De Novo Sequencing of Neuropeptides Using Reductive Isotopic

Anal. Chem. 2005, 77, 7783-7795

De Novo Sequencing of Neuropeptides Using Reductive Isotopic Methylation and Investigation of ESI QTOF MS/MS Fragmentation Pattern of Neuropeptides with N-Terminal Dimethylation Qiang Fu† and Lingjun Li*,†,‡

School of Pharmacy and Department of Chemistry, University of Wisconsin at Madison, Madison, Wisconsin 53705-2222, USA

A stable-isotope dimethyl labeling strategy was previously shown to be a useful tool for quantitative proteomics. More recently, N-terminal dimethyl labeling was also reported for peptide sequencing in combination with database searching. Here, we extend these previous studies by incorporating N-terminal isotopic dimethylation for de novo sequencing of neuropeptides directly from tissue extract without any genomic information. We demonstrated several new sequencing applications of this method in addition to the identification of the N-terminal residue using the enhanced a1 ion. The isotopic labeling also provides easier and more confident de novo sequencing of peptides by comparing similar MS/MS fragmentation patterns of the isotopically labeled peptide pairs. The current study on neuropeptides shows several distinct fragmentation patterns after N-terminal dimethylation which have not been reported previously. The y(n-1) ion is enhanced in multiply charged peptides and is weak or missing in singly charged peptides. The MS/MS spectra of singly charged peptides are simplified due to the enhanced N-terminal fragments and suppressed internal fragments. The neutral loss of dimethylamine is also observed. The mechanisms for the above fragmentations are proposed. Finally, the structures of the immonium ion and related ions of Nr, NE-tetramethylated lysine and NEdimethylated lysine are explored. Neuropeptides encompass the largest and the most diverse group of signaling molecules in the nervous system. They can act as neurotransmitters, neuromodulators, and hormones.1 This complex array of bioactive molecules controls and influences many types of behaviors.1-5 Elucidation of peptide messengers is a * To whom correspondence should be addressed. Phone: (608) 265-8491. Fax: (608) 262-5345. E-mail: [email protected]. † Department of Chemistry. ‡ School of Pharmacy. (1) Strand, F. L. Neuropeptides, regulators of physiological processes, 1st ed.; MIT Press: Cambridge; 1999. (2) Schwartz, M. W.; Woods, S. C.; Porte, D., Jr; Seeley, R. J.; Baskin, D. G. Nature 2000, 404, 661-671. (3) Gulpinar, M. A.; Yegen, B. C. Curr. Protein Pept. Sci. 2004, 5, 457-473. (4) Nassel, D. R. Prog. Neurobiol. 2002, 68, 1-84. (5) Keller, R. Experientia 1992, 48, 439-448. 10.1021/ac051324e CCC: $30.25 Published on Web 11/04/2005

© 2005 American Chemical Society

crucial step toward understanding how the neural circuitry functions. Biological mass spectrometry (MS) has become the method of choice for neuropeptide analysis due to its speed, high sensitivity, chemical specificity, and capability for complex mixture analysis.6-12 In contrast to the tryptic peptides utilized in “bottomup” proteomics, most neuropeptides do not contain lysine or arginine at the C-termini, resulting in the suppression of ionization and fragmentation efficiencies.13 Furthermore, many neuropeptides are extensively modified. In addition, many organisms used to study neuropeptide functionality lack a complete genomic database, thus making the database searching strategy unsuitable for these species. Consequently, de novo sequencing represents one of the biggest challenges in neuropeptide discovery. A major problem in de novo sequencing analysis is the determination of the respective N-terminal and C-terminal fragment ion series. Many chemical derivatization schemes have been developed to facilitate the de novo sequencing by introducing a mass shift at either the N-terminus14-19 or the C-terminus20-22 of (6) Nilsson, C. L.; Karlsson, G.; Bergquist, J.; Westman, A.; Ekman, R. Peptides 1998, 19, 781-789. (7) Desiderio, D. M.; Zhu, X. J. Chromatogr., A 1998, 794, 85-96. (8) Baggerman, G.; Verleyen, P.; Clynen, E.; Huybrechts, J.; De Loof, A.; Schoofs, L. J. Chromatogr., B 2004, 803, 3-16. (9) Andren, P. E.; Caprioli, R. M. Brain Res. 1999, 845, 123-129. (10) Kennedy, R. T.; Watson, C. J.; Haskins, W. E.; Powell, D. H.; Strecker, R. E. Curr. Opin. Chem. Biol. 2002, 6, 659-665. (11) Che, F. Y.; Yuan, Q.; Kalinina, E.; Fricker, L. D. J. Biol. Chem. 2005, 280, 4451-4461. (12) Jakubowski, J. A.; Sweedler, J. V. Anal. Chem. 2004, 76, 6541-6547. (13) Haskins, W. E.; Watson, C. J.; Cellar, N. A.; Powell, D. H.; Kennedy, R. T. Anal. Chem. 2004, 76, 5523-5533. (14) Yew, J. Y.; Dikler, S.; Stretton, A. O. Rapid Commun. Mass Spectrom. 2003, 17, 2693-2698. (15) Liu, P. R.; Regnier, F. E. J. Proteome Res. 2002, 1, 443-450. (16) Zhang, R.; Sioma, C. S.; Wang, S.; Regnier, F. E. Anal. Chem. 2001, 73, 5142-5149. (17) Munchbach, M.; Quadroni, M.; Miotto, G.; James, P. Anal. Chem. 2000, 72, 4047-4057. (18) Keough, T.; Lacey, M. P.; Youngquist, R. S. Rapid Commun. Mass Spectrom. 2000, 14, 2348-2356. (19) Lee, Y. H.; Han, H.; Chang, S. B.; Lee, S. W. Rapid Commun. Mass Spectrom. 2004, 18, 3019-3027. (20) 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. (21) Lindh, I.; Hjelmqvist, L.; Bergman, T.; Sjovall, J.; Griffiths, W. J. J. Am. Soc. Mass Spectrom. 2000, 11, 673-686.

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a peptide, thus enabling differentiation between b- and y-type fragment ion series in a complex MS/MS spectrum. Most of these derivatization methods work well for tryptic peptides, but their utility for native neuropeptide sequencing is somewhat problematic and requires further evaluation. For example, acetylation of basic amino groups changes the ionic states of peptides. This may lead to substantial loss of sensitivity in peptides due to reduced ionization and fragmentation efficiencies for lower charge state ions. Likewise, a C-terminal methylation method would not work for peptides lacking free carboxyl termini. Because C-terminal amidation is the most common posttranslational modification in neuropeptides, the utility of this labeling approach for neuropeptide de novo sequencing is limited. Recently, a reductive methylation labeling method using formaldehyde has shown great promise for de novo sequencing.23 This derivatization method labels the N-terminus and -amino group of lysine in peptides through reductive alkylation and produces peaks differing by 28 Da for each derivatized site. The intensity of the a1 ion is also substantially enhanced upon labeling, which is beneficial for de novo peptide sequencing because the b1 ion is usually missing in the MS/MS spectra.24 Furthermore, the ionic state of the modified peptides is not changed.25 The use of isotope-based formaldehyde labeling for quantitative proteomics was also demonstrated.25-27 In this paper, we expand upon these prior studies and investigate the use of a reductive isotopic dimethylation method for neuropeptide de novo sequencing. We apply this method for de novo sequencing of neuropeptides directly from the pericardial organ tissue extract of Cancer borealis, a model organism whose genomic sequence is currently unavailable. Several unique advantages of the isotope-based reductive methylation for neuropeptide de novo sequencing are demonstrated. We also propose mechanisms for the fragmentation pattern changes caused by the N-terminal dimethyl modification. Several fragmentation ions unique to this modification are explored. EXPERIMENTAL SECTION Animal Dissection and Tissue Extraction. Jonah crabs (Cancer borealis) were shipped from Marine Biological Laboratories (Woods Hole, MA) and maintained in an artificial seawater tank at 10-12 °C. The animal dissection and tissue extraction was performed using the same procedures published previously.28,29 Briefly, 30 pericardial organs (POs) were homogenized and peptides were extracted using ice-cold acidified methanol (90% methanol/9% glacial acetic acid/1% water). The extract was dried down and resuspended with 100 µL of water. Peptide Standards. The peptide KHKNYLRFamide was synthesized at the Biotechnology Center of the University of (22) Shevchenko, A.; Chernushevich, I.; Ens, W.; Standing, K. G.; Thomson, B.; Wilm, M.; Mann, M. Rapid Commun. Mass Spectrom. 1997, 11, 10151024. (23) Hsu, J. L.; Huang, S. Y.; Shiea, J. T.; Huang, W. Y.; Chen, S. H. J. Proteome Res. 2005, 4, 101-108. (24) Papayannopoulos, I. A. Mass Spectrom. Rev. 1995, 14, 49-73. (25) Hsu, J. L.; Huang, S. Y.; Chow, N. H.; Chen, S. H. Anal. Chem. 2003, 75, 6843-6852. (26) Ji, C.; Li, L. J. Proteome Res. 2005, 4, 734-742. (27) Ji, C.; Li, L.; Gebre, M.; Pasdar, M.; Li, L. J. Proteome Res. 2005, published online. (28) Fu, Q.; Christie, A. E.; Li, L. Peptides 2005, in press. (29) Fu, Q.; Kutz, K. K.; Schmidt, J. J.; Hsu, Y. A.; Messinger, D. I.; Cain, S. D.; de la Iglesia, H. O.; Christie, A. E.; Li, L. J. Comput. Neurol. 2005, in press.

