C-Terminal Peptide Sequencing via Multistage Mass Spectrometry

Under typical low-energy collision-induced dissociation conditions of quadrupole ion trap and ion cyclotron resonance mass spectrometers, lithium- and...
0 downloads 0 Views 128KB Size
Download PDF
Anal. Chem. 1998, 70, 5162-5165

C-Terminal Peptide Sequencing via Multistage Mass Spectrometry Tong Lin and Gary L. Glish*

Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290

Results are presented showing the ability to obtain Cterminal sequence information from peptides by multiple stages of mass spectrometry. Under typical low-energy collision-induced dissociation conditions of quadrupole ion trap and ion cyclotron resonance mass spectrometers, lithium- and sodium-cationized peptides dissociate predominantly by reaction at the C-terminal peptide bond or an adjacent bond. For the majority of cases studied, the dominant reaction is a rearrangement process that results in the loss of the C-terminal residue and formation of a product ion that is one amino acid shorter than the original peptide ion. Using the multistage MS/MS capabilities of quadrupole ion trap and ion cyclotron resonance mass spectrometers, a subsequent stage of MS/MS can be performed to determine the identity of the new Cterminal residue. Up to eight stages of MS/MS have been performed with both quadrupole ion trap and ion cyclotron resonance mass spectrometers. In general, the same dissociation pathways are observed with both instruments, although occasionally there are significant differences in the branching ratios of competing pathways. Primary sequence determination of a protein is very important in solving the problems of structure-function relationships of proteins. The primary sequence is typically determined by cleaving the protein into peptides and then using one or more of several methods to sequence the peptides. These methods include Edman degradation1 and various types of mass spectrometry. Under the category of mass spectrometry, two general approaches are used: ladder sequencing2-5 and tandem mass spectrometry (MS/MS).6,7 The Edman degradation and ladder sequencing by mass spectrometry both rely on chemical degradations of the peptides in the condensed phase, with subsequent analysis of the degradation products. For the Edman technique, * Address correspondence to this author at Department of Chemistry, Kenan Laboratories, CB #3290, University of North Carolina, Chapel Hill, NC 275993290. Phone: 919-962-2303. E-mail: [email protected]. (1) Edman, P. Acta Chem. Scand. 1950, 4, 283-293. (2) Aldrich, C. J.; DeCloux, A.; Woods, A. S.; Cotter, R. J.; Soloski, M. J.; Forman, J. Cell 1994, 79, 649-658. (3) Chait, B. T.; Wang, R.; Beavis, R. C.; Kent, S. B. H. Science 1993, 262, 89-92. (4) Bartlet-Jones, M.; Jeffery, W. A.; Hansen, H. F.; Pappin, D. J. C. Rapid Commun. Mass Spectrom. 1994, 8, 737-742. (5) Woods, A. S.; Huang, A. Y. C.; Cotter, R. J.; Pasternack, G. R.; Pardoll, D. M.; Jaffee, E. M. Anal. Biochem. 1995, 226, 15-25. (6) Hunt, D. F.; Yates, J. R. I.; Shabanowitz, J.; Winston, S.; Hauer, C. R. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 6233-6237. (7) Biemann, K.; Scoble, H. A. Science 1987, 237, 992-998.

