Accelerated Article pubs.acs.org/ac
Ion Mobility Separation of Variant Histone Tails Extending to the “Middle-Down” Range Alexandre A. Shvartsburg,*,† Yupeng Zheng,‡ Richard D. Smith,† and Neil L. Kelleher‡ †
Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352, United States Department of Chemistry, Department of Molecular Biosciences, and the Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois 60208, United States
‡
S Supporting Information *
ABSTRACT: Differential ion mobility spectrometry (FAIMS) can baseline-resolve multiple variants of posttranslationally modified peptides extending to the 3−4 kDa range, which differ in the localization of a PTM as small as acetylation. Essentially orthogonal separations for different charge states expand the total peak capacity with the number of observed states that increases for longer polypeptides. This potentially enables resolving localization variants for yet larger peptides and even intact proteins.
P
species frequently masks multiple variants in CID data. However, chromatographic and electrophoretic separations of localization variants ordinarily require lengthy gradients and often fail, especially for cases of alternative PTMs on proximate residues where MS/MS is most problematic.14 Localization variants of modified peptides were recently resolved employing ion mobility spectrometry (IMS), including conventional (drift-tube) IMS15 based on absolute mobility (K) at low electric field intensity (E) and differential or field asymmetric waveform IMS (FAIMS)13,16−18 that exploits the difference between K values at high and low E. Unlike dispersive drift-tube IMS, FAIMS is a filtering technique: species with a given derivative of K(E) over a range of E are selected while pulled through a gap between two electrodes carrying the waveform.19,20 Scanning a dc voltage (the compensation voltage) superposed on the waveform, commonly expressed20 as the compensation field (EC), generates the FAIMS spectrum. The initial efforts13,16,17 have focused on phosphorylation, perhaps the most common and important PTM. Drift-tube IMS (with ion funnel interfaces to the preceding ion source and subsequent MS stage) is exceptionally sensitive but, at the resolving power (R) of ∼80, separated the phosphopeptide variants only in part.15 The commercial FAIMS stage with cylindrical gap geometry and thus inhomogeneous electric field provides a much lower21 R ∼ 10 (for peptides) and, despite FAIMS being more orthogonal to MS than conventional IMS is,22,23 the separation is even worse.13 Planar-geometry FAIMS systems utilize homogeneous fields that permit much greater R, up to ∼300 for multiply charged peptides using the He/N2 or H2/N2 gas mixtures.24−26
roteins are replete with various post-translational modifications (PTM) that often govern their biological function.1−4 As proteomic technologies mature, interest shifts to the determination, detection, and quantification of 3-D protein structures (conformations) and PTMs. Complete characterization of PTMs includes their precise localization on the protein, as multiple variants (some with a PTM transposed by just one residue) generally coexist and have differing activity.5−9 Proteolytic digestion in “bottom-up” or “middle-down” proteomic methods translates such variants into isomeric peptides. Variants are normally identified by tandem mass spectrometry (MS/MS) via collision-induced dissociation (CID) and/or electron capture/transfer dissociation (EC/TD). Constraints of this approach are (1) major sensitivity losses due to a common preference for facile PTM elimination (over informative backbone severance) in CID and low efficiency and largely indiscriminate fragmentation in EC/TD that partitions the useful ion signal into multiple channels,4 (2) zero or very low EC/TD yield for smaller peptides with 1+ and 2+ charge states or peptides with “electron predator” PTMs such as nitrate,10 (3) difficulty of distinguishing the PTMs on a basic and adjacent residue (including the two N-terminal residues), as the bond between them is hard to cleave,11 (4) possibility of missing or mistaken assignments caused by shifts of labile PTMs or other isomerizations during the CID process,12 and (5) fundamental inability to distinguish some variants when the number of options n exceeds two because (n − 2) peptides yield no unique-mass fragments in either CID or EC/TD. For example, a mixture of X1mZX2ZX3ZX4, X1ZX2mZX3ZX4, and X1ZX2ZX3mZX4 (where X1−4 and Z are arbitrary amino acids and m is a modification of Z) produces unique fragments for the first and third, but not the second peptide.13 Variant mixtures are more challenging and usually must be separated prior to MS analysis, as the competitive fragmentation of same © 2012 American Chemical Society
Received: March 5, 2012 Accepted: April 27, 2012 Published: May 3, 2012 4271
dx.doi.org/10.1021/ac300612y | Anal. Chem. 2012, 84, 4271−4276
Analytical Chemistry
Accelerated Article
Figure 1. Normalized FAIMS spectra for the localization variants of H4 histone tail (color-coded on the top) with z = 4−7, measured using He/N2 with up to 46% He (v/v), as labeled. Peaks for different conformers are marked by letters. The widths (w, V/cm) are shown for the well-shaped major peaks at 46% He, with R values added for z = 6 and 7.