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Wisconsin at Madison. DFSAWAamide (Pev-kinin 2) and pyrTSFTPRLamide (Lem-PK) were generous gifts from Prof. Michael Nusbaum (School of Medicine, University of Pennsylvania). YGGFL (Leu-enkephalin), FMRFamide, NRNFLRFamide, pyrLYENK (Gallus gallus neurotensin [1-6]), PFCNAFTGCamide (CCAP), EGVYVHPV (angiotensin II antipeptide), and CYFQNCPRGamide ([Arg8] vasopressin] were purchased from American Peptide Company (Sunnyvale, CA). Reductive Methylation of Peptide Standards and Tissue Extract. Reductive methylation was performed using a combination of previously published procedures23,25,30 with modifications. Ten microliters of crude extract was mixed with 15 µL of acetate buffer (pH 4.8, 1 M), followed by the addition and mixing of 5 µL of formaldehyde (37% in H2O v/v, Sigma-Aldrich, St. Louis, MO) and subsequent addition of 2 µL of 5 M NaBH3CN (SigmaAldrich). The labeling reaction was allowed to take place for 6 h at room temperature. Excess formaldehyde was quenched via the addition of 4 µL of ammonia (37% in H2O v/v, Sigma-Aldrich). The resulting solution was stored at -20 °C before LC-MS/MS analysis. Formaldehyde-d2 labeling was performed in a similar manner with the exception that 7 µL of formaldehyde-d2 (25% in H2O v/v, Sigma-Aldrich) was used instead. The detailed experimental conditions for the labeling of the peptide standards are listed in the Supporting Information (Table S-1). Both formaldehyde and sodium cyanoborohydride are very toxic by inhalation, in contact with skin, or if swallowed and may cause cancer and heritable genetic damage (Sigma-Aldrich Rick Statements 23/24/25-34-40-43 and 26-28-36/37/39-45-60-61). Special caution must be taken when handling these chemicals (SigmaAldrich Safety Statements 26-36/37/39-45 and 26-28-36/37/39-4560-61). Capillary LC Q-TOF MS/MS. NanoLC-MS/MS analysis was performed using a Waters capillary LC system coupled to a Q-TOF Micro mass spectrometer (Waters Corporation, Milford, MA). Chromatographic separations were performed on a reversed-phase capillary column (Atlantis dC18, 75 µm i.d. × 100 mm length, 3-µm particle size, Waters). The mobile phases used were (A) deionized H2O with 5% acetonitrile and 0.1% formic acid, (B) acetonitrile with 5% deionized H2O and 0.1% formic acid, and (C) deionized H2O with 0.1% formic acid. An aliquot of sample was injected and loaded onto the trap column (PepMap C18, 300 µm column i.d. × 1 mm, 5-µm particle size, LC Packings, Sunnyvale, CA) for desalting the sample using mobile phase C at a flow rate of 30 µL/min for 3 min. Following this, the stream-select module was switched to a position where the trap column became inline with the analytical capillary column, and a linear gradient of mobile phases A and B was initiated. A splitter was added between the mobile phase mixer and the stream-select module to reduce the flow rate from 18 µL/ min to 200 nL/min. For tissue samples, 1.4 µL of crude PO extract and 5.0 µL of labeled extract was used in each analysis. The gradient was from 5% B to 45% B in 120 min. To correct for mass drift during data collection and improve the mass accuracy, a Nano LockSprayer was employed. Briefly, a syringe pump was used to introduce 1 ng/µL Leu-enkephalin into the reference spray at a flow rate of 0.5 µL/min. (30) Metz, B.; Kersten, G. F.; Hoogerhout, P.; Brugghe, H. F.; Timmermans, H. A.; de Jong, A.; Meiring, H.; ten Hove, J.; Hennink, W. E.; Crommelin, D. J.; Jiskoot, W. J. Biol. Chem. 2004, 279, 6235-6243.

Scheme 1. Reductive Methylation Reaction

The nanoflow electrospray ionization (ESI) source conditions were set as follows: capillary voltage, 3800 V; sample cone voltage, 40 V; extraction cone voltage, 1 V; source temperature, 120 °C; cone gas, 13 L/h. For the reference spray, the same settings were used except that the sample cone voltage was set at 10 V and reference scans were performed every 10 s. A data-dependent acquisition was employed for the MS survey scan and the selection of precursor ions and subsequent tandem MS of the selected parent ions. The MS/MS threshold was set at 20 counts. Each MS/MS scan from m/z 50 to 2000 took 1.9 s. The collision energy was set according to the ion charge state and the mass-to-charge ratio and varied from 16 to 55 eV. The MS/MS scan of each precursor ion was switched to MS scan after 6 s, and the precursor ion was excluded from the next MS/MS scan for 3 min. De Novo Sequencing. De novo sequencing was performed using a combination of the PepSeq peptide sequencer of MassLynx 4.0 (Waters) and manual sequencing. The MS/MS spectra were manually calibrated using the lock mass of Leuenkephalin (m/z 556.2771) in the LockSpray and processed using MaxEnt 3. The multiply charged ions were converted into their singly charged form after MaxEnt process. The resulting spectra were then pasted into the PepSeq window for automatic sequencing or manual sequencing. To verify the sequence assignments, the raw MS/MS spectra were compared with the predicted fragmentation pattern (using the MS-Product software at http:// prospector.ucsf.edu/) of the proposed sequences. RESULTS AND DISCUSSION Study of the Reductive Methylation Using Peptide Standards. The reaction mechanism of reductive methylation of peptides was investigated previously.30,31 As shown in Scheme 1, formaldehyde reacts with the amine group at the N-terminus or -amino group of lysine to produce a methylol group, which can subsequently lose water to form an imine. The imine can then be reduced to a methyl group by adding sodium cyanoborohydride. The formations of the methylol and the imine are reversible reactions, but the reduction of the imine to a methyl group is irreversible.30 Previous studies of reductive methylation showed different reaction products. In Hsu’s work,23,25 only dimethylation at the N-terminus or lysine was observed. The reaction was performed at pH 5.0, and ammonia was added to the solution to quench the excess formaldehyde after 5 min, whereas in an earlier work by Metz et al. using synthetic peptide standards, formaldehyde was found to react with not only the N-terminus and lysine but also with cysteine, arginine, histidine, and tryptophan in the peptide to form either methylol or imine adducts.30 The reaction was (31) Means, G. E.; Feeney, R. E. J. Food Biochem. 1998, 22, 399-425.

