Study of the Fragmentation Patterns of the Phosphate-Arginine

Synopsis. We investigated gas-phase stability and dissociation pathways of the NCX of a basic peptide VLRRRRKRVN, with the phosphopetide SVSTDpTpSAE...
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Study of the Fragmentation Patterns of the Phosphate-Arginine Noncovalent Bond Shelley N. Jackson, Hay-Yan J. Wang, and Amina S. Woods* NIDA IRP, National Institutes of Health, 5500 Nathan Schock Drive, Baltimore, Maryland 21224 Received August 10, 2005

Abstract: Our previous work has highlighted the role of certain amino acid residues, mainly two or more adjacent arginine on one peptide and two or more adjacent glutamate, or aspartate, or a phosphorylated residue on the other in the formation of noncovalent complexes (NCX) between peptides. In the present study, we employ ESI-MS to investigate the gas-phase stability and dissociation pathways of the NCX of a basic peptide VLRRRRKRVN, an epitope from the third intracellular loop of the dopamine D2 receptor, with the phosphopetide SVSTDpTpSAE, an epitope from the cannabinoid CB1 carboxyl terminus. ESI-MS/MS analysis of the NCX between VLRRRRKRVN and SVSTDpTpSAE suggests two dissociation pathways for the NCX. The major pathway is the disruption of the electrostatic interactions between the Arg residues and the phosphate groups, while an alternative pathway is also recorded, in which the complex is dissociated along the covalent bond between the oxygen from either Thr or Ser and HPO3. To verify the alternative pathway, we have used an ion trap instrument to conduct MS3 analysis on the product ions of both dissociation pathways. Keywords: phosphate-arginine interaction • receptor heteromerization • gas phase stability • fragmentation pathways • noncovalent complexes

Introduction Biological mass spectrometry has emerged as an important technique for the study of noncovalent complexes (NCX). Several studies have been conducted using either electrospray ionization mass spectrometry (ESI-MS) or matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the analysis of NCXs.1-5 One of the main motivations for these studies is the possibility that the structure, stability, and conformations of NCX gas-phase ions may provide information pertaining to their formation in biological systems. Schug and Lindner’s comprehensive review6 has addressed NCXs formed between the cationic guanidinium and the anionic phosphate group. In biological systems, a cationic guanidinium group is located at the terminus of the amino acid arginine (R) side chain and has a delocalized positive charge that is distributed * To whom correspondence should be addressed. Tel: (410) 550-1507. Fax: (410) 550-6859. E-mail: [email protected].

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over the entire group. One source of anionic phosphate groups in biological organisms is the phosphorylated amino acid residues: serine (S), threonine (T), and tyrosine (Y). Phosphorylation/dephosphorylation events usually alter a protein conformation thus influencing its function. As structure and function are interrelated phosphorylation/dephosphorylation events govern numerous biochemical and physiological processes, which when interfered with can result in serious pathology.7,8 In previous studies9-12, we have shown that motifs composed of a minimum of two adjacent arginine “RR” or of “RKR” on one peptide and one phosphorylated residue on the other, were sufficient to generate stable NCXs between the two peptides. In one study of epitopes involved in the formation of receptor heteromers using ESI-MS we attempted to fragment the NCX formed between VLRRRRKRVN, a Dopamine D2 receptor epitope, and SAQEpSQGNT, an Adenosine A2A receptor epitope.12 The peptides dissociated, and in addition a new molecular ion, 80 amu larger than the VLRRRRKRVN epitope was detected suggesting the covalent bond between the serine’s oxygen and phosphorus was cleaved and that the electrostatic bond between the guanidinium group of arginine and the phosphate, could survive the CID. In addition, a study of the interaction of a peptide with adjacent Arg and peptides containing six adjacent acidic residues and a phosphate or six adjacent acidic residues or just one phosphate on the acidic peptide showed variations in the collision energies needed to induce dissociation.13 The peptide containing six acidic residues and a phosphate needed 25% higher collision energy voltages than the ones containing just one phosphate or just six acidic residues. A similar weaker pattern for the latter two peptides implies that the interaction involving just one phosphate is almost as stable as the one involving multiple acidic residues, and that disruption of interactions involving a phosphate proceeds in a biphasic fashion. In the present work, we use ESI-MS to investigate the gasphase stability and dissociation pathways of the NCX of a basic peptide VLRRRRKRVN, an epitope from the third intracellular loop of the dopamine D2 receptor, with the phosphopetide SVSTDpTpSAE, an epitope from the cannabinoid CB1 carboxyl terminus. ESI-MS/MS analysis of the NCX between VLRRRRKRVN and SVSTDpTpSAE revealed two dissociation pathways for the NCX. The major pathway is the disruption of the electrostatic interactions between the Arg residues and the phosphate groups, while a minor pathway is also observed, in which the complex is dissociated along the covalent bond between the oxygen from either Thr or Ser and HPO3. To 10.1021/pr050261d CCC: $30.25

