crayfish O. limosus.31,34 Furthermore, the application of exogenous Ala13-orcokinin to the stomatogastric ganglion of the lobster Homarus americanus results in changes in the pyloric rhythm.32 These myotropic neuropeptides exhibit the conserved N-terminus sequence NFDEIDR, with C-terminal variations occurring at residues 8 and 9 or most commonly residue 13.37,44 Distinct from most of the other neuropeptides in crustaceans with amidated C-termini, orcokinins have a free carboxylic acid at the C-terminus. Despite extensive biochemical characterization, the physiological roles of this unique family are not well understood. Hence, the comprehensive profiling and characterization of the orcokinin family represents an important first step toward a better understanding of the structure and function relationship of this widely distributed neuropeptide family. In this study, we explore the utility of a methyl esterification reaction for the identification and characterization of the orcokinin neuropeptide family by screening and localizing the acidic amino acid residues D/E characteristic of the orcokinin sequence. We also study the reaction mechanism and kinetics of the side reaction that occurs at the amide side chain of N. This side reaction facilitates the identification and localization of N/Q. Furthermore, we describe the potential application of this two-step deamidationmethyl esterification reaction for locating and sequencing Cterminally amidated neuropeptides in both LC fractions from complex tissue extracts and in situ direct tissue analysis. MATERIALS AND METHODS Materials. The peptides Ala13-orcokinin (NFDEIDRSGFGFA) and Val13-orcokinin (NFDEIDRSGFGFV) were synthesized at the Biotechnology Center of the University of Illinois at Urbanas Champaign. The peptide N (U-413C, U-215N) FDEIDR (labeled as N*FDEIDR throughout the paper) was synthesized at the Biotechnology Center of the University of Wisconsin at Madison. Methanol, acetonitrile, and glacial acetic acid were purchased from Fisher Scientific (Pittsburgh, PA). Acetyl chloride was purchased from Sigma-Aldrich (St. Louis, MO). 2, 5-dihydroxybenzoic acid (DHB) was obtained from ICN Biomedicals Inc. (36) Yasuda-Kamatani, Y.; Yasuda, A. Gen. Comp. Endocrinol. 2000, 118, 161172. (37) Skiebe, P.; Dreger, M.; Borner, J.; Meseke, M.; Weckwerth, W. Cell. Mol. Biol. (Noisy-le-grand) 2003, 49, 851-871. (38) Huybrechts, J.; Nusbaum, M. P.; Bosch, L. V.; Baggerman, G.; De Loof, A.; Schoofs, L. Biochem. Biophys. Res. Commun. 2003, 308, 535-544. (39) Pascual, N.; Castresana, J.; Valero, M. L.; Andreu, D.; Belles, X. Insect Biochem. Mol. Biol. 2004, 34, 1141-1146. (40) Hofer, S.; Homberg, U. Cell Tissue Res. 2006, 325, 589-600. (41) Hofer, S.; Dircksen, H.; Tollback, P.; Homberg, U. J. Comp. Neurol. 2005, 490, 57-71. (42) Nathoo, A. N.; Moeller, R. A.; Westlund, B. A.; Hart, A. C. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 14000-14005. (43) Liu, F.; Baggerman, G.; D’Hertog, W.; Verleyen, P.; Schoofs, L.; Wets, G. Mol. Cell. Proteomics 2006, 5, 510-522.