Figure 1. The MS/MS fragmentation spectra of (A) KHKNYLRFamide (552.842+), (B) (CH3)2K(CH3)2HK(CH3)2NYLRFamide (594.952+), (C) (CH3)2K(CH3)2HK(CH3)2NYLR(CH3OH)Famide (609.982+), and (D) (CHD2)2K(CHD2)2HK(CHD2)2NYLRFamide (600.932+). Major sequence-specific ions are labeled with identities. The immonium ion and related ions are labeled in the spectra. In panel C, the a-, b-, and y-ion series are labeled according to the MS/MS of (CH3)2K(CH3)2HK(CH3)2NYLRFamide (panel B). In panel C, the apostrophe indicates the modification of imine to arginine residue. The half quote mark stands for the modification of methylol group to arginine side chain. In panel D, the low mass region is enlarged to show the immonium ion and related ions in the inset. The neutral losses of dimethylamine ions are marked by black squares in panels B, C, and D. In panels B and C: 277.14, b2 - 45 (-45 means -NH(CH3)2); 294.15, HK*; 547.21, K*NYL; 823.35, HK*NYLR-NH3. In panel B: 481.712+, (b7 + H - NH3 - 45)2+; 572.242+, (M + 2H - 45)2+; 959.34, Y7 - 45. In panel C: 481.762+, (b7 + H - NH3 - CH2O - 45)2+; 572.33, (M + 2H - CH2O - 45)2+; 959.50, Y7 - CH2O - 45. In panel D: 281.22, b2 - 49 (-49 stands for -NH(CHD2)2); 298.25, HK*; 485.852+, (b7 + H - NH3 - 49)2+; 551.42, K*NYL; 576.442+, (M + 2H - 49)2+; 660.51, HK*NYL - 28; 827.58, HK*NYLR - NH3; 959.67, Y7 - 49. The asterisk represents the dimethyl (or di-D2-methyl, panel D) modification. The neutral loss from the y7 ion is from the side chain of lysine. The other neutral losses from the b ions and the precursor ions can result from either the side chain or the N-terminus. In panel C, the 594.872+ is loss of CH2O from the precursor ion. In panel D, the peak between 84 and 88 peaks is 86 Da, which is the immonium ion of L.

conducted at pH 7.2 and lasted for 48 h. Following the addition of reducing agent, a mass increment of 28/26 or 28/24 Da was observed for peptides with a free N-terminus, lysine or both lysine and arginine. To investigate the reason behind the discrepancy between the above observations and to determine the optimum labeling conditions for neuropeptides in tissue extract, we performed a systematic investigation of reaction conditions using a set of neuropeptide standards. We observed complex fragmentation patterns of two side products, the imine or methylol adduction at Analytical Chemistry, Vol. 77, No. 23, December 1, 2005

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Scheme 2. Structures of the Immonium Ion and Related Ions of Tryptophana

Figure 2. The MS/MS fragmentation spectra of (A) DFSAWAamide (695.29+), (B) (CH3)2DFSAWAamide (723.29+), (C) (CH3)2DFSAW(CH3OH)Aamide (753.44+) and (D) (CH3)2DFSAW(dCH2)Aamide (735.45+). The imine adducted peptide is the neutral loss of water from the methylol-modified peptide because they eluted at the same time. In panels C and D, the apostrophes and half quotation marks are labeled in comparison with the MS/MS of (CH3)2DFSAWAamide (panel B). The b-ions are enhanced in panel B as compared with panel A. The ions in italic font (panels B and C) are possibly a result of intramolecular reaction.

the side chain of arginine and tryptophan residues. Furthermore, we also investigated the HPLC retention time behavior of the labeling products. The Relationship between the Labeling Products and Experimental Condition. Our experiments (Table S-1) showed that pH had very little effect on the modification products from pH 3.0 to 8.2. A peptide with two lysine residues, KHKNYLRFamide, was used to study the effect of insufficient formaldehyde or reducing reagent on modification. When an insufficient amount of formaldehyde was used, the peptide was incompletely modified, and mass increments of +28, +42, +56, +70, and +84 Da were detected. When an insufficient amount of NaBH3CN was used, mass increments of +76, +78, +80, and +82 Da were seen. The 2-Da mass decrease is due to the incomplete reduction of imine. Our study showed that the arginine and tryptophan residues can be labeled with a methylol group after formaldehyde incubation. The labeling of arginine and tryptophan is relatively slow when compared with the N-terminus and lysine side chain. Almost no labeling of arginine or tryptophan was detected using a 5-min reaction time followed by the addition of ammonia to quench excess formaldehyde. Longer reaction times and higher concentrations of formaldehyde increase the labeling of arginine and tryptophan. In contrast, reductive methylation of the N-terminus and lysine side chains was completed in 5 min, suggesting that this is a fast derivatization reaction. 7786


a (A), imine-modified tryptophan (B) and methyl-modified trytophan (C) observed in the current study (Figure 2). The structure of imine-modified tryptophan was previously reported by Metz et al.30 The two structures for the 130 ion are equivalent. The two structures for the 144 ion are equivalent.

The MS/MS Fragmentation Pattern of Peptides with Imine or Methylol Modified Arginine/Tryptophan. The methylol-modified arginine-containing peptides yielded complex fragmentation patterns. For example, in the MS/MS spectrum of (CH3)2K(CH3)2HK(CH3)2NYLR(CH3OH)Famide (Figure 1C), the loss of both water and formaldehyde from the precursor ion and the fragment ions can be seen. The relative intensity order of those ions is as follows: loss of formaldehyde ion > loss of water ion (imine) > intact methylol ion. This phenomenon was also seen in other peptides with methylol-modified arginine. In the previous study by Metz et al.,30 the presence of the neutral loss of water in the MS/MS spectra of peptides with methylol-modified arginine was also observed; however, the complete loss of the methylol ion was not reported. The order of relative intensity in the MS/MS spectrum is different for methylol-modified tryptophan ((CH3)2DFSAW(methylol)Aamide, Figure 2C). Here, the imine-modified ions are the strongest (y3′, AW′, and immonium ion W′). The loss of formaldehyde ions can be seen (y3, AW, and W) and weaker than the imine-modified ions. The prevalence of tryptophan (imine) during MS/MS is probably due to the stability provided by delocalization of the positive charge over the indole ring (Scheme 2). The imine-modified peptide (Figure 2D) has the same MS/ MS fragmentation pattern as the methylol-modified peptide. The spectra of the methylol and imine-modified peptides are more complicated than the peptides with only N-terminal dimethyl modification. There are several pairs of ions with 2-Da mass difference in the MS/MS spectra (Figure 2C and D). Several ions correspond to the addition of 2 Da to the ions with imine-modified tryptophan, such as 173.10 (W′ + 2), 272.11 (AW′ + 2), and 360.14

(y3′ + 2). In addition, several ions are detected with 2 Da less than the corresponding N-terminal fragment ions without tryptophan, such as 261.10 (a2 - 2), 289.10(b2 - 2), 348.13 (a3 - 2), 376.12 (b3 - 2), 429.13 (b4 - H2O - 2), and 447.13 (b4 - 2). The observation of these ions suggests an intramolecular reaction which converts the imine to the methyl group, and the two hydrogen atoms are from the N-terminus. The losses of H2 from the N-terminal fragment ions are strong, which indicates that the product ions are more stable than the regular a and b ions. The mechanism for this reaction is currently under investigation. The proposed structures of the 173 and 144 ions are shown in Scheme 2. Investigation of the Retention Time after Reductive Methylation. Our experiments show that the methylation of the N-terminus or lysine side chain only slightly increases the peptide’s retention time. For example, the retention times of FMRFamide, its dimethyl-modified form, and its acetylated form are 27.12, 27.71, and 38.40 min, respectively. The retention times of KHKNYLRFamide, its triple dimethylated (+84 Da) and tetraacetylated forms (acetylation also labels Y32) are 18.78, 19.28, and 29.64 min, respectively. Compared with acetylation, the changes in retention time caused by dimethylation are negligible. The methylol modification on arginine and tryptophan decreases the peptide’s retention time by less than 1 min (data not shown). The imine adduct will elute at the same time as the methylol-modified peptide if it is produced by the loss of water from a methylol-modified peptide in the gas phase. However, for an imine product formed at the N-terminus or lysine side chain, the retention time will increase, as compared to the unmodified peptide. For example, the retention times of KHKNYLRFamide with 84-, 82-, 80-, and 78-Da modifications are 18.69, 23.16, 26.63, and 29.86 min, respectively. On the basis of the mass shift, the retention time change, and the fragmentation patterns of the imine- and methylol-modified peptides, these two modifications can be easily differentiated from other peptides. These two side products can provide additional information about the peptide sequence, as demonstrated later. The Reductive Methylation Does Not Label Peptides with NTerminal Pyroglutamte Modification. We did not observe methylation at the N-terminus of either pyrTSFTPRLamide or pyrLYENK under the experimental conditions we used (Table S-1). This is because the amide at the N-terminus of pyroglutamte is a poor nucleophile. Characterization of the Neuropeptidome of C. borealis Pericardial Organ. Using a combination of N-terminal isotopic methylation and nanoLC-MS/MS de novo sequencing, 54 neuropeptides, including 24 novel ones, are sequenced from the crab C. borealis PO tissue extract (Table 1). When compared to a previous MS study of this neuronal tissue,33 our new strategy combining N-terminal isotopic dimethyl labeling and nanoscale HPLC MS/MS de novo sequencing enables a more complete elucidation of the neuropeptidome of this important neuroendocrine organ. Several unique advantages of isotope-based formaldehyde labeling for de novo sequencing of neuropeptides are noted and highlighted in the following sections. Specifically, the isotopic (32) Silberring, J.; Nyberg, F. J. Chromatogr. 1991, 562, 459-467. (33) Li, L.; Kelley, W. P.; Billimoria, C. P.; Christie, A. E.; Pulver, S. R.; Sweedler, J. V.; Marder, E. J. Neurochem. 2003, 87, 642-656.

