Electrospray Ionization Mass Spectrometry of Highly Heterogeneous

Aug 23, 2010 - Ion Charge State Assignment via Incomplete .... strongly denaturing conditions failed (it was impossible to resolve the individual char...
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Letters to Analytical Chemistry Electrospray Ionization Mass Spectrometry of Highly Heterogeneous Protein Systems: Protein Ion Charge State Assignment via Incomplete Charge Reduction Rinat R. Abzalimov and Igor A. Kaltashov* Department of Chemistry, University of Massachusetts-Amherst, Amherst, Massachusetts 01003 Correct mass and charge assignment for large highly heterogeneous macromolecular ions (e.g., large glycoproteins with significant carbohydrate content) presents a great challenge in native electrospray ionization mass spectrometry (ESI MS). A new approach to this problem combines complexity reduction (mass-selection of a narrow distribution of ionic species from a heterogeneous mixture) and gas-phase ion chemistry (electron-transfer reactions) to induce partial reduction of the ionic charge. The resulting spectra are devoid of complexity and are easy to interpret, leading to correct mass assignment. The new method is tested using several glycoproteins and their complexes, for which standard deconvolution approaches do not work. Characterization of intact biopolymers and macromolecular assemblies using electrospray ionization mass spectrometry (ESI MS) has become an indispensable tool in structural biology, as meaningful data can be obtained for complexes in excess of 1 MDa.1 However, even in the case of relatively homogeneous species (e.g., ESI MS analysis of proteins expressed in bacteria), ESI MS data interpretation becomes considerably more challenging with the increase of physical size. One of the major contributing factors is the noncovalent adduct formation, which leads to ion peak broadening and less-than-desirable resolution. Several methods have been introduced in recent years that deal with this problem, most of which rely upon finding the best fit for the experimental data using various computational algorithms.2-4 While these methods have been utilized successfully to interpret ESI MS data of homogeneous proteins and protein complexes, they are not readily applicable to large heterogeneous * To whom correspondence should be addressed. Igor A. Kaltashov Department of Chemistry University of Massachusetts-Amherst 710 North Pleasant Street, LGRT 701 Amherst, MA 01003. Phone: (413) 545-1460. Fax: (413) 5454490. E-mail: [email protected]. (1) Heck, A. J. R. Nat. Methods 2008, 5, 927–933. (2) McKay, A. R.; Ruotolo, B. T.; Ilag, L. L.; Robinson, C. V. J. Am. Chem. Soc. 2006, 128, 11433–11442. (3) Liepold, L.; Oltrogge, L. M.; Suci, P. A.; Young, M. J.; Douglas, T. J. Am. Soc. Mass Spectrom. 2009, 20, 435–442. (4) Hernandez, H.; Robinson, C. V. Nat. Protoc. 2007, 2, 715–726. 10.1021/ac101848z  2010 American Chemical Society Published on Web 08/23/2010

systems. The upper mass limit for heterogeneous biopolymers (e.g., glycoproteins) for which meaningful ESI MS data can be acquired is dependent on the extent of heterogeneity. The structural heterogeneity of glycoproteins with high carbohydrate content and other polydisperse macromolecules poses a serious challenge to ESI MS analysis for two reasons. First, the population of ionic species with different masses distributed over a relatively narrow m/z range often gives rise to unresolved or poorly resolved ionic signals and may lead to significant overlap of ion peaks representing different charge states. Second, the extent of multiple charging in ESI MS depends on the physical size of macromolecules.5-7 In the case of polydisperse systems, this may result in a situation when the measured average ionic mass (or mass distribution) depends on its charge, which obviously complicates deconvolution of ESI MS data. Currently, there are no reliable methods for extracting meaningful information from such mass spectra short of enzymatic removal of carbohydrate chains. While partial protein deglycosylation does provide an opportunity to reduce complexity in ESI mass spectra and therefore enhance the information content, it may alter protein solubility and other properties to a very significant extent.8 Unlike ESI, matrix-assisted laser desorption ionization (MALDI) produces low charge density ions (mostly singly charged), making the interpretation of the MS data of glycoproteins easier. However, noncovalent complexes typically do not survive the MALDI process. Preservation of noncovalent complexes in MALDI MS using chemical cross-linking has met with only limited success due to the low yields of the cross-linking products and uncertainty regarding the binding stoichiometry.9 Finally, methods employing charge reduction schemes based on proton-transfer reactions were demonstrated to enhance the mass (5) Kaltashov, I. A.; Mohimen, A. Anal. Chem. 2005, 77, 5370–5379. (6) Konermann, L. J. Phys. Chem. B 2007, 111, 6534–6543. (7) Kaltashov, I. A.; Abzalimov, R. R. J. Am. Soc. Mass Spectrom. 2008, 19, 1239–1246. (8) Byrne, S. L.; Leverence, R.; Klein, J. S.; Giannetti, A. M.; Smith, V. C.; MacGillivray, R. T. A.; Kaltashov, I. A.; Mason, A. B. Biochemistry 2006, 45, 6663–6673. (9) Pimenova, T.; Pereira, C. P.; Schaer, D. J.; Zenobi, R. J. Sep. Sci. 2009, 32, 1224–1230.