5162 Analytical Chemistry, Vol. 70, No. 24, December 15, 1998

the chemical degradation is done sequentially from the N-terminus of the peptide, with subsequent analysis by liquid chromatography. In ladder sequencing, chemical degradation can be done from the N-terminus3,4 (using modified Edman chemistry) or Cterminus2,5 (using carboxypeptidase) and is performed so that a mixture of peptides of various length is obtained and analyzed by mass spectrometry. A requirement of any of these chemical methods is that the peptide be pure. MS/MS differs from the Edman and ladder sequencing methods in that a mixture of peptides can be analyzed, i.e., the sample does not have to be pure. When doing MS/MS, the first stage of mass spectrometry acts as a separation technique to select from a mixture the peptide ion to be analyzed. After the selection, the peptide ion is activated (typically via collision(s) with gas molecule(s),8-11 or by collisions with surfaces,12-15 or by absorption of a photon16-18). The activated peptide ion can dissociate, and the products of the dissociation are analyzed by the second stage of mass spectrometry. The resulting MS/MS spectrum can then, in favorable cases, be interpreted to deduce the complete amino acid sequence of the peptide. The most informative dissociations are generally along the peptide backbone, e.g., the peptide bond or the C-C bond. However, the current state of knowledge is insufficient to predict or understand routinely which of these bonds will be broken and, when a bond is broken, which end of the dissociated peptide will retain the charge. A nomenclature to describe ions formed by dissociation along the peptide backbone has been developed.19,20 The product ions that are formed can be divided into two general categories: an, bn, and cn (8) Biemann, K.; Martin, S. A. Mass Spectrom. Rev. 1987, 6, 1-76. (9) Cooks, R. G.; Beynon, J. H.; Caprioli, R. M.; Lester, G. R. Metastable Ions; Elservier Scientific Publishing Co.: New York, 1973. (10) McLafferty, F. W.; Bente, I. P. F.; Kornfeld, R.; Tsai, S. C.; Howe, I. J. Am. Chem. Soc. 1973, 95, 2120. (11) Poulter, L.; Taylor, L. C. E. Int. J. Mass Spectrom. Ion Processes 1989, 91, 183-197. (12) Mabud, M. A.; DeKrey, M. J.; Cooks, R. G. Int. J. Mass Spectrom. Ion Processes 1985, 67, 285-294. (13) McCormack, A. L.; Jones, J. L.; Wysocki, V. H. J. Am. Soc. Mass Spectrom. 1992, 3, 859-862. (14) Williams, E. R.; Henry, K. D.; McLafferty, F. W.; Shabanowitz, J.; Hunt, D. F. J. Am. Soc. Mass Spectrom. 1990, 1, 413-416. (15) Lammert, S. A.; Cooks, R. G. J. Am. Soc. Mass Spectrom. 1991, 2, 487. (16) Hunt, D. F.; Shabanowitz, J.; Yates, J. R. I. J. Chem. Soc., Chem. Commun. 1987, 16, 548-550. (17) Lebrilla, C. B.; Wang, D. T. S.; Mizoguchi, T. J.; McIver, R. T. J.; Russell, D. H. J. Am. Chem. Soc. 1989, 111, 8593-8598. (18) Tecklenburg, R. E. J.; Russell, D. H. Mass Spectrom. Rev. 1990, 9, 405451. (19) Roepstorff, P.; Fohlman, J. Biomed. Mass Spectrom. 1984, 11, 601. (20) Biemann, K. Annu. Rev. Biochem. 1992, 61, 977-1010. 10.1021/ac980823v CCC: $15.00

© 1998 American Chemical Society Published on Web 11/13/1998

Figure 1. Quadrupole ion trap MS/MS spectrum of protonated YGPFL.

ions contain the N-terminus of the peptide, while xn, yn, and zn ions contain the C-terminus. When using MS/MS for peptide sequencing, almost without exception, the protonated peptide is the species which is dissociated in an attempt to obtain the sequence. This is due, in part, to the ease of protonating peptides and the long history in mass spectrometry of using protonated species.21 MS/MS of a protonated peptide typically results in formation of a variety of product ions, from both the C- and N-terminus, regardless of the type of mass spectrometer used (i.e., sectors using high collision energies, quadrupole(s), and ion trapping instruments using low collision energies). While some general trends in the dissociation have been observed, e.g., facile cleavage on the N-terminal side of proline residues,22 in general it is hard to predict what type of product ions are going to be formed. Consequently, it is often very hard to interpret the MS/MS spectra to obtain the amino acid sequence. Figure 1 shows a MS/MS spectrum of a small protonated peptide (YGPFL, [M + H+] ) m/z 596) obtained with a quadrupole ion trap mass spectrometer. As is typical, several classes of product ions are observed in the MS/MS spectrum, including N-terminal product ions (e.g., a5, m/z 550, a4, m/z 437; and b5, m/z 578, b4, m/z 465) and C-terminal product ions (e.g., y3 ions, m/z 376) as well as product ions that have dissociated from both termini (e.g., (b4y3)2, m/z 245). While the sequence probably could be determined if this was an unknown, even this relatively short peptide gives a variety of different types of product ions in the MS/MS spectrum. A number of years ago, several groups investigated the dissociation of alkali-cationized peptides using sector mass spectrometers.23-30 These studies did not find MS/MS of alkalicationized peptides to have any advantages over MS/MS of (21) Harrison, A. G. Chemical Ionization Mass Spectrometry; CRC Press: Boca Raton, FL, 1983. (22) Loo, J. A.; Edmonds, C. G.; Smith, R. D. Anal. Chem. 1993, 65, 425-438. (23) Mallis, L. M.; Russell, D. H. Anal. Chem. 1986, 58, 1076-1080. (24) Tang, X.; Ens, W.; Standing, K. G.; Westmore, J. B. Anal. Chem. 1988, 60, 1791-1799. (25) Renner, D.; Spiteller, G. Biomed. Mass Spectrom. 1988, 15, 75-77. (26) Grese, R. P.; Cerny, R. L.; Gross, M. L. J. Am. Chem. Soc. 1989, 111, 28352842. (27) Grese, R. P.; Gross, M. L. J. Am. Chem. Soc. 1990, 112, 5098-5104. (28) Leary, J. A.; Zhou, Z.; Ogden, S. A.; Williams, T. D. J. Am. Soc. Mass Spectrom. 1990, 1, 473-480. (29) Teesch, L. M.; Adams, J. J. Am. Chem. Soc. 1990, 112, 4110-4120. (30) Teesch, L. M.; Adams, J. J. Am. Chem. Soc. 1991, 113, 812-820.