Table 1. Neuropeptides Identified in a Pericardial Organ Extract from Cancer borealis precursor ions

monoisotopic mass

m/zc

qd

meas

theor

926.43 469.72 483.23 511.77 454.74 573.77 574.28 591.26 483.74 586.79 481.74 636.25

1 2 2 2 2 2 2 2 2 2 2 2

926.43 938.43 965.44 1022.54 908.46 1146.52 1147.55 1181.52 966.48 1172.57 962.46 1271.49

926.52 938.53 965.54 1022.56 908.52 1146.61 1147.65 1181.62 966.53 1172.63 962.53 1271.69

QRAYSFGLa PRDYAFGLa YSFGLa DRPYSFGLa AGGAYSFGLa YAFGLa GGAYSFGLa GSGQYAFGLa SSGQYAFGLa GQYAFGLa pyrRTYSFGLa pyrRAYSFGLa ERPYSFGLa APQPYAFGLa AGPYSFGLa EPYAFGLa GDPYAFGLa DPYAFGLa PDMYGFGLa PSMYAFGLa PDMYAFGLa PADLYEFGLa PATDLYAFGLa

Allatostatins A-type 30.30 940.42 35.09 937.39 38.96 585.28 39.33 953.38 45.58 841.33 45.70 569.24 46.32 770.29 46.53 898.33 46.55 928.36 47.33 754.29 48.84 953.37 49.07 923.30 49.23 967.66 50.42 962.36 51.30 810.35 52.77 795.56 53.76 838.30 55.74 781.53 62.97 898.28 63.04 884.32 65.19 912.31 68.22 1023.39 70.55 1066.44

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

940.42 940.50 937.39 937.49 585.28 585.30 953.38 953.48 841.33 841.42 569.24 569.31 770.29 770.38 898.33 898.44 928.36 928.45 754.29 754.39 953.37 953.48 923.30 923.47 967.66 967.50 962.36 962.51 810.35 810.41 795.56 795.40 838.30 838.41 781.53 781.39 898.28 898.41 884.32 884.43 912.31 912.43 1023.39 1023.52 1066.44 1066.56

GNWNKFQGSWa NWNKFQGSWa NNWSKFQGSWa NNNWSKFQGSWa TSWGKFQGSWa STNWSSLRSAWa

Allotostatins B-type 42.59 611.73 43.61 583.20 45.76 626.72 46.02 683.74 48.14 591.72 48.66 647.26

2 2 2 2 2 2

1222.46 1165.40 1252.44 1366.57 1182.43 1293.51

1222.61 1165.59 1252.62 1366.67 1182.57 1293.63

Allatostatin Combos EPYAFGLGKRPATDL 46.70 817.88 DPYAFGLGKRPADL 50.75 760.34 GSGQYAFGLGKKAGGAYSFGLa 57.95 1017.94 DPYAFGLGKRPDMYGFGLa 73.45 1001.87 EPYAFGLGKRPATDLYAFGLa 75.74 1092.96 DPYAFGLGKRPDMYAFGLa 76.01 1008.84 DPYAFGLGKRPADLYEFGLa 76.68 1064.40

2 2 2 2 2 2 2

1634.75 1519.66 2034.88 2002.73 2184.91 2016.67 2127.79

1634.85 1519.79 2035.04 2002.98 2185.14 2017.00 2128.09

Other Neuropeptides 14.91 422.73 16.77 649.36 17.13 810.31 19.30 976.34 37.81 1030.35 52.52 956.25

2 1 1 1 1 1

844.46 844.48 649.36 649.37 810.31 832.41 976.34 976.46 1030.35 1030.51 956.25 956.37

peptide sequencea SKNYLRFa NRSFLRFa GGRNFLRFa GNRNFLRFa GRNFLRFa GYSKNYLRFa APQRNFLRFa SENRNFLRFa DRNFLRFa AYNRSFLRFa pyrRNFLRFa PELDHVFLRFa

HL/IGSL/IYRa RYLPT FYANRYa SGFYANRYa PAFYSQRYa PFCNAFTGCa

tRb FLRFamides 18.80 20.76 21.32 21.93 22.30 24.08 27.18 27.22 31.19 31.86 43.72 67.15

a C-terminal amidation is indicated by the character “a” at the C terminus of a given peptide sequence. “pyr” stands for the N-terminal pyroglutamate residue. The peptide sequences in bold are novel peptides reported in this study. b Chromatographic retention time of the peptide. c Mass-to-charge ratio of the precursor ion. d Charge state of the precursor ion.


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Figure 3. The MS/MS sequencing of (A) PADLYEFGLamide (1023.39+), (B) (CH3)PADLYEFGLamide (1037.28+), and (C) (CHD2)PADLYEFGLamide (1039.63+). The methyl or D2-methyl group modification at the N-terminus is labeled by an asterisk. After modification, the a- and b-ion series are enhanced. The internal fragment ions, DL, LY/EF, and YE are suppressed.

labeling enables resolving sequence ambiguities both at the N-terminus and in the middle of the sequence. This strategy allows differentiation of isobaric amino acid residues K versus Q and overlapping masses composed of a single amino acid residue versus two amino acids. The reductive methylation method also enables the characterization of neuropeptides with N-terminal blockage, such as pyroglutamate modification due to its inability to react at the N-terminus. Overall, the isotopic formaldehyde labeling provides more confident sequence assignment than using dimethyl modification alone. Determining the N-Terminal Sequence via Isotopic Formaldehyde Labeling. As demonstrated by Hsu et al., the substantial signal enhancement of the a1 ions can be used to resolve the ambiguities at the N-terminus.23,25 However, several complications exist for interpretation of these low-mass fragment ions using de novo sequencing. The dimethyl labeling increases the masses of a1 ions by 28 Da, which could coincide with the masses of immonium ions or their related ions from other amino acid residues in the same peptide. For example, the dimethyl labeled a1 ion for an N-terminal alanine shares the identical mass of 72 Da with that of the immonium ion for valine. Similarly, N-terminal serine would produce a dimethylated a1 ion of 88 Da, which overlaps with the immonium ion of glutamic acid. To address these sequencing ambiguities, we incorporated a pair of isotopically labeled reagents, formaldehyde-h2 and formaldehyde-d2, to produce peaks that differ by 4 mass units for each derivatized isotopic pair. This strategy enhances and confirms the detection of a1 ion and provides critical 7788 Analytical Chemistry, Vol. 77, No. 23, December 1, 2005

Figure 4. The MS/MS fragmentation spectra of (A) GGRNFLRFamide (483.232+), (B) (CH3)2 GGRNFLRFamide (497.302+), (C) (CHD2)2 GGRNFLRFamide (499.242+), and (D) (CH3)2 NRNFLRFamide (497.162+). The neutral losses of dimethylamine ions are highlighted by black squares in panels B, C, and D. 474.72+ in panels B, C, and D corresponds to the loss of dimethylamine (or di-D2methylamine) from the precursor ion. The 767.3+ ion corresponds to the loss of dimethyl (or di-D2-methyl) amine from the b7 - NH3 ion (panels B and C) or b6 - NH3 ion (panel D). The 739.20 (panel D) is a6 - NH3 - NH(CH3)2. The y(n-1) - NH3 ions are also in high abundance in these spectra and are all singly charged.