H4 permits ∼3 × 106 combinations for just the known histone PTMs and attachment sites, of which 60% H2 exceeds that at the maximum He fraction, inducing further transitions such as the vanishing a feature for 6+ ions of K5 (Figure 3). The folding distinctions between variants elicited by altering the gas influence the separation outcomes. For example, K5 can be fully resolved from K12 as 6+ ion in 46% He or 50−60% H2 because the K12 b feature that overlaps with the dominant K5 b peak at lower He or H2 fractions is destroyed by field heating.
PTMs, although it does not isolate the two effects. Seeking to quantify this, we note that the PTM makes 1.3% of the peptide mass here versus 5.0% for phosphorylation of the above τpeptide, and the maximum EC spans (∼0.13 and 0.4, respectively) are roughly proportional to those percentages. How broadly this relationship holds remains to be determined. Ions have comparable mobilities in H2 and He, and He/N2 and H2/N2 buffers with equal N2 fractions provide close FAIMS resolving powers.26 However, much greater electrical breakdown resistance of H2 permits lower N2 fractions (down to ∼10% versus ∼50% with He/N2 at the presently maximum DV = 5.4 kV) and thus higher R values.20,26 The gain for multiply charged peptides was less than that for large singly charged ions but still significant.26 In agreement with those findings, here the EC values and peak widths at 46% H2 (⟨w⟩ of 1.0 V/cm for 6+ and 7+ ions, 1.5 V/cm for 5+, and 1.6 V/cm for 4+ from interpolation of the data at 40% and 50% H2) about match those at 46% He, leading to the same range of R ∼ 140−190 for z = 6 and 7 (Figure 3). As H2 content grows to 70%, the peaks narrow (to ⟨w⟩ of 0.9 V/cm for z = 6 and 1.3 V/cm for z = 4) while EC continue rising, and the resolving power (for z = 6) increases to ∼230−310. This does not enhance the variant resolution as all four variants can be 4274
dx.doi.org/10.1021/ac300612y | Anal. Chem. 2012, 84, 4271−4276
Analytical Chemistry
Accelerated Article
Figure 4. FAIMS spectra for the K5Ac/K8Ac, K8Ac/K12Ac, and K8Ac/K16Ac mixtures with z = 4−7, measured using 46:54 He/N2. The vertically scaled spectra for individual components are overlaid.
their unfolding. The PTM position should intuitively make a lesser difference for longer peptides, and indeed the separation range in any charge state is narrower for these histones than for shorter phosphopeptide variants. However, separations in different charge states are virtually orthogonal as for sequence inversions;31 hence, the total peak capacity approximately scales with the number of available states. With standard ESI sources, that number grows roughly in proportion to (peptide mass)1/2 or faster:40 e.g., from sole z = 1 for leucine enkephalin41 (0.56 kDa) to 5 states for present histone tails to 24 states (z = 15− 38) for carbonic anhydrase (29 kDa).39 These ranges can be expanded to higher z via supercharging42 and lower z by proton stripping.36 This dramatically raises the odds for resolving larger peptide variants for at least one charge state. These findings suggest the possibility that such localization variants, even involving smaller PTMs like acetylation, may be separated for intact proteins.
However, the major peaks of K5 and K12 move closer at higher He or H2 concentrations and coincide at 70% H2 (Figure 3), rendering them inseparable (for z = 6) despite the greater resolving power. Other species such as K5 (z = 7) and K12 (z = 4 and 7) are eliminated at higher He or H2 fractions, presumably by FAIMS “self-cleaning” upon extensive unfolding.20,32 This process may limit the room for higher H2 fractions to ranges below the electrical breakdown threshold, depending on the analytical targets. One path around this obstacle may be thermal or collisional heating of ions before FAIMS analysis. As previously,16,17,31 separations using all He/N2 and H2/N2 compositions tried were confirmed by analyses of 1:1 binary mixtures of K8 with K5, K12, or K16 (Figure 4 and Figures S1−S4 in the Supporting Information). All significant spectral features were attributed employing the data for individual variants, which validates the calibration procedure and verifies the utility of FAIMS for separation of histone variants.