Cancer borealis Brain Extract and H. americanus Thoracic and Abdominal Ganglion Extract HPLC Fractionation. C. borealis brains and H. americanus thoracic and abdominal ganglion were separately pooled, homogenized, and extracted with acidified methanol (90% methanol (Fisher Scientific), 9% glacial acetic acid (Fisher Scientific), and 1% deionized water). Extracts were dried in a Speedvac concentrator (Thermo Electron) and resuspended with a minimum amount of 0.1% formic acid. The resuspended extracts were then vortexed and briefly centrifuged. The supernatants were then used for HPLC separation. HPLC separations were performed using a Rainin Dynamax HPLC system equipped with a Dynamax UV-D II absorbance detector (Rainin Instrument Inc., Woburn, MA). The mobile phases included the following: (solution A) deionized water containing 0.1% formic acid, and (solution B) acetonitrile (HPLC grade, Fisher Scientific) containing 0.1% formic acid. A 20-µL aliquot of extract was injected onto a Macrosphere C18 column (2.1 mm i.d. × 250 mm length, 5-µm particle size; Alltech Assoc. Inc., Deerfield, IL). The separations consisted of a 120-min gradient of 5-95% solution B. Fractions were automatically collected every 2 min using a Rainin Dynamax FC-4 fraction collector. Methyl Esterification of Orcokinin Standards. An aliquot of 20 µL each of 10-5 M Ala13-orcokinin and Val13-orcokinin and was transferred to a clean tube, respectively, and lyophilized to dryness in a Speedvac followed by addition of 50 µL of methanolic HCl to each tube. Esterification was performed at room temperature or 37 °C for 2 h. The reaction solution was then concentrated to dryness in a Speedvac. Esterified peptides were resuspended in 10 µL of 0.1% formic acid in 30% methanol. The methanolic HCl solution was prepared freshly before use by slow drop addition of 160 µL of acetyl chloride to 1 mL of methanol with stirring on ice. After addition of acetyl chloride, the solution was kept on ice for 5 min. Methyl Esterification of C. borealis Brain, H. americanus Thoracic and Abdominal Ganglion HPLC Fractions and Brain Tissue. Aliquots of 5 µL each of HPLC fractions from C. borealis brain and H. americanus thoracic and abdominal ganglion extracts were transferred to clean tubes. The fractions were lyophilized, followed by addition of 25 µL of methanolic HCl. The solutions were allowed to react at room temperature or 37 °C for 2 h. Then, the reaction mixtures were concentrated to dryness and resuspended in 2 µL of 0.1% formic acid in 30% methanol. A piece of C. borealis brain tissue was dissected and rinsed in the acidified methanol (90:9:1 methanol/acetic acid/deionized water). The dried tissue piece was then transferred to a tube with 25 µL of methanolic HCl and allowed to react at 37 °C for 2 h. The esterified tissue was then transferred onto the MALDI plate followed by the addition of 0.3 µL of 150 mg/mL DHB on the top. MALDI FTMS and SORI-CID. Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI FTMS) experiments were performed on an IonSpec ProMALDI Fourier transform mass spectrometer (Lake Forest, CA) equipped with a 7.0-T actively shielded superconducting magnet. The FTMS instrument consists of a high-pressure MALDI source where the ions from multiple laser shots can be accumulated in the external hexapole storage trap before the ions are transferred to the ICR