information for the unambiguous identification of the N-terminal amino acid residue. The isotopically labeled peptides also produce the same MS/MS fragmentation patterns, thus facilitating the identification of the N-terminal fragment ions via comparison of the spectra from labeled pairs. Figure 3 shows an example of the MS/MS sequencing of a novel A-type allatostatin (AST) peptide, PADLYEFGLamide, and its N-terminal methyl and D2-methyl-labeled products. Since it is relatively uncommon to have proline occupying the N-terminus of a neuropeptide and a previously identified AST peptide in C. borealis has the sequence APTDMYSFGLamide,34 one might assign the first two amino acid residues as AP rather than PA. However, as evident in Figure 3B and C, the detection of monomethylated a1 ions in both N-terminal methyl and D2-methyllabeled peptides supports the assignment of proline as the first N-terminal amino acid. Using the same strategy, several other novel ASTs containing proline at the N-terminus are de novo sequenced, including PSMYAFGLamide, PDMYGFGLamide, and PATDLYAFGLamide (Table 1). Isotope-Based Labeling Enables Differentiation of Overlapping Masses with One Amino Acid from the Combination of Two Amino Acids. In addition to sequence ambiguity at the N-terminus of a (34) Huybrechts, J.; Nusbaum, M. P.; Bosch, L. V.; Baggerman, G.; De Loof, A.; Schoofs, L. Biochem. Biophys. Res. Commun. 2003, 308, 535-544.

Figure 5. The MS/MS sequencing of (A) GGAYSFGLamide (770.29+), (B) (CH3)2 GGAYSFGLamide (798.45+), and (C) (CHD2)2 GGAYSFGLamide (802.51+). The dimethyl or di-D2-methyl modification at the N-terminus is labeled by an asterisk. In panel A, some fragment ions which cannot be identified are labeled with m/z only. After modification, the y3 ion, immonium ions (Y, F, L), and the internal fragment ions (e.g., SF - 28, SF/AY, YS, SFG/GAY - 28, SFG/GAY) are suppressed. The a-, b-ion series and their neutral loss ions are enhanced.

peptide, another challenging aspect of peptide de novo sequencing is the fact that the combined residue mass of two amino acids (e.g., A1 and A2) may equal the residue mass of a single amino acid (A3). The A1A2 can be misassigned to A3 if the fragment ions between these two amino acid residues are weak. Similarly, the A3 can also be assigned incorrectly to A1A2 if one ion is misassigned to the fragment ion between A1 and A2. For example, the following amino acid residue(s) have very close molecular masses: K (128.09496)/Q (128.05858 Da)/G + A (128.05858 Da), N (114.04293 Da)/G + G (114.08292 Da), R (156.10111 Da)/G + V (156.08988 Da), W (186.07931 Da)/G + E (186.06405 Da)/A + D (186.06405 Da)/S + V (186.08479 Da). As noted, many of these two amino acid residue combinations involve glycine. It was previously reported that glycine in the sequence generally produces less abundant fragment ions upon cleavage and could be difficult to detect.24 The N-terminal isotopic dimethyl labeling strategy presented here aids in solving some of these ambiguities involving glycine, both at the N-terminus and in the middle of the sequence. As an example, Figure 4 shows the MS/MS sequencing of GGRNFLRFamide, a peptide that shares an identical mass with that of NRNFLRFamide. NRNFLRFamide was previously sequenced in the brain and thoracic ganglion of the same species, C. borealis.34 The MS/MS of the native peptide (Figure 4A) shows no ions corresponding to the y7 ion of GGRNFLRFamide. The y7

Figure 6. The MS/MS sequencing of (A) TSWGKFQGSWamide (591.722+), (B) (CH3)2TSWGK(CH3)2FQGSWamide (619.942+), and (C) (CHD2)2TSWGK(CHD2)2FQGSWamide (623.922+). The asterisks indicate the modification of the N-terminus or lysine residue by a dimethyl or di-D2-methyl group. Due to space limitations, some of the peaks are labeled with mass-to-charge ratio only. In panel A: 357.16, b3 - H2O; 588.25, GKFQGS - NH3; 773.36, WGKFQGS H2O; 949.43, y8 - 45. In panel B: 791.39, WGK*FQGS - 28; 1064.51, y9 - 45; 1074.57, y9 - NH3 - H2O; 1092.51, y9 - NH3; 1193.58, (M + H)+ - 45. In panel C: 806.44, WGK*FQGS - NH3; 1068.55, y9 - 45; 1096.59, y9 - NH3; 1201.61, (M + H)+ - 45. The -45 Da neutral loss is possibly related to the glutamine residue because the immonium ion -45 Da (-CO-NH3) has been reported for glutamine.24,50 The intensities of the a1 and y9 ions increased after modification (panels B and C). The detection of the internal fragment ions confirms the assignment of K5 and Q.7

and y7 - NH3 ions are seen, however, after reductive dimethylation, with the y7 - NH3 being significantly enhanced (Figure 4B and C). Moreover, the isotopically labeled a1 ions, m/z 58 (Figure 4B) and 62 (Figure 4C), in the MS/MS of the modified peptides reveal the presence of glycine at the N-terminus. As a control experiment, we performed a reductive methylation experiment for a synthetic peptide standard, NRNFLRFamide. The MS/ MS of (CH3)2NRNFLRFamide (Figure 4D) displayed the presence of an a1 ion at m/z 115. The absence of 58+, 908.5+ (y7 ion of (CH3)2GGRNFLRFamide)or891.5+ (y7 -NH3 ionof(CH3)2GGRNFLRFamide) further supports the assignment of GGRNFLRFamide. Another example is the differentiation of Q/K vs AG/GA. Figure 5 shows the MS/MS sequencing of GGAYSFGLamide. As seen, the native peptide displays a highly complex MS/MS spectrum that is difficult to interpret, even with the assistance of de novo sequencing software (Figure 5A). However, the formaldehyde labeling results in much cleaner fragmentation spectra (Figure 5B and C). The correlation between Figure 5A and B is nearly impossible due to dramatically different fragmentation Analytical Chemistry, Vol. 77, No. 23, December 1, 2005

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Figure 7. The MS/MS sequencing of (A) pyrRAYSFGLamide (923.30+) and (B) pyrR(methylol)AYSFGLamide (953.28+). “pyr” stands for pyroglutamate residue. The a-, b-, and y-ion series in panel B are labeled according to the MS/MS of pyrRAYSFGLamide. In panel B, the apostrophe indicates the modification of imine to arginine residue. The ions with imine modification are highlighted with black dots. In panel B, some of the ions are labeled with m/z only, due to space limitation: 485.22, b4 - NH3; 554.25, b5 - H2O - NH3; 673.32, a6 - H2O - NH3; 701.30, b6 - H2O - NH3; 758.34, b7 - H2O NH3. In panel B, the 923.49+ is loss of formaldehyde from the precursor ion. The 935.46+ is loss of water from the precursor ion.

patterns. The peptide sequencing can be performed, however, by comparing the MS/MS spectra of the N-terminal dimethyl and di-D2-methyl-labeled peptides, which display almost identical fragmentation patterns. The b- and a-ion series of isotopically labeled peptides are 4 Da different between each isotopic pair and can be easily assigned due to similar intensities in the light- and heavy-isotope-labeled spectra. For example, the detection of dimethylated a1 ions at m/z 58 and 62 reveals the presence of glycine at the N-terminus. Furthermore, the b2-ion in the modified spectra which is missing in that of the native peptide enables the identification of a second glycine at the N-terminus. Therefore, the peptide sequence is assigned to be GGAYSFGLamide instead of GQYSFGLamide. Interestingly, a different AST peptide was also sequenced in our study using the formaldehyde labeling technique, and the amino acid sequence was determined to be GQYAFGLamide instead of GGAYAFGLamide, even though a clear homology between the latter sequence and GGAYSFGLamide is noted. Clearly, the N-terminal isotopic dimethylation provides excellent chemical specificity to allow resolving some of these sequencing ambiguities. Differentiation of Isobaric Amino Acids Q/K. The reductive methylation can be used to differentiate Q and K because the side chain of lysine can be modified to produce mass shift, but Q cannot. Figure 6 shows de novo sequencing of a B-type allatostatin, TSWGKFQGSWamide. The N-terminal T is determined via the detection of paired intense a1 ions (102/106 Da) and y9 (1109.5/ 1113.6 Da) ions upon isotopic dimethyl labeling. The presence of K5 and Q7 is determined by comparing the corresponding fragmentation spectra of the native peptide (Figure 6A) and the modified peptides (Figure 6B, C). Several other neuropeptides with ambiguities of Q/K in the sequences are identified in a similar 7790 Analytical Chemistry, Vol. 77, No. 23, December 1, 2005