 2005 American Chemical Society

technical notes

Figure 1. (a) ESI-MS spectra of a peptide mixture consisting of 1 pmol/µL of VLRRRRKRVN and 15 pmol/µL of SVSTDpTpSAE and (b) ESI-MS/MS spectra of [NCX+3H]3+ mass peak in part a at a collision energy of 30 V.

confirm this minor pathway, we have used an ion trap instrument to perform MS3 analysis on the product ions of the two dissociation pathways.

Experimental Section Mass Spectrometers. A Q-TOF Global Ultima mass spectrometer (Waters, Milford, MA) was used for electrospray analysis. A flow rate of 5 µL/min was used to introduce the sample into the mass spectrometer. The mass spectrometer was operated in positive ion mode with a capillary voltage of 2.9 kV, a sampling cone voltage of 50 V, a source temperature of 100 °C, a desolvation temperature of 200 °C, a desolvation gas flow rate of 650 L/Hr, and a cone gas flow of 100 L/Hr. For MS/MS analysis, a selection mass window of 6 Da with a collision gas (Argon) pressure of 7 psi was employed. Collision energies between 5 and 35 V were used in the collision cell for ion fragmentation. Mass spectra presented are the sum of 50 consecutive 1-second scans. For MSn analysis, a LCQ Deca XP MAX ion trap mass spectrometer (ThermoFinnigan) was used. The mass spectrometer was operated in positive ion mode with a source voltage of 2.2 kV, a capillary voltage of 44 V, and a capillary temperature of 200 °C. A nanoflow capillary was employed to introduce the sample at 200 nL/minute. Collision energies between 0 and 35 V were used to produce ion fragmentation. Peptides. The basic peptide VLRRRRKRVN (MW ) 1351.87 Da) and SVSTDpTpSAE (MW ) 1055.31 Da), where pS is a phosphorylated serine and pT is a phosphorylated threonine, were synthesized at the John Hopkins School of Medicine

Jackson and Woods

Figure 2. ESI-MS/MS spectra of a peptide mixture consisting of 1 pmol/µL of VLRRRRKRVN and 15 pmol/µL of SVSTDpTpSAE. Ion chromatographs of (a) [NCX+3H]3+ and (b) [VLRRRRKRVN+2H]2+,[VLRRRRKRVN+HPO3+2H]2+, and [NCXH3PO4+3H]3+ at collision energies of 5, 10, 15, 20, 25, 30, 35 V.

Peptide Synthesis Core Facility. Sample mixtures, consisting of VLRRRRKRVN at 1 pmol/µL and SVSTDpTpSAE at 15 pmol/ µL in water, were employed for mass analysis.