cell via a quadrupole ion guide. A 337-nm nitrogen laser (Laser Science, Inc., Franklin, MA) was used for ionization/desorption. The ions were excited prior to detection with an rf sweep beginning at 7050 ms with a width of 4 ms and amplitude of 150 V base to peak. The filament and quadrupole trapping plates were initialized to 15 V, and both were ramped to 1 V from 6500 to 7000 ms to reduce baseline distortion of peaks. Detection was performed in broadband mode from m/z 108.00 to 4500.00. Fragmentation of the peptides was accomplished by sustained off resonance irradiation and collision-induced dissociation (SORICID). An arbitrary waveform with a (10-Da isolation window was introduced to isolate the ion of interest during the period of 20002131 ms. Ions were excited with a SORI burst excitation (2.648 V, 2500-3000 ms). A pulse of N2 was introduced through a pulse valve from 2500 to 2750 ms to induce collision activation. RESULTS AND DISCUSSION Screening for D/E and C-Terminus Free Acid Containing Peptides. Methyl esterification of a peptide converts carboxylic acids, such as those present on the side chains of aspartic and glutamic acid as well as the free carboxyl terminus, to their corresponding methyl esters. A mass increment of 14 Da is introduced by each conversion. Figure 1 shows the MALDI FTMS spectra of native orcokinin standards Ala13-orcokinin (Figure 1a), Val13-orcokinin (Figure 1c) and their corresponding methyl esterification products (Figure 1b and d). In Figure 1b and d, the absence of the native orcokinins indicates a complete reaction. The mass increments of 56 Da are seen in Figure 1b and d with m/z 1530.75 and 1558.79 representing the methylated Ala13-orcokinin and Val13-orcokinin, respectively. The mass shift of 56 Da in both derivatized orcokinins thus indicates the formation of four methyl esters in each orcokinin: two at the side chain of D, one at E, and one at the C-terminus. As seen, the number of the carboxylic acid groups in a peptide can be easily deduced by dividing the total mass increments upon methyl esterification by 14. This simple reaction thus provides a fast and effective way to screen for the presence of and determine the number of carboxylic acid groups in a peptide. Improving MS/MS Fragmentation Efficiency via Methyl Esterification. The MALDI SORI-CID fragmentation pattern of orcokinin methyl esterification products was drastically improved following the reaction compared to their native counterparts. It was previously shown that SORI-CID tandem MS analysis of orcokinins by MALDI FTMS only produced limited fragmentation with predominant formation of several y-series ions resulting from preferential cleavage at the peptide bond C-terminal to the aspartate residue.44 Similar results are seen in the SORI-CID fragmentation spectra of two native orcokinin standards. The y7 and y10 fragment ions governed by Asp-directed cleavage are predominantly detected (Figure 2a and c). Such selective cleavage at C-terminal to Asp has been reported extensively, especially for singly charged MALDI ions with an arginine residue sequestering the ionizing proton.45,46 After the carboxylic acid groups of aspartic (44) Stemmler, E. A.; Provencher, H. L.; Guiney, M. E.; Gardner, N. P.; Dickinson, P. S. Anal. Chem. 2005, 77, 3594-3606. (45) Gu, C.; Tsaprailis, G.; Breci, L.; Wysocki, V. H. Anal. Chem. 2000, 72, 58045813. (46) Wysocki, V. H.; Tsaprailis, G.; Smith, L. L.; Breci, L. A. J. Mass Spectrom. 2000, 35, 1399-1406.

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Figure 1. MALDI FTMS spectra of the native orcokinin standards and their methyl esterification products. (a) Native Ala13-orcokinin standard (NFDEIDRSGFGFA, m/z 1474.64). (b) Methyl esterification products of Ala13-orcokinin (+56-Da product at m/z 1530.75 and +71Da product at m/z 1545.76). (c) Native Val13-orcokinin standard (NFDEIDRSGFGFV, m/z 1502.70). (d) Methyl esterification products of Val13-orcokinin (+56-Da product at m/z 1558.79, +71-Da product at m/z 1573.79).

acid are converted to esters, this preferential cleavage pathway is blocked. Thus, the SORI-CID spectra of the derivatized peptides produce more complete b- and y-fragment ion coverage resulting from nonselective peptide backbone cleavage. This enhanced sequence coverage facilitates the localization of the carboxylic acid groups and improves sequence derivation. In contrast to the exclusive formation of only a few y-ions in SORI-CID of native orcokinin peptides, a contiguous series of b- and y-type fragment ions are detected in the methyl esterified counterparts (Figure 2b and d). By inspecting the b-ion series in the MS/MS spectra of derivatized peptides (Figure 2b and d), the localization of the 14-Da mass shifts is easily deduced with additions to D/E and C-terminus. Furthermore, the y-ions present in the native orcokinin MS/MS spectra (Figure 2a and c) were compared to those in the spectra of their methyl ester counterparts (Figure 2b and 676