Figure 8. The MS/MS sequencing of (A) PFCNAFTGCamide (CCAP, 956.25+), (B) (CH3)PFCNAFTGCamide (970.23+), and (C) (CHD2)PFCNAFTGCamide (972.20+). The methyl or D2-methyl modification at the N-termini is labeled by an asterisk in the sequence. The a-, b-, and y-ions are labeled as the peptide without disulfide bond. The 665.26 (B)/667.24 (C) is equal to b6 - 29. The 823.38 (B) and 825.29 (C) is equal to b8 - 29. The identity of the -29 Da is unclear yet. The intensities of the a1 ions increase after N-terminal dimethyl modification (panels B and C). The y3 ion, immonium ions (P, F), and the internal fragments (NA, FT, NAF) are suppressed after modification.

manner, such as GYSKNYLRFamide, APQRNFLRFamide, GNWNKFQGSWamide, and NNNWSKFQGSWa. As discussed above, the tryptophan residue has a molecular mass isobaric to the combination of two amino acid residues, such as GE, AD, SV, or GK/Qamide at the C-terminus. The 1092.51+ peak in Figure 6B is 57 Da higher than b9 (1035.49+). If based solely on N-terminal dimethyl labeling, the sequence could have been assigned as TSWGKFQGSGQamide. Via the use of isotopic labeling, however, it is evident that the expected b10 of formaldehyde-d2-labeled peptide at 1100.6+ for the sequence of TSWGKFQGSGQamide is missing in Figure 6C. On the other hand, the 4-Da difference between the 1096.59+ peak in Figure 6C and 1092.51+ peak in Figure 6B indicates that they are y9 - NH3 in heavy and light isotopic forms, respectively. Therefore, the isotopic labeling aids in solving the sequence ambiguity at the C-terminus, yielding the amino acid identity of W instead of GQamide. Peptides with an additional imine modification at W residues are also seen. Two peptides with m/z at 625.932+ and one peptide with the m/z at 631.942+ are found to elute earlier than the peptide with two dimethyl modifications (619.942+, Figure 6B). They are assigned as the (CH3)2TSWGK(CH3)2FQGSWamide with single and double imine modifications, respectively. These three peptides provide evidence for the presence of two tryptophan residues in

Figure 10. The MS/MS fragmentation spectra of (A) FMRFamide (599.37+), (B) (CHD2)2FMRFamide (316.182+), and (C) (CHD2)2FMRFamide (631.37+). The a1 and y3 ions in panel B are enhanced as compared with panel A. The a1 ion is enhanced from panels A to C. The y3 ion in panel C is weaker than in panel B. The 582.30+ ion in panel C is a neutral loss of di-D2-methylamine from the precursor ion, which is highlighted by a black square.

Figure 9. The MS/MS de novo sequencing of (A) SQPALAEVAL (998.57+), (B) (CH3)2SQPALAEVAL (1026.60+), and (C) (CHD2)2SQPALAEVAL (1030.72+). The dimethyl group modification at the N-terminus is labeled by an asterisk in the sequence. The internal fragments (PA, PAL, PALA, PALAE, PALAEV, PALAEVA, QPAL, LAE) and neutral loss ions (such as b4 - H2O, b5 - H2O, b6 - H2O, b7 - H2O) are suppressed after N-terminal dimethyl modification. The intensities of the a1 ion and the b ion series are increased.

this peptide. These ions are produced from the methylol-modified peptides due to their shorter retention times, as compared with that of the dimethyl-modified peptide. The methylol adducted peptides are not detected due to their low abundance. Characterization of N-Terminal Pyroglutamate (pyr) Modification. Amino pyroglutamate modification is one of the most common posttranslational modifications of neuropeptides. As shown in the peptide standard labeling experiments, pyroglutamate cannot be modified by reductive methylation. A peptide is known to be N-terminally blocked (i.e., pyr modification) under the following two conditions: (1) identical masses are observed in the native and modified samples with similar retention times; (2) modification (such as dimethyl, methylol, or imine modification) only occurs in the middle of the peptide sequence. Using this guideline, we sequenced three neuropeptides with N-terminal pyroglutamate: pyrRNFLRFamide, pyrRTYSFGLamide, and pyrRAYSFGLamide. Figure 7 shows the MS/MS spectra of pyrRAYSFGLa and pyrR(methylol)AYSFGLa. The modified peptide displays a peak at m/z 953.52+ as well as an intense nonmodified counterpart at m/z 923.49+. The identical a-/b-ion series and lowabundance imine-modified products (+12 Da) in the MS/MS of 953.52+ confirm the methylol modification at arginine. Mechanistic Study of the Fragmentation of Neuropeptides with N-Terminal Dimethylation Modification. Previous work by Hsu et al. showed enhanced detection of the a1 and y(n-1) ions

for tryptic peptides after reductive methylation.23,25 Unlike tryptic peptides containing either arginine or lysine C-terminus and being multiply charged, neuropeptides usually do not have basic residue at their C-termini, and many neuropeptides are singly charged. We discovered several unique fragmentation patterns for neuropeptides with reductive methylation. For example, we observed that the MS/MS spectra of the singly charged peptides were simplified after N-terminal dimethylation. We also noted that the y(n-1) ions were enhanced in the MS/MS fragmentation spectra of multiply charged peptides, but they were missing or weak in the MS/MS of singly charged peptides. Furthermore, we reported the neutral loss of dimethylamine and structures of the immonium ion and related ions of NR,N-tetramethylated lysine and Ndimethylated lysine. The mechanisms for the above fragmentations were proposed. Simplification of the MS/MS Fragmentation Pattern of Singly Charged Peptides after N-Terminal Dimethylation. As shown in Figures 3 and 5, formaldehyde labeling increases the signals of the a-/b-ion series and reduces the complexity of the MS/MS fragmentation patterns of two singly charged peptides. The same simplification effect is also observed in other singly charged peptides. Figure 8 shows the MS/MS sequencing of the crustacean cardioactive peptide (CCAP). This peptide consists of an internal disulfide bond between Cys2 and Cys7. The MS/MS of the native peptide displayed a complicated fragmentation pattern that could not be deciphered (Figure 8A). The isotopic formaldehyde-labeled peptides, however, yielded much cleaner MS/MS spectra containing primarily a- and b-ion series. The N-terminal fragment ions associated with the disulfide bond (such as b4 H2S and b4 + S) are also readily detected. To the best of our knowledge, this is the first demonstration of MS/MS sequencing of the CCAP without reduction and alkylation of the disulfide bond. Figure 9 shows another example of a simplified MS/MS profile upon reductive methylation. Extensive proline-directed internal fragmentation35 is observed in the MS/MS spectrum of SQAnalytical Chemistry, Vol. 77, No. 23, December 1, 2005

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Scheme 3. Two Pathways for the Formation of the a1 and y(n-1) Ions by Charge Direct Fragmentationa

a (A) The formation of the a and y 1 (n-1) ions of a peptide with N-terminal dimethylation by charge direct fragmentation. The resulting C-terminal fragment has one positive charge less than the precursor ion. If there is a charge (or more than one) on the side chain of the C-terminal fragment, the C-terminal fragment will be detected as a y(n-1) ion. Otherwise, it will be neutral and will not be detected. (B) The formation of the y(n-1) ion through rearrangement. The hydrogen at the R-carbon is transferred to the C-terminal fragment. As a result, an N-terminal neutral fragment (ketene) and the y(n-1) ion are formed. The C-terminal fragment retains the same number of charges as the precursor ions. For peptide precursors with the number of arginine residues equal to or more than the number of charges (e.g., NRNFLRFamide, 2+; FMRFamide, 1+), the charge will be located at the arginine side chain instead of the N-terminus before the fragmentation (not drawn in the scheme).48