■
■
CONCLUSIONS Planar FAIMS analyzers using He/N2 or H2/N2 mixtures can separate peptides in the ∼3−4 kDa size range and charge states of 4−7 with resolving power similar to that for smaller peptides in the “bottom-up” range. Here, we baseline-resolved all four biological monoacetylated H4 histone tail variants with differing modification sites. The exact separation mechanism is unclear, but both the initial 3-D peptide geometry and its evolution under the field heating likely matter. Moving a PTM may strongly affect both factors by modulating the charge distribution along the backbone and/or steric hindrances that control the relative energies of competing folds and barriers to
ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest. 4275
dx.doi.org/10.1021/ac300612y | Anal. Chem. 2012, 84, 4271−4276
Analytical Chemistry
■
Accelerated Article
(29) Phanstiel, D.; Brumbaugh, J.; Berggren, W. T.; Conard, K.; Feng, X.; Levenstein, M. E.; McAlister, G. C.; Thomson, J. A.; Coon, J. J. Proc. Natl Acad. Sci. U.S.A. 2008, 105, 4093−4098. (30) Pesavento, J. J.; Bullock, C. R.; Leduc, R. D.; Mizzen, C. A.; Kelleher, N. L. J. Biol. Chem. 2008, 283, 14927−14937. (31) Shvartsburg, A. A.; Creese, A. J.; Smith, R. D.; Cooper, H. J. Anal. Chem. 2011, 83, 6918−6923. (32) Shvartsburg, A. A.; Li, F.; Tang, K.; Smith, R. D. Anal. Chem. 2007, 79, 1523−1528. (33) Purves, R. W.; Barnett, D. A.; Ells, B.; Guevremont, R. J. Am. Soc. Mass Spectrom. 2000, 11, 738−745. (34) Shvartsburg, A. A.; Li, F.; Tang, K.; Smith, R. D. Anal. Chem. 2006, 78, 3304−3315. (35) Pierson, N. A.; Chen, L.; Valentine, S. J.; Russell, D. H.; Clemmer, D. E. J. Am. Chem. Soc. 2011, 133, 13810−13813. (36) Shelimov, K. B.; Jarrold, M. F. J. Am. Chem. Soc. 1997, 119, 2987−2994. (37) Hudgins, R. R.; Woenckhaus, J.; Jarrold, M. F. Int. J. Mass Spectrom. Ion Processes 1997, 165/166, 497−507. (38) Shelimov, K. B.; Clemmer, D. E.; Hudgins, R. R.; Jarrold, M. F. J. Am. Chem. Soc. 1997, 119, 2240−2248. (39) Shvartsburg, A. A.; Bryskiewicz, T.; Purves, R. W.; Tang, K.; Guevremont, R.; Smith, R. D. J. Phys. Chem. B 2006, 110, 21966− 21980. (40) de la Mora, J. F. Anal. Chim. Acta 2000, 406, 93−104. (41) Guevremont, R.; Purves, R. W. J. Am. Soc. Mass Spectrom. 1999, 10, 492−501. (42) Iavarone, A. T.; Jurchen, J. C.; Williams, E. R. Anal. Chem. 2001, 73, 1455−1460.
ACKNOWLEDGMENTS We thank Ron Moore, Dr. Keqi Tang, and Dr. Zhixin Tian for experimental help and useful discussions. Parts of this research were supported by NCRR (Grant RR18522), NIGMS (Grants GM 067193-09 and GM 103493-10), NCI (Grant CA 155252), Northwestern University Physical Sciences Oncology Center (Grant CA 143869), the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust, and a gift from the Zell Family to the Robert H. Lurie Comprehensive Cancer Center. Work was performed in the Environmental Molecular Sciences Laboratory, a U.S. DoE OBER national scientific user facility at PNNL.