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d). As shown, a 14-Da mass increment is observed between pairs of y7 ions, 28 Da for y9 ions, 42 Da for y10 ions, and 56 Da for y11 ions. Such corresponding mass shifts enable the determination of the number and the site of methyl esters. Screening and Localizing Carboxamide-Containing Residues. In addition to the 56-Da mass increment products upon methyl esterification, a minor peak with a mass increment of 71 Da is observed in Figure 1b and d at m/z 1545.76 and 1573.79 for Ala13-orcokinin and Val13-orcokinin, respectively. Figure 3 shows the effect of reaction temperature on the formation of these methyl esterification products using the orcokinin standard Val13-orcokinin. By elevating the reaction temperature from room temperature (Figure 3a) to 37 °C (Figure 3b), the relative abundance of the product at m/z 1573.79 is dramatically enhanced with inversed ratio of the +56- and +71-Da products. The SORI-CID spectra of the 71-Da mass increment products of Ala13-orcokinin (m/z 1545.76) and Val13-orcokinin (m/z 1573.79) provide relatively complete coverage of the b- and y-series of fragment ions for de novo sequencing (Figure 4). The comparison of the MS/MS spectra of m/z 1545.76 and 1573.79 with their corresponding 56Da increment products (Figure 2b and d) reveals identical y-ion pairs and a consistent mass shift of 15 Da for all the b-ions. This observation strongly suggests a mass increment of 15 Da occurs at the N-terminal Asn residues of these orcokinin peptides. In order to probe the reaction mechanism that occurs at Asn, an isotopic encoded peptide standard N*FDEIDR was used for the methyl esterification reaction. Figure 5 shows the MS spectra of the heavy isotope (15N,13C-) encoded standard N*FDEIDR (Figure 5a), methyl-esterified product at room temperature (Figure 5b) and 37 °C (Figure 5c). The derivatization at room temperature produced the main product at m/z 970.5 with mass increment of 56 Da from the original isotope-encoded standard (m/z 914.4). The minor product was m/z 984.5 with further mass increment of 14 Da compared to m/z 970.5. When the reaction took place at 37 °C, the minor product at m/z 984 was promoted. The SORICID spectrum of m/z 984.5 showed that the addition of 14 Da occurred at the N-terminus Asn (data not shown), which was inconsistent with the 15-Da increments observed in the native form. We propose a two-step reaction mechanism to account for such a mass increment. First, deamidation occurs by amide acid hydrolysis. Second, methyl esterification occurs at the free carboxylic acid group to form an ester. This reaction mechanism explains the addition of 14 Da at Asn in the heavy-isotope-encoded standard via deamidation (lost -N*H2, -17 Da, instead of -16 Da for regular amide group) followed by subsequent methyl esterification (addition of -OCH3, +31 Da), whereas in the unlabeled orcokinin standard the mass increment is 15 Da. Elevation of the reaction temperature promoted the formation of the deamidation-methyl esterification product of Val13-orcokinin (m/z 1573.85) with mass shift of 71 Da, becoming a dominant peak compared to m/z 1558.85 at the room temperature. Such an inverse ratio of relative product abundance can be explained by the endothermic process of the amide acid hydrolysis reaction, which is the first step of the carboxamide methyl esterification. With temperature elevation, the reaction equilibrium is shifted toward the formation of the deamidation product and is therefore favored for the formation of the deamidation-methyl esterification product. To assist in the identification and localization of the amide

Figure 2. MALDI FTMS SORI-CID fragmentation spectra of the native orcokinin standards and their +56-Da methyl esterification products. The peptide sequences are shown on the left with detected sequence-specific fragment ions labeled. The asterisks in the sequence indicate sites of methyl esterification. (a) Native Ala13-orcokinin standard (m/z 1474.6). (b) +56-Da methyl esterification product of Ala13-orcokinin standard (m/z 1530.8). (c) Native Val13-orcokinin standard (m/z 1502.7). (d) +56-Da methyl esterification product of Val13-orcokinin standard (m/z 1558.8).