PALAEVAL, which complicates sequence interpretation. After formaldehyde labeling, b- and a-type ions are significantly enhanced, and internal fragment ions are suppressed, thus yielding a simplified fragmentation pattern. The simplification of MS/MS spectra of singly charged peptides after dimethylation can be explained by the pathway in competition model.36 According to this model, the b- and y-ions are produced from the same transient state in competition for one proton. The relative abundance of b-and y-ions is determined by the relative proton affinities (PA) of the C-terminal and N-terminal fragments. After reductive methylation, the N-terminal primary amine becomes a tertiary amine. Since the PA of a tertiary amine is higher than a primary amine,37 the PA of the modified N-terminus is higher than the unmodified form. The resulting fragmentation spectra of modified peptides show a high relative abundance of b-ions as compared with the corresponding y-ions. The internal fragmentation requires a minimum of one b-type and one y-type bond cleavage.38,39 Since the y-type cleavage is suppressed, less internal fragmentation is produced, thus yielding a simplified MS/MS fragmentation pattern. (35) Breci, L. A.; Tabb, D. L.; Yates, J. R., 3rd; Wysocki, V. H. Anal. Chem. 2003, 75, 1963-1971. (36) Paizs, B.; Suhai, S. Mass Spectrom. Rev. 2005, 24, 508-548. (37) Raabe, G.; Wang, Y.; Fleischhauer, J. Z. Naturforsch. 2000, 55a, 687-694. (38) Ballard, K. D.; Gaskell, S. Int. J. Mass Spectrom., Ion Processes 1991, 111, 173-189. (39) Wysocki, V. H.; Resing, K. A.; Zhang, Q.; Cheng, G. Methods 2005, 35, 211-222.

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Scheme 4a

a (A) The proposed fragmentation pathway for the neutral loss of dimethylamine from the N-terminus of the N-terminal dimethylated peptide. (B) The proposed fragmentation pathway for the neutral loss of dimethylamine from the N-dimethyl lysine side chain. The loss of dimethylamine will cause a 45-Da mass decrease of the precursor ion mass. The loss of di-D2-methylation will cause a 49-Da mass decrease. The curved arrows represent double electron transfer.

The increased N-terminal fragment ions of tryptic peptides after dimethylation have been reported by Hsu et al. 23,25 When compared to their observation and our current results, the fragmentation pattern changes for multiply charged peptides after

Table 2. Immonium Ion and Related Ions of Lysine Detected in the MS/MS of Neurotensin and Its Modified Forms sequence

m/z

Ea (eV)

immonium ion and related ionsb

y1 ionsb

pyrLYENK pyrLYENK(CH3)2 pyrLYENK(CH3)2 pyrLYENK(CHD2)2 pyrLYENK(CHD2)

777.29+ 805.33+ 805.39+ 809.35+ 793.32+

48.0 41.3 52.7 54.7 49.4

84 (67), 101 (11), 129 (269), 130 (52) 84 (83), 129 (66), 130 (31) 84 (87), 129 (60), 130 (100) 84 (155), 130 (58), 133 (29) 84 (52), 100 (103), 117 (15), 130 (64)

147 (89, y1) 175 (270, y1) 175 (277, y1) 179 (125, y1) 145 (229, y1 - H2O), 163 (177, y1)

a Collision energy. b The number inside the parenthesis is the ion counts. Fragment ions with ion count less than 10 counts were not included in the table. The masses in italics are the immonium ions. The y1 and y1 - H2O ions are also listed in the table.

dimethylation are less dramatic than those for singly charged peptides. This is likely due to the fact that the PA of tertiary amine is still lower than that of basic residues.38,40,41 Thus, the increase of the relative abundance of the N-terminal fragment ions is less significant if there is a basic residue in the C-terminal fragment. Formation of the a1 and y(n-1) Ions of N-Terminally Dimethylated Peptides. As shown previously, the intensities of the a1 and y(n-1) ions increased significantly after dimethylation.25 In our study, we find that the a1 ions are generally enhanced. A signal enhancement of the y(n-1) ion is observed in the MS/MS spectra of doubly charged peptides after modification but is absent or weak in the MS/MS spectra of singly charged peptides. For example, the doubly charged (CHD2)2FMRFamide displays strong y3 ion (Figure 10B), but this product ion is weak in the singly charged precursor ion (Figure 10C). The a1 ions cannot be formed from b1 ions because b1-ions are absent in most MS/MS spectra. The formation of the b-ions requires nucleophilic attack of the carbonyl carbon by the adjacent N-terminal carbonyl oxygen atom.36,42,43 Due to the lack of adjacent N-terminal carbonyl group of native peptide and peptide with dimethylation, the b1 ion cannot be observed. The formation of the a1 and y(n-1) ions of peptides with a free N-terminus was studied previously. The a1 ion of peptides with a free N-terminus can be produced from the a1-y(n-1) fragmentation pathway,44 b2,45 or a246 ions. The y(n-1) ions of peptides with a free N-terminus is formed via either the a1-y(n-1)44 or the aziridinone pathway.47 In both pathways, two hydrogen atoms are transferred from the N-terminus of the peptides to the N-terminus of the C-terminal fragments to form the y(n-1) ions. The resulting y(n-1) ions retain the same charge as the precursor ions. Due to the lack of hydrogen at the N-terminus of peptides with dimethylation, this hydrogen transferring reaction is unlikely to happen. On the basis of our experimental observation, we propose the fragmentation pathway for the formation of the a1 and y(n-1) ions as shown in pathway A (Scheme 3). It involves a concerted cleavage at the amide and CR-Camide bonds in a similar manner (40) Bouchoux, G.; Buisson, D.; Colasa, C.; Sabliera, M. Eur. J. Mass Spectrom. 2004, 10, 977-992. (41) Rak, J.; Skurski, P.; Simons, J.; Gutowski, M. J. Am. Chem. Soc. 2001, 123, 11695-11707. (42) Schlosser, A.; Lehmann, W. D. J. Mass Spectrom. 2000, 35, 1382-1390. (43) Paizs, B.; Suhai, S. J. Am. Soc. Mass Spectrom. 2004, 15, 103-113. (44) Paizs, B.; Suhai, S. Rapid Commun. Mass Spectrom. 2001, 15, 651-663. (45) Harrison, A. G.; Csizmadia, I. G.; Tang, T. H. J. Am. Soc. Mass Spectrom. 2000, 11, 427-436. (46) Harrison, A. G.; Young, A. B.; Schnoelzer, M.; Paizs, B. Rapid Commun. Mass Spectrom. 2004, 18, 1635-1640. (47) Harrison, A. G.; Csizmadia, I. G.; Tang, T. H.; Tu, Y. P. J. Mass Spectrom. 2000, 35, 683-688.

as the a1-y(n-1) pathway. The cleavage produces the a1 ion, carbon monoxide, and the C-terminal fragment. For singly charged peptides in which the charge is retained at the N-terminus, the C-terminal fragment will be neutral, and thus, no y(n-1) ion can be formed from this pathway. For peptides with another charge in the sequence (e.g., the doubly or multiply charged peptide precursor ions), the C-terminal fragments will be charged due to the remaining charge at the basic residue. Therefore, the y(n-1) ion can be produced, with one charge state lower than the charge state of the precursor ions. For example, the y7 and y72+ ions can be seen in the MS/MS of KHKNYLRFamide (Figure 1A). After dimethylation modification, only strong y7 ion was observed (Figure 1B, D). The a1 ion of the dimethylated peptide is stabilized by the electron-donating methyl group. The energy of the transient state is also lowered after dimethylation due to the stabilization of the protonated N-terminal fragment in the transient state complex. Consequently, the activation energy required for the fragmentation is lower after dimethylation, and thus, signals of the a1 and y(n-1) ions are significantly enhanced. The y(n-1) or y(n-1) - NH3 ion can be seen in some singly charged peptides, such as (CH3)2FMRFamide and (CH3)2EGVYVHPV (data not shown). The formation of the y(n-1) ions of these peptides is from pathway B shown in Scheme 3. Pathway B involves transferring Hb to the N-terminus of the newly formed C-terminal fragment to form the y(n-1) ion. Pathway B requires higher activation energy than pathway A because in the MS/MS of singly charged FMRFamide, the y(n-1) and y(n-1) - NH3 ions (from pathway B) are much weaker than the a1 ions (from pathway A and B). Neutral Loss of Dimethylamine. The neutral losses of dimethylamine from the precursor ion and the b-ions can be seen in Figures 1, 4, and 10 (labeled as black squares in the spectra). For dimethyl-labeled peptides, -45 Da was seen, whereas for diD2-methyl-labeled peptides, -49 Da was seen for corresponding ions. The mechanism for this neutral loss from the N-terminus is shown in Scheme 4A. The neutral loss of dimethylamine from the side chain of N-dimethyl lysine is also observed (pyrLYENK(CH3)2, Figure S-1). The mechanism for this neutral loss is shown in Scheme 4B. The Immonium Ion Related Ions of NR,N-Tetramethylated Lysine and N-Dimethylated Lysine. N-terminal lysine residue plays a unique role in the interpretation of MS/MS fragmentation spectra of peptides modified with N-terminal reductive methylation because it is the only residue that can also be labeled at the side chain. Therefore, the lysine at the N-terminus and in the middle of the sequence or C-terminus will be labeled differently and Analytical Chemistry, Vol. 77, No. 23, December 1, 2005