Results Figure 1a illustrates a typical ESI-MS spectrum for a sample mixture of VLRRRRKRVN and SVSTDpTpSAE. The major peaks observed are [VLRRRRKRVN + 3H]3+ and [SVSTDpTpSAE + H]+. Due to the presence of six positively charged amino acid residues (Arg and Lys), which favor ionization in positive ion mode, [VLRRRRKRVN + 3H]3+ is the base peak despite being at a lower concentration than SVSTDpTpSAE. Multi-charged mass peaks (2+, 3+, 4+) corresponding to the NCX formed by the electrostatic interactions between VLRRRRKRVN and SVSTDpTpSAE are also observed. Figure 1b shows a ESI-MS/MS spectrum at a collision energy of 30 V for the [NCX + 3H]3+ mass peak observed in Figure 1a. The base peak in this mass spectrum is the [NCX + 3H]3+ corresponding to the intact NCX, a peak corresponding to [NCX - H3PO4 + 3H]3+ is also observed. The loss of one phosphate group did not disrupt the NCX between VLRRRRKRVN and SVSTDpTpSAE, which is held together by one phosphate-guanidinium interaction. The dissociation of the [NCX + 3H]3+ peak resulted in singly charged ions corresponding to SVSTDpTpSAE and doubly charged ions attributed to VLRRRRKRVN. The acidic peptide and its fragment ions SVSTDpTpSAE - HPO3, SVSTDpTpSAE - H3PO4, SVSTDpTpSAE - HPO3 - H3PO4, and SVSTDpTpSAE - 2H3PO4 (all singly charged) were observed. The loss of HPO3 (80 Da) and H3PO4 (98 Da) is expected since previous ESI-MS analysis of phosJournal of Proteome Research • Vol. 4, No. 6, 2005 2361

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technical notes

Figure 3. ESI-ion trap mass spectra of a peptide mixture consisting of 1 pmol/µL of VLRRRRKRVN and 15 pmol/µL of SVSTDpTpSAE. (a) MS spectrum, (b) MS2 spectrum of [NCX+3H]3+ mass peak in part a, and MS3 mass spectra of (c) [VLRRRRKRVN+2H]2+, and (d) [VLRRRRKRVN+HPO3+2H]2+.

phopeptides has shown a similar fragmentation pathway.14,15 For the basic peptide, the following ions [VLRRRRKRVN + 2H]2+ and [VLRRRRKRVN - NH3 + 2H]2+ were observed. In addition a fragment from the complex [VLRRRRKRVN + HPO3+ 2H]2+ was recorded. The major dissociation pathway for the NCX is the disruption of the salt bridges formed by the interaction of the phosphate groups in SVSTDpTpSAE with the Arg residues in VLRRRRKRVN, resulting in [VLRRRRKRVN + 2H]2+ and [SVSTDpTpSAE + H]+. The presence of [VLRRRRKRVN + HPO3 + 2H]2+ in the mass spectrum suggests an alternative dissociation pathway for the NCX in which the covalent bond between the oxygen from either Thr or Ser and HPO3 is broken. To gain a better understanding of the fragmentation pathway for the NCX of VLRRRRKRVN and SVSTDpTpSAE, the [NCX + 3H]3+ ion was fragmented at collision energies of 5, 10, 15, 20, 25, 30, and 35 V. Figure 2(a) contains an ion chromatograph of the [NCX + 3H]3+ ion at increasing collision energy. The plot reveals that the NCX is stable with little to no fragmentation up to 25 V. However, at 25 V the complex starts to dissociate and is almost completely dissociated at 35 V. Figure 2b shows an ion chromatograph of the [NCX - H3PO4 + 3H]3+, [VLRRRRKRVN + 2H]2+ and [VLRRRRKRVN + HPO3 + 2H]2+ mass peaks for the same sample run in Figure 2a. The ion chromatograph for [NCX-H3PO4+ 3H]3+ suggests a two-step dissociation of the complex, in which the two phosphorylated residues of SVSTDpTpSAE interacting with the Arg residues in VLRRRRRKRVN dissociate at different collision energies. The [NCX - H3PO4+ 3H]3+ mass peak starts to appear as the NCX 2362