groups in a peptide, the reaction temperature can be elevated for the methyl esterification reaction to promote the deamidationmethyl esterification product. However, the instability of a peptide at higher temperature could be a concern. For instance, further attempts to elevate reaction temperature to 70 °C caused degradation of the orcokinin standards (data not shown). This issue could be more problematic when dealing with biological tissue samples. Application for Characterization of C-Terminal Amidated Neuropeptides. Although methyl esterification is often considered as a bad choice for derivatizing neuropeptides because the

carboxyl terminus is often blocked by amidation, the utility of this reaction for locating Asn- and Gln-containing peptides prompts us to explore its application for studying C-terminal amidated neuropeptides. The amide group at the C-terminus can theoretically undergo the same two-step deamidation-methyl esterification reaction proposed to produce characteristic mass shifts upon derivatization. In order to probe the methyl esterification reaction with a C-terminal amidated peptide, a pevkinin-2 neuropeptide standard DFSAWAamide was used. Figure 6 shows MS spectra of the C-terminus amidated neuropeptide standard DFSAWAamide Analytical Chemistry, Vol. 79, No. 2, January 15, 2007

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Figure 3. Effects of reaction temperature on the products of the methyl esterification reaction with Val13-orcokinin (m/z 1502.7). (a) Val13-orcokinin methyl esterification at room temperature showing the +56-Da peak (m/z 1558.8) as major product. (b) Val13-orcokinin methyl esterification at 37 °C, with the enhancement of +71-Da product at m/z 1573.8.

Figure 4. MALDI FTMS SORI-CID fragmentation spectra of deamidation-methyl esterification products of (a) Ala13-orcokinin (m/z 1545.8) and (b) Val13-orcokinin (m/z 1573.8). Esterification sites are highlighted with asterisks in the peptide sequence.

(m/z 695.3, Figure 6a) and the methyl esterification product at room temperature (Figure 6b) and 37 °C (Figure 6c). Upon the methyl esterification reaction, a product with a mass increment of 14 Da is observed at m/z 709.3, suggesting the presence of a carboxylic acid in the peptide. In addition, another product with a further mass increment of 15 Da (m/z 724.3) is readily detected. Elevation of the derivatization temperature to 37 °C promotes the 678 Analytical Chemistry, Vol. 79, No. 2, January 15, 2007

Figure 5. Methyl esterification of the 15N-, 13C-isotope-encoded standard N*FDEIDR. (a) The native isotope-encoded standard (m/z 914.44). (b) Methyl esterification at room temperature with the detection of +56- and +70-Da products. (c) Methyl esterification at 37 °C with enhanced detection of a +70-Da product at m/z 984.47.

detection of this latter product, which indicates a deamidationmethyl esterification reaction occurring at the amide group. MS/MS analysis of derivatized pevkinin-2 (Figure 6d) further confirmed the presence of a C-terminal amide group with corresponding y-ions showing a characteristic mass shift of 15 Da. Thus, the methyl esterification reaction can be successfully applied to screening for C-terminal amidation, which is useful in neuropeptide sequencing and characterization because a large number of neuropeptides are C-terminally amidated. Utility for Biological Samples. One of the C. borealis brain HPLC fractions containing putative orcokinins (Figure 7a) at m/z 1502.54 and 1532.59 was used in this study. Panels b and c in Figure 7 show the MALDI FTMS spectra of the fraction after methyl esterification at room temperature and 37 °C, respectively. Derivatized products with mass increments of 56 and 71 Da are observed for both peptides, suggesting that both peptides contain four carboxylic acid groups and one amide group. Elevation of the derivatization reaction temperature to 37 °C promotes the formation of the 71-Da mass increment products, suggesting that m/z 1573.8 and 1603.8 peaks are products of the deamidationmethyl esterification reaction occurring at the amide group. The results presented herein highlight the utility of this simple derivatization method as a screening tool for discovering and characterizing orcokinin-like peptides in an unknown sample. The methyl esterification reaction was further applied to in situ direct tissue orcokinin neuropeptide family analysis. Figure 8a shows a mass spectrum of a piece of C. borealis brain with several orcokinins present in the spectrum, which were identified by accurate mass measurements with internal calibration (data not