■
REFERENCES
(1) Ficarro, S. B.; McCleland, M. L.; Stukenberg, P. T.; Burke, D. J.; Ross, M. M.; Shabanowitz, J.; Hunt, D. F.; White, F. M. Nat. Biotechnol. 2002, 20, 301−305. (2) Mann, M.; Jensen, O. N. Nat. Biotechnol. 2003, 21, 255−261. (3) Savitski, M. M.; Nielsen, M. L.; Zubarev, R. A. Mol. Cell. Proteomics 2006, 5, 935−948. (4) Molina, H.; Horn, D. M.; Tang, N.; Mathivanan, S.; Pandey, A. Proc. Natl. Acad. Sci. U.S.A 2007, 104, 2199−2204. (5) Daly, N. L.; Hoffmann, R.; Otvos, L.; Craik, D. J. Biochemistry 2000, 39, 9039−9046. (6) Chi, A.; Huttenhower, C.; Geer, L. Y.; Coon, J. J.; Syka, J. E. P.; Bai, D. L.; Shabanowitz, J.; Burke, D. J.; Troyanskaya, O. G.; Hunt, D. F. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 2193−2198. (7) Sweet, S. M. M.; Mardakheh, F. K.; Ryan, K. J. P.; Langton, A. J.; Heath, J. K.; Cooper, H. J. Anal. Chem. 2008, 80, 6650−6657. (8) Langlais, P.; Mandarino, L. J.; Yi, Z. J. Am. Soc. Mass Spectrom. 2010, 21, 1490−1499. (9) Cunningham, D. L.; Sweet, S. M. M.; Cooper, H. J.; Heath, J. K. J. Proteome Res. 2010, 9, 2317−2328. (10) Sohn, C. H.; Chung, C. K.; Yin, S.; Ramachandran, P.; Loo, J. A.; Beauchamp, J. L. J. Am. Chem. Soc. 2009, 131, 5444−5459. (11) Winter, D.; Pipkorn, R.; Lehmann, W. D. J. Sep. Sci. 2009, 32, 1111−1119. (12) Palumbo, A.; Reid, G. E. Anal. Chem. 2008, 80, 9735−9743. (13) Xuan, Y.; Creese, A. J.; Horner, J. A.; Cooper, H. J. Rapid Commun. Mass Spectrom. 2009, 23, 1963−1969. (14) Singer, D.; Kuhlmann, J.; Muschket, M.; Hoffman, R. Anal. Chem. 2010, 82, 6409−6414. (15) Ibrahim, Y.; Shvartsburg, A. A.; Smith, R. D.; Belov, M. E. Anal. Chem. 2011, 83, 5617−5623. (16) Shvartsburg, A. A.; Creese, A. J.; Smith, R. D.; Cooper, H. J. Anal. Chem. 2010, 82, 8327−8334. (17) Shvartsburg, A. A.; Singer, D.; Smith, R. D.; Hoffmann, R. Anal. Chem. 2011, 83, 5078−5085. (18) Creese, A. J.; Cooper, H. J. Anal. Chem. 2012, 84, 2597−2601. (19) Guevremont, R. J. Chromatogr., A 2004, 1058, 3−19. (20) Shvartsburg, A. A. Differential Ion Mobility Spectrometry: Nonlinear Ion Transport and Fundamentals of FAIMS; CRC Press: Boca Raton, FL, 2008. (21) Shvartsburg, A. A.; Tang, K.; Smith, R. D. Anal. Chem. 2010, 82, 32−35. (22) Guevremont, R.; Barnett, D. A.; Purves, R. W.; Vandermey, J. Anal. Chem. 2000, 72, 4577−4584. (23) Tang, K.; Li, F.; Shvartsburg, A. A.; Strittmatter, E. F.; Smith, R. D. Anal. Chem. 2005, 77, 6381−6388. (24) Shvartsburg, A. A.; Prior, D. C.; Tang, K.; Smith, R. D. Anal. Chem. 2010, 82, 7649−7655. (25) Shvartsburg, A. A.; Smith, R. D. Anal. Chem. 2011, 83, 23−29. (26) Shvartsburg, A. A.; Smith, R. D. Anal. Chem. 2011, 83, 9159− 9166. (27) Jenuwein, T.; Allis, C. D. Science 2001, 293, 1074−1080. (28) Strahl, B. D.; Allis, C. D. Nature 2000, 403, 41−45. 4276
dx.doi.org/10.1021/ac300612y | Anal. Chem. 2012, 84, 4271−4276