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begins to dissociate and increases up to 25 V. However, between 25 and 30 V the ion count for [NCX - H3PO4 + 3H]3+ is stable suggesting that the collision energy in this range is insufficient to dissociate the interaction between the remaining phosphate group and Arg residues. At 35 V, the ion count for [NCX - H3PO4 + 3H]3+ is significantly reduced and as can be seen in Figure 2a at 35 V the ion counts of [NCX + 3H]3+ also decrease drastically. This result suggests that at 35 V, the electrostatic interactions between VLRRRRKRVN and SVSTDpTpSAE are almost completely disrupted. Comparing the chromatograph in Figure 2b with the one in Figure 2a, illustrates that as the NCX starts to dissociate both [VLRRRRKRVN + 2H]2+ and [VLRRRRKRVN + HPO3 + 2H]2+ mass peaks start to appear at similar ion counts and increase in counts up to 30 V. However, at 35 V the [VLRRRRKRVN + 2H]2+ ion count continues to increase, but the ion count for [VLRRRRKRVN+ HPO3 + 2H]2+ starts to decrease. The decrease in ion count for [VLRRRRKRVN + HPO3 + 2H]2+ at 35 V is most likely attributed to the dissociation of the noncovalent interaction between HPO3 and VLRRRRKRVN at the higher collision energy. MSn experiments were conducted on the [NCX + 3H]3+ ion using an ion trap MS to further study the fragmentation of the NCX complex. Figure 3a displays a ESI-MS spectrum for a mixture of VLRRRRKRVN and SVSTDpTpSAE. The major peaks observed for the peptides and NCXs are similar to the mass spectrum in Figure 1a. Figure 3b shows a MS2 spectrum of the [NCX+3H]3+ ion at a collision energy of 25 V. A similar series of fragmentation ions are observed for the NCX as seen in

technical notes Figure 1b including the presence of [VLRRRRKRVN + 2H]2+ and [VLRRRRKRVN + HPO3 + 2H]2+. To confirm the assignment of the [VLRRRRKRVN + HPO3 + 2H]2+ mass peak, MS3 analysis was conducted. Figure 3c contains a MS3 spectrum of [VLRRRRKRVN + 2H]2+ at a collision energy of 25 V, whereas Figure 3d illustrates a MS3 spectrum of [VLRRRRKRVN + HPO3+2H]2+ at the same collision energy. In both of these mass spectra, the major fragment peak is [VLRRRRKRVN-NH3 + 2H]2+, confirming the assignment of the [VLRRRRKRVN + HPO3 + 2H]2+ mass peak. We have previously studied the NCX of VLRRRRKRVN and SAQEpSQGNT, although the acidic peptide has only one phosphate it generated a similar fragmentation pattern, including the [VLRRRRKRVN + HPO3 + 2H]2+ mass peak.12 MSn analysis of this mass peak gave similar results (data not shown).

Discussion The above results suggest two dissociation pathways for the noncovalent complex between VLRRRRKRVN and SVSTDpTpSAE. The major pathway is the disruption of the electrostatic interactions between the Arg residues and the phosphate groups, which results in intact VLRRRRKRVN and SVSTDpTpSAE mass peaks. An alternative pathway is also observed, in which the complex is dissociated along the covalent bond between the oxygen from either Thr or Ser and HPO3. The observation of this pathway demonstrates the stability of the electrostatic interaction between phosphorylated residues and Arg residues in the gas phase. Electrostatic interactions are know to be greatly strengthen in the gas phase when compared to the solution phase.1,6 This is due to the dielectric constant of the solvent, which weakens electrostatic interactions in solution phase. The experimental data shows that the complex is very stable and that voltages >25 V are required for the CID to dissociate the complex into its two components. At a voltage >30 V minor peaks due to the loss of one HPO3 and H3PO4 from the acidic peptide and a small one from the loss of NH3 from the basic peptide are seen but no fragmentation of the backbone of the peptides (Figure 2a). The ion-chromatogram in Figure 2b shows that the new peak [VLRRRRKRVN + HPO3 + 2H]2+ (green), starts appearing at a CID of 20V and its relative abundance gradually increases and parallels that of [VLRRRRKRVN + 2H]2+ (blue), with higher voltages up to 35 V. However at voltages >35 V, the relative abundance of the basic peptide continues to increase while that of the complex between HPO3 and