protonated peptides for sequence determination. In fact, the main focus of most of these studies involved investigating a novel rearrangement in which an ion corresponding to [bn-1 + Cat. + OH]+ was a major product from an n-residue peptide (Cat. ) alkali atom, typically Li or Na). This reaction was observed not only under CID conditions but also as a metastable dissociation, suggesting it is a low-energy process. The structure of [peptide(n) + Cat.]+ (n ) the number of amino acid residues) and the mechanism of dissociation are still open to debate.26,29,31 One proposal is that the alkali ion replaces the carboxy proton to form the salt, with the proton located elsewhere in the ion (the amino terminus is the most basic site for peptides without basic residues).26,31 The alternative proposed structure has the alkali ion coordinated to carbonyl oxygens toward the N-terminus.29 In either case, the proposed mechanism involves a charge remote process, transferring either -OH or OCat. to the carbonyl oxygen of the residue adjacent to the C-terminal residue. The resulting product ion [bn-1 + Cat. + OH]+ is equivalent to [peptide(n - 1) + Cat.]+, i.e., an alkali-cationized peptide ion one residue shorter than the original alkali-cationized peptide. This suggests that this product ion could undergo the same reaction to produce [bn-2 + Cat. + OH]+ (equivalent to [peptide(n - 2) + Cat.]+), and indeed this ion was observed in MS3 spectra.26,27 Because the rearrangement reaction is a low-energy process and produces a product ion that seems likely to have a structure that can subsequently undergo the same reaction,26,27,30 a mass spectrometer that favors low-energy reactions and can perform multiple stages of MS/MS (MSn) would appear to have potential to provide C-terminal sequence information. Under typical operating conditions, quadrupole ion trap mass spectrometers favor lowenergy CID processes32,33 and are capable of multiple stages of MS/MS.34,35 In addition, Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers offer similar capabilities, especially when sustained off-resonance irradiation (SORI)36 is used to effect CID. In this report, the capability of using multiple stages of MS/MS to obtain amino acid sequence information from peptides is demonstrated with these two types of instruments. EXPERIMENTAL SECTION Quadrupole ion trap experiments were performed with a modified Finnigan (San Jose, CA) quadrupole ion trap mass spectrometer controlled with Finnigan Rev. B ITMS or ICMS-FL software.37 Alkali-cationized peptide ions were generated using a custom-built electrospray ionization source similar to one previously described.38 Solutions (50/50 H2O/MeOH) containing 1:1 mixtures of peptides and sodium or lithium salts were used. (31) Lee, S.-W.; Kim, H. S.; Beauchamp, J. L. J. Am. Chem. Soc. 1998, 120, 3188-3195. (32) Louris, J. N.; Cooks, R. G.; Syka, J. E. P.; Kelley, P. E.; Stafford, G. C. J.; Todd, J. F. J. Anal. Chem. 1987, 59, 1677. (33) Cooks, R. G.; Glish, G. L.; McLuckey, S. A.; Kaiser, R. E. J. Chem. Eng. News 1991, 69 (12), 26-41. (34) Louris, J. N.; Brodbelt-Lustig, J. S.; Cooks, R. G.; Glish, G. L.; Van Berkel, G. J.; McLuckey, S. A. Int. J. Mass Spectrom. Ion Processes 1990, 96, 117137. (35) McLuckey, S. A.; Glish, G. L.; Van Berkel, G. J. Int. J. Mass Spectrom. Ion Processes 1991, 106, 213-235. (36) Gauthier, J. W.; Trautman, T. R.; Jacobson, D. B. Anal. Chim. Acta 1991, 246, 211-225. (37) Yost, R. A.; Yates, N. A. University of Florida, Gainsville, FL, unpublished. (38) Van Berkel, G. J.; Glish, G. L.; McLuckey, S. A. Anal. Chem. 1990, 62, 1284-1295.