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Scheme 5. Proposed Structures of the Immonium Ion and Related Ions of Lysine, Nr,NE-Tetramethyl Lysine, and Nr,NE-Tetra-D2-methyl Lysinea

Scheme 6. Proposed Mechanism for the Formation of Several Immonium Ion Related Ions of Nr,NE-Tetramethyl Lysine and Nr,NE-Tetra-D2-methyl Lysinea

a The curved arrows represent double electron transfer. The 98/100 ions cannot be produced from 114/118/119 ion because only one mass was observed in the D form (100 Da).

a The structures of the 101 and 84 ions of lysine were proposed by Fenaille et al.51 The rest of the ions are proposed in this study on the basis of MS/MS experiments of these modified peptides. The 86 ion of lysine is not detected in the MS/MS spectrum. The nitrogen atoms in the ions 112/140/144 are from the side chain. The origin of the nitrogen in 84/112/116, 114, 118/119, 98 and 100 cannot be determined by the current study because elimination of the side chain amine or N-terminal amine produces the same mass.

possibly produce different immonium ion and related ions. Clearly, the characterization of these ions is useful to understand the lowmass region of the MS/MS spectra and will be helpful for de novo sequencing. Several immonium ion and related ions of nonmodified lysine were previously documented, including 84, 101, 112, and 129 Da.24,49,50 Recently, two studies on the side chain alkylated lysines proposed different structures and fragmentation mechanisms.51,52 7794


Their difference was whether the side chain amine or the N-terminal amine should be eliminated. We did MS/MS experiments on pyrLYENK and its modified forms to determine the immonium ion and related ions of side chain dimethylated lysine. The result is summarized in Table 2. The immonium ions are observed for native and side chain modified lysines. The 84 and 130 ions are observed for all four peptides. The 130 ion is produced from elimination of the side chain amine49 instead of the N-terminal amine.52 The formation of the 130 ion is favored at higher fragmentation energy (Table 2, pyrLYENK(CH3)2 at 41.3 vs 52.7 eV). Two different structures have been reported for the 84 ion.51,52 One is shown in Scheme 5 with the nitrogen originating from the side chain.52 The other one has similar structure except that the double bond is located between the nitrogen and the carbon near to it.51 In the second structure, the nitrogen is from the N-terminus. For N-mono-D2-methyl lysine, the 100 Da (a six member ring with one methyl group connecting to the nitrogen atom) is observed. However, the six-member ring structure with a dimethyl group connecting to the nitrogen atom (112/116 Da for H/D form) is not detected for N-dimethyl lysine. Fenaille previously postulated that this observation could be due to the high PA of the (48) Wysocki, V. H.; Tsaprailis, G.; Smith, L. L.; Breci, L. A. J. Mass Spectrom. 2000, 35, 1399-1406. (49) Yalcin, T.; Harrison, A. G. J. Mass Spectrom. 1996, 31, 1237-1243. (50) Dookeran, N. D.; Yalcin, T.; Harrison, A. G. J. Mass Spectrom. 1996, 31, 500-508. (51) Zhang, K.; Yau, P. M.; Chandrasekhar, B.; New, R.; Kondrat, R.; Imai, B. S.; Bradbury, M. E. Proteomics 2004, 4, 1-10. (52) Fenaille, F.; Tabet, J. C.; Guy, P. A. Rapid Commun. Mass Spectrom. 2004, 18, 67-76.

dimethylamine group, which leads to fragmentation that favors the formation of protonated dialkylate amines.52 However, the mechanism for the formation of dialkylated amine ions and the subsequent formation of the 84 ions was not reported.52 A further study using different isotopic labeled nitrogen at the N-terminus and the side chain of lysine will be able to test their assumptions. The structures of the immonium ion and its related ions for lysine with both side chain and N-terminal dimethylation modification have not been previously studied. As shown in Figure 1, the masses of 84, 98, 112, 114, 140, and 157 are seen in the MS/MS spectrum of (CH3)2K(CH3)2HK(CH3)2NYLRFamide. The fragment ions, 84, 88, 100, 116, 118, 119, 144, and 165, are seen in the MS/ MS spectrum of (CHD2)2K(CHD2)2HK(CHD2)2NYLRFamide. As mentioned above, the 84 ions are from the side chain modified K3. The rest of the ions are from the NR, N-tetramethylated (or tetra-D2-methylated) K1. The proposed structures of those ions are shown in Scheme 5. The proposed mechanisms for the formation of the 112/116, 114/(118, 119), and 98/100 ions are shown in Scheme 6. The 112 (H form) and 116 (D form) ions result from neutral loss of dimethylamine (CH3)2NH (45 Da) from the a1 ion. During the formation of 114/118(119) ion, one proton (or D for D form) is transferred from the leaving amine group to the six-member ring. For D2-methylated lysine, the ratio of the D/H atoms in the leaving amine group is 2:1, so the 119 ion is stronger than the 118 ion. The formation of 98/100 ion is due to the loss of trimethylamine from the a1 ion. The identity of the 88 Da ion in Figure 1D is still unclear. The 4-Da mass difference from the 84 ion may correspond to one dimethylated (or di-D2-methylated) amine group. Further study is required to determine its structure and formation mechanism. After N-terminal dimethyl modification, the immonium ion related ions of other amino acid residues can also be observed at high abundance, such as the a1 - HSCH3 ion from NR-dimethylated methionine (Figure S-2). Even though the immonium ion related ions of the first amino acid residue are relatively weak compared with the a1 ions of these N-terminal dimethylated peptides, an improved understanding of the identities of these lowmass diagnostic ions will provide additional confirmation of the assignment of the a1 ion and reduce the likelihood of incorrect sequence assignment. CONCLUSIONS In this study, we have significantly expanded the scope of previous work on peptide de novo sequencing by reductive

methylation.23 The reductive isotopic methylation provides more confident de novo sequencing results than using reductive methylation alone. Furthermore, we report the simplification of the MS/MS fragmentation patterns of singly charged peptides, which highlights the unique advantages of this method for neuropeptide sequencing because many neuropeptides are singly charged. Comparing this work to previous work that focused on the application of this labeling method,23,25-27 we present the first detailed mechanistic investigation of the fragmentation pattern changes and the formation of several signature ions resulting from the N-terminal dimethylation. This information will be important for further development of this and related labeling techniques and the application for peptide de novo sequencing. This study is also the first application of the isotope-assisted formaldehyde labeling for de novo sequencing of neuropeptides from neuronal tissue extract. The novel peptides reported in C. borealis will provide the neurochemical basis for future physiological and functional studies, defining the roles that neuropeptides play in controlling behaviors in this model system. Collectively, our study greatly extended the utility of the reductive methylation as a powerful tool for neuropeptide research. ACKNOWLEDGMENT We thank Kimberly Kutz and Joshua Schmidt in the Li lab for assistance in tissue dissection and sample preparation. We thank Dr. Qiang Han from the Department of Chemistry, University of Pennsylvania, for helpful discussion. We also thank Heidi Behrens, Stephanie DeKeyser, and James Dowell for critical reading and helpful comments on an earlier draft of the manuscript. We gratefully acknowledge startup funds from the School of Pharmacy and Wisconsin Alumni Research Foundation at the University of Wisconsin, a research award from the American Society for Mass Spectrometry (sponsored by Thermo Electron Corp.), and National Science Foundation CAREER Award (CHE-0449991). SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review July 25, 2005. Accepted September 27, 2005. AC051324E


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De Novo Sequencing of Neuropeptides Using Reductive Isotopic

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