Jackson and Woods

VLRRRRKRVN starts to decrease, suggesting that increased availability of peptide from dissociation and free phosphate does not result in the formation of the complex in the gas phase, but rather that the complex between the basic peptide and the phosphate is due to the cleavage of the bond between the serine’s or threonine’s oxygen and phosphorus. To ascertain the composition of the complex, the same experiments were repeated using an ESI-Ion trap instrument. The results confirmed those obtained with the ESI-Q-TOF (Figures 3a,b). Additionally, MS3 analysis on the [VLRRRRKRVN + 2H]2+ and [VLRRRRKRVN + HPO3 + 2H]2+ mass peaks yielded complimentary mass spectra confirming the assignment of the [VLRRRRKRVN + HPO3 + 2H]2+ mass peak.

Acknowledgment. This research was supported by the Intramural Research Program of the National Institute on Drug Abuse, NIH. We would like to thank Dr. Robert Cole and Mr. Robert O’Meally of the Johns Hopkins Mass Spectrometry facility for their help and ONDCP for instruments funding, without which this and other projects could not have been done. References (1) Loo, J. A. Int. J. Mass Spectrom. 2000, 200, 175-186. (2) Veenstra, T. D. Biochem. Biophys. Res. Commun. 1999, 257, 1-5. (3) Woods, A. S.; Koomen, J. M.; Ruotolo, B. T.; Gillig, K. J.; Fuhrer, K.; Gonin, M.; Egan, T. F.; Schultz, J. A. J. Am. Soc. Mass Spectrom. 2002, 13, 166-169. (4) Woods, A. S., Huestis, M. A. J. Am. Soc. Mass Spectrom. 2001, 12, 88-96. (5) Shields, S. J.; Oyeyemi, O.; Lightstone, F. C.; Balhorn, R. J. Am. Soc. Mass Spectrom. 2003, 14, 460-470. (6) Schug, K. A.; Lindner, W. Chem. Rev. 2005, 105, 67-114. (7) Kim, C. H.; Braud, S.; Isacc, J. T.; Roche, K. W. J Biol Chem. 2005, 280, 25409-25415. (8) Ray, K. Int. Arch. Biosci. 2001, 1027-1035. (9) Woods, A. S. J. Proteome Res. 2004, 3, 478-484. (10) Ciruela, F.; Burgueno, J.; Casado, V.; Canals, M.; Marcellino, D.; Goldberg, S. R.; Bader, M.; Fuxe, K.; Agnati, L. F.; Lluis, C.; Franco, R.; Ferre´, S.; Woods, A. S. Anal. Chem. 2004, 76, 5354-5363. (11) Woods, A. S.; Ciruela, F.; Fuxe, K.; Agnati, L. F.; Lluis, C.; Franco, R.; Ferre´. J. Mol. Neurosci. 2005, 26, 125-132. (12) Woods, A. S. And Ferre S. J. Proteome Res. 2005, 4, 1397-1402. (13) Woods, A. S.; Jackson, S. N.; Wang, H.-Y. J.; Yergey, A. Proc. 53rd ASMS Conf. Mass Spectrometry and Allied Topics, San Antonio, TX, 2005. (14) Moyer, S. C.; Cotter, R. J.; Woods, A. S. J. Am. Soc. Mass Spectrom. 2002, 13, 274-283. (15) DeGnore, J. P.; Qin, J. J. Am. Soc. Mass Spectrom. 1998, 9, 1175-1188.

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