Analytical Chemistry, Vol. 70, No. 24, December 15, 1998

5163

Helium was used as the bath gas and to effect CID at a typical pressure of 1 × 10-3 Torr in the ion trap. FT-ICR experiments were performed at the National ICR User Facility at Florida State University with a custom-built FT-ICR mass spectrometer.39 Alkali-cationized peptide ions were generated from a ESI needle made from polished fused silica capillaries of 25-50 µm inner diameter, sharply tapered (to 5 µm exit i.d.). Stored-waveform inverse Fourier transform (SWIFT) radial ejection was used to mass select the parent ion, and sustained offresonance irradiation (SORI) was used for dissociation. Helium was used as the collision gas and was introduced through a pulse valve. RESULTS AND DISCUSSION Figure 2 shows the MS/MS, MS3, MS4, and MS5 spectra of the sodiated pentapeptide YGPFL. Figure 2a is the MS/MS spectrum of the parent ion [M + Na]+ at m/z 618. All the product ions formed are the result of cleavage of the C-terminal peptide bond or bonds adjacent to that bond. By far the dominant peak is due to the rearrangement reaction producing the [b4 + Na + OH]+ ion (m/z 505), which is empirically the same as the sodiated tetrapeptide YGPF. The loss of 113 Da indicates that one of the leucine isomers was the C-terminal residue. The other peaks observed are [b5 + Na - H]+ (m/z 600, relative intensity ) 14), [b4 + Na - H]+ (m/z 487, relative intensity ) 10), and [a4 + Na - H]+ (m/z 459, relative intensity ) 3), and these data support the identification of the C-terminal residue as a leucine isomer. The MS/MS efficiency40 for this stage of analysis is greater than 99%, and the conversion efficiency35 to the rearrangement ion is 70%. For the MS3 spectrum shown in Figure 2b, the [b4 + Na + OH]+ product ion from the MS/MS spectrum was selected as the parent ion (618 f 505 f MS3 products). The dominant product ion in the MS3 spectrum is [b3 + Na + OH]+ (m/z 358), due to the rearrangement reaction. The MS/MS efficiency in this stage and the conversion efficiency to the rearrangement ion are both 67%. Thus, after two stages of MS/MS, the intensity of the product ion that corresponds to the sodiated tripeptide YGP is 47% of the original pentapeptide. The loss of 147 Da in the formation of the rearrangement ion in this stage of analysis indicates that phenylalanine is the second residue from the C-terminus. The MS4 spectrum with the sodiated YGP, [b3 + Na + OH]+, as the parent ion (618 f 505 f 358 f MS4 products) shows only the rearrangement ion (b2 + Na + OH)+ (m/z 261) as the product ion (Figure 2c). The MS/MS efficiency and the conversion efficiency of this stage are 25%. The 97 Da loss indicates that proline is the third residue from the C-terminus of the pentapeptide. Figure 2d is the MS5 spectrum with the sodiated YG, (b2 + Na + OH)+, as the parent ion (618 f 505 f 358 f 261 f MS5 products). Not only is the rearrangement ion observed, but also other dissociation pathways become competitive. The (a1 + Na - H)+ (m/z 158) is observed as the most abundant peak in the (39) Senko, M. W.; Hendrickson, C. L.; Pasa-Tolic, L.; Marto, J. A.; White, F. M.; Guan, S.; Marshall, A. G. Rapid Commun. Mass Spectrom. 1996, 10, 1824-1828. (40) Yost, R. A.; Enke, C. G.; McGilvery, D. C.; Smith, D.; Morrison, J. D. Int. J. Mass Spectrom. Ion Phys. 1979, 30, 127-136.