Figure 7. MALDI FTMS spectra showing methyl esterification of a C. borealis brain HPLC fraction. (a) C. borealis brain HPLC fraction with two putative orcokinin peptides highlighted with asterisks. (b) Methyl esterification of C. borealis brain HPLC fraction at room temperature. (c) Methyl esterification of C. borealis brain HPLC fraction at 37 °C. Open circles represent the esterificated products of Val13-orcokinin (m/z 1502.54) and closed circles represent the esterified products of Ser9-Val13-orcokinin (m/z 1532.59).

Figure 6. Methyl esterification of C-terminal amidated neuropeptide DFSAWAamide. (a) The native neuropeptide standard DFSAWAamide (m/z 695.25). (b) Methyl esterification of the standard DFSAWAamide at room temperature with detection of +14- and +29Da products. (c) Methyl esterification of the standard DFSAWAamide at 37 °C with significantly enhanced detection of +29-Da product at m/z 724.30. (d) SORI-CID spectrum of esterificated product at m/z 724.30. Sequence-specific b- and y-type fragment ions are detected and labeled. Esterification sites are highlighted with asterisks in the peptide sequence.

shown). Furthermore, their identities were further confirmed by SORI-CID fragmentation analysis of LC fractions showing the corresponding peaks. Figure 8b shows the FTMS spectrum of C. borealis brain tissue after methyl esterification. The +56- and +71Da products of each orcokinin are marked with the same symbol as the corresponding unmodified counterparts in Figure 8a. Both products are observed for Ala13-orcokinin (m/z 1474.63) and Ser9Val13-orcokinin (m/z 1532.72), whereas for Val13-orcokinin (m/z 1502.72) and orcokinin1-11 (m/z 1256.56), only the 56-Da mass increment products are present. This could be due to the lower abundances of these particular orcokinins and the heterogeneity of the tissue sample for complete reaction. In addition to its utility for orcokinin peptide analysis, we also explore the application of methyl esterification for profiling other neuropeptides with D/E residue and/or C-terminal amidation. An

Figure 8. In situ analysis of orcokinin neuropeptides via methyl esterification of C. borealis brain tissue. (a) C. borealis brain direct tissue MALDI FTMS spectrum with four putative orcokinin peptides highlighted with different symbols. (b) MALDI FTMS spectrum of methyl esterified C. borealis brain tissue with symbols representing the methylated products that correspond to specific orcokinins in (a).

aliquot of HPLC fraction from the lobster H. americanus thoracic and abdominal ganglion extract was used in this study. Figure 9 shows MALDI FTMS and SORI-CID fragmentation spectra of both Analytical Chemistry, Vol. 79, No. 2, January 15, 2007

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side of Asp (Figure 9c). The fragmentation is drastically improved for the esterificated product at m/z 1314.68 (Figure 9d). With a contiguous series of y-ions and abundant b-ions, the sequence of the peptide is derived as pyrDLDHVFLRFamide. A mass increment of 15 Da for the y2-y6 ions suggests the deamidationmethyl esterification occurring at the C-terminus. The b5-b9 ions display 28-Da mass increments, which suggest that there are two acidic residues in the first five amino acid residues in the peptide sequence. When this information is combined with the detection of mass-shifted y6-y8 ions, the location of two D residues are determined. Assisted with reductive methylation and acetylation reactions, the first two amino acid residues at the N-terminus are determined to be a Gln-derived pyroglutamate modification followed by an Asp residue (data not shown). A similar RFamide of PELDHVFLRFamide neuropeptide was previously detected in the C. borealis brain and thoracic ganglion extracts.38 Our data described here suggest that a highly homologous peptide is also present in the lobster central nervous system. Overall, the methyl esterification not only greatly improves the fragmentation efficiency of Asp-containing neuropeptides but also can be used as a tool to screen for the presence of C-terminal carboxamide, which is one of the hallmarks of neuropeptides. The reaction is compatible with complex biological samples such as tissue samples that contain relatively low abundance peptides for a quick determination of the number of residues D/E, N/Q, and Cterminus amide. For a complex biological sample, prefractionation with HPLC is not required as we demonstrated applicability of this method for direct tissue derivatization. However, preseparation of tissue extracts results in a less complex mixture, facilitates the acquisition of higher quality MS/MS data for specific peptides, and enables more confident de novo sequencing. This technique thus facilitates neuropeptide discovery and sequence characterization.