5164 Analytical Chemistry, Vol. 70, No. 24, December 15, 1998

Figure 2. (a) Quadrupole ion trap MS/MS spectrum of sodiated YGPFL. (b) Quadrupole ion trap MS3 spectrum of sodiated YGPFL with the [b4 + Na + OH]+ from (a) as the second-generation parent ion. (c) Quadrupole ion trap MS4 spectrum of sodiated YGPFL with the [b3 + Na + OH]+ from (b) as the third-generation parent ion. (d) Quadrupole ion trap MS5 spectrum of sodiated YGPFL with the [b2 + Na + OH]+ from (b) as the fourth-generation parent ion.

Figure 4. Composite MSn spectrum of sodiated GVAYFMVHPD using a FT-ICR mass spectrometer. Figure 3. Composite MSn spectrum of sodiated GVYVHPV using a quadrupole ion trap.

spectrum, while the (b1 + Na + (m/z 204) formed by the rearrangement reaction is observed with only 16% relative intensity. YGPFL is characteristic of almost all alkali-cationized peptides studied thus far (>100 peptides) in that the dissociation observed for either CID in the quadrupole ion trap or SORI in an FT-ICR occurs only around the C-terminal peptide bond. In the majority of cases, the rearrangement ion is the dominant product ion, as shown in the case of YGPFL, where the complete amino acid sequence can be obtained by multiple stages of mass spectrometry. However, this is not the case for all peptides. For example, if the proline in the above example is replaced with leucine or tryptophan, similar results are obtained; however, if it is replaced with glycine (which is the peptide leucine enkephalin, YGGFL), differences appear at the MS4 stage of the experiment. It is more difficult to determine the sequence of the last two residues (YG) because the MS4 spectrum does not have the rearrangement ion as the major product ion. This change of dissociation pathways is not uncommon when only three residues remain in the peptide ion, and it appears to be dependent upon the sequence. In part, this may be due to the cation being able to coordinate to both the C- and N-termini.27 Figure 3 shows the composite MS7 spectra of the sodiated heptapeptide GVYVHPV, obtained with the quadrupole ion trap. Sequential dissociation of the amino acid residues from the C-terminus is again observed. A complete series of (bn-x + Na + OH)+ ions is obtained, and the entire sequence of this peptide can be readily determined. Dissociation up to MS8 has been observed with the FT-ICR for the sodiated decapeptide GVAYFMVHPD, shown in Figure 4. The results are similar to those obtained with the quadrupole ion trap, except that the most abundant product ion in the MS3 spectrum in the FT-ICR is not the rearrangement ion but the [b6 + Na - H]+ ion. The rearrangement ion is still observed and was used to continue the sequencing, but at reduced sensitivity. This indicates that the dissociation pathways can be sensitive not only to the amino acid

MS5

OH)+

sequence but also to the time frame/energetics of the activation process. SORI occurs over a longer time frame compared with CID in the quadrupole ion trap (1 s versus 30 ms in these experiments), but the collision gas density is several orders of magnitude lower in the ICR, so there are many fewer, but more energetic, collisions between the ions and collision gas. The results presented here show that ion-trapping instruments using low collisional activation processes with alkali-cationized peptides lead to dissociation related only to the C-terminal residue. The capability of ion-trapping instruments to do multiple stages of MS/MS allows sequential cleavage of the peptide ion from the C-terminus, which provides the amino acid sequence from the C-terminus. The specific sequence of amino acids can affect the dissociation pathway, and this is currently being investigated in detail. The length of peptide that can be sequenced is currently limited to seven or eight residues. This is not a fundamental limit but is due to the difficulty of forming singly cationized peptide ions for peptides with more than seven or eight residues via electrospray ionization. Work is underway to generate alkalicationized peptide ions with more residues via matrix-assisted laser desorption/ionization or liquid secondary ionization mass spectrometry. For work in proteomics, this approach will allow ready identification of the last several amino acid residues of peptides obtained from protein digests. This will provide a high level of confidence in identifying the correct protein from a database search. ACKNOWLEDGMENT This work was supported by NIH Grant GM49852. FT-ICR work was done at the National ICR User Facility at Florida State University. We thank Dr. Chris Hendrickson, Professor Alan Marshall, and their colleagues at the facility for their guidance in operation of the ICR. Received for review July 24, 1998. Accepted September 29, 1998. AC980823V

Analytical Chemistry, Vol. 70, No. 24, December 15, 1998

5165