Figure 9. Methyl esterification assisted MALDI FTMS identification of a FLRFamide-related neuropeptide in an HPLC fraction from the H. americanus thoracic/abdominal ganglion extract. (a) MALDI FTMS spectrum of H. americanus thoracic/abdominal ganglion HPLC fraction showing the presence of a putative neuropeptide at m/z 1271.67. (b) Methyl esterification of the H. americanus thoracic/abdominal ganglion HPLC fraction at 37 °C with enhanced detection of a +43-Da methyl esterification product at m/z 1314.68. (c) SORI-CID fragmentation spectrum of the peptide at m/z 1271.67 in the LC fraction. (d) SORICID fragmentation spectrum of the methyl-esterificated product at m/z 1314.68 with multiple sequence-specific b- and y-type fragment ions detected and labeled. The methyl esterification sites are highlighted with asterisks in the peptide sequence.

the native peptide and methyl esterification product of a major component at m/z 1271.67 (Figure 9a) in the fraction. Upon methyl esterification at 37 °C, a main product m/z 1314.68 is observed with a mass increment of 43 Da (Figure 9b). MS/MS spectrum of the native peptide exhibits few fragment ions with a major peak at m/z 817.47 resulting from selective cleavage at the C-terminal 680

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CONCLUSIONS In this report, we have demonstrated a novel application of methyl esterification for rapid screening and characterization of acidic residue-containing neuropeptides using MALDI FTMS. The significant improvement of fragmentation efficiency upon methyl esterification of the acidic residues enabled more complete sequence coverage due to the production of complementary b-type and y-type fragment ion series. In addition, the side reaction involving deamidation followed by subsequent methyl esterification provides potential utility for profiling and sequencing amidecontaining residues such as Asn- and Gln-containing peptides as well as a diverse array of neuropeptides with C-terminal amidation. Collectively, the methyl esterification reaction coupled with highresolution, high-accuracy MALDI FTMS, offers enhanced chemical information and confident identification of neuropeptides in complex biological samples. ACKNOWLEDGMENT The authors thank Xin Wei for helpful discussion and Stephanie DeKeyser for critical reading of the manuscript. We thank members from the laboratory of Professor Michael P. Nusbaum (Department of Neuroscience, University of Pennsylvania School of Medicine) for providing some of the C. borealis brain tissue used in the HPLC fractionation. Dr. Andrew E. Christie (Department of Biology, University of Washington) is acknowledged for

providing lobster tissue used in HPLC fractionation. We are also grateful to the UW School of Pharmacy Analytical Instrumentation Center for access to the MALDI FTMS instrument. This work was supported in part by the School of Pharmacy and the Wisconsin Alumni Research Foundation at the University of WisconsinsMadison, a National Science Foundation CAREER Award (CHE-0449991), and the National Institutes of Health through grant 1R01DK071801. L.L. acknowledges an Alfred P.

Sloan Research Fellowship. K.K.-N. acknowledges the National Institutes of Health Chemistry-Biology Interface Training Grant T32 GM008505.

Received for review August 17, 2006. Accepted October 11, 2006. AC061536R

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