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resolution by generating low charge density ions of nucleic acids10 and proteins.11 More recently, a similar approach was applied to highly heterogeneous populations of protein-polymer conjugates, with significant reduction of their charge states induced by proton transfer reactions occurring in the ESI interface region.12 This method, however, is unlikely to become practical in the field of native mass spectrometry, where significant reduction of the ionic charge density would push the signal to a very high m/z range, which is out of the reach of most commercially available mass analyzers. In this work, we present an alternative approach to the problem of ESI MS data interpretation for highly heterogeneous biopolymers, which is based on mass selecting a narrow distribution of ionic species followed by their incomplete charge reduction using fluoranthene, a standard reagent for electron transfer dissociation (ETD). The progeny ions have the same narrow mass distribution as the parent ions, but their charges differ from parent ions by one unit, giving rise to very distinct and easily interpretable signals in mass spectra. In some instances, a second generation of progeny ions are also seen, where the ionic charge is reduced by two units. The utility of the new method is demonstrated using extensively glycosylated proteins R-galactosidase (52 kDa, carbohydrate content 12% by weight) and human haptoglobin 1-1 (90.6 kDa, carbohydrate content 19%), as well as the haptoglobinhemoglobin complex. EXPERIMENTAL SECTION Human hemoglobin and haptoglobin 1-1 were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO), and human R-galactosidase was generously provided by Shire Human Genetic Therapies, Inc. (Cambridge, MA). All other chemicals were of analytical grade or higher. Protein solutions for ESI MS analyses were prepared in 50 mM ammonium acetate to a final concentration of 1-3 µM. All full-range ESI MS data were acquired with a QStar-XL (ABI-Sciex, Toronto, Canada) quadrupole-TOF mass spectrometer equipped with a nano-ESI source. Fitting of the experimental data to obtain ionic mass and charge information was carried out using a procedure developed by McKay et al.2 Protein ion charge reduction was carried out with a SolariX 70 (Bruker Daltonics, Billerica, MA) Fourier transform ion cyclotron resonance (FTICR) MS. Mass selection was carried out in a frontend quadrupole analyzer and limited charge reduction was induced by allowing the mass-selected protein polycations to react with fluoranthene anions for 6-10 ms, followed by ion introduction to the ICR cell. RESULTS AND DISCUSSION Human R-galactosidase is a glycoprotein which exhibits a high degree of structural heterogeneity due to significant carbohydrate content (∼12% of total weight). The functional form of the protein in solution is a noncovalent homodimer. Despite the high signalto-noise ratio, interpretation of the ESI MS data is not straightforward due to the poorly defined and partially overlapping peaks corresponding to different charge states (Figure 1). Deconvolution of this mass spectrum using standard software provided by the (10) McLuckey, S. A.; Goeringer, D. E. Anal. Chem. 1995, 67, 2493–2497. (11) Stephenson, J. L., Jr.; McLuckey, S. A. J. Mass Spectrom. 1998, 33, 664– 672. (12) Bagal, D.; Zhang, H.; Schnier, P. D. Anal. Chem. 2008, 80, 2408–2418.

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Figure 1. ESI MS of R-galactosidase (3 µM in 50 mM ammonium acetate) acquired with a hybrid quadrupole-TOF MS (gray trace) and the results of incomplete charge reduction of a narrow population of protein ions mass-selected from one of the charge states (black trace). A mass spectrum of the denatured protein (in 50/47/3 water/methanol/ acetic acid by volume) is shown in the inset.

instrument manufacturer (Analyst) and the algorithm described by McKay et al.2 leads to the charge state assignment for the most abundant peak as +24 and the ionic mass as 126 kDa, while the expected mass of the protein dimer is 104 kDa. This discrepancy could be accounted for by any of the following: (i) significant deviation of the protein mass from that calculated based on the published sequence and glycosylation pattern, (ii) presence of yet unknown high-mass cofactor(s) in the dimer, and (iii) incorrect charge state assignment. Although an attempt to verify the monomer mass by acquiring ESI MS of R-galactosidase under strongly denaturing conditions failed (it was impossible to resolve the individual charge states, see inset in Figure 1), MALDI MS analysis did yield 51.9 kDa as the average monomer mass, thereby narrowing down the possibilities mentioned above to (ii) and (iii). We approached the task of charge assignment by reducing the complexity of the ESI MS data. This was accomplished by mass-selecting a very narrow subpopulation of R-galactosidase ions in the gas phase followed by fluoranthene-induced limited charge reduction. A one-step ionic charge reduction can be clearly observed (Figure 1), and the charge states of both precursor and progeny ions can be confidently assigned as +21 and +20 (based on their m/z values), which corresponds to a mass of 105 kDa. This number is only slightly higher than that expected for a homodimer, and the small observed difference is likely due to formation of nonspecific adducts, an almost universal phenomenon in native ESI MS.13 A similar approach was taken to interpret the results of ESI MS measurements of human haptoglobin 1-1 (Hp), a heavily glycosylated plasma protein (90.6 kDa, 19% carbohydrate content) and its unsaturated complex with hemoglobin (Hb) under nearnative conditions. The significant degree of structural heterogeneity is evident in native ESI mass spectra of Hp, as the ion peaks corresponding to different charge states are very broad and (13) Loo, J. A. Mass Spectrom. Rev. 1997, 16, 1–23.

partially overlap with each other (Figure 2A). Deconvolution of this mass spectrum using conventional approaches (built-in deconvolution procedure and a fitting algorithm2) resulted in assigning charge state +22 to the most abundant peak in the spectrum and yielding the average protein mass of 98 kDa, more than 6 kDa higher than the mass calculated based on the Hp amino acid sequence and glycosylation pattern. Mass-selecting a narrow population of Hp ions with subsequent incomplete charge reduction generates two more ionic subpopulations, which represent progeny ions lacking one and two charges compared to the parent ions. On the basis of the measured m/z values of the parent and progeny ions, their charge states are confidently assigned as +21, +20, and +19, which corresponds to the protein mass of 92 kDa (once again, a small increase over the calculated mass is due to formation of nonspecific adducts). A mixture of Hp and Hb presents an even more challenging case vis-a`-vis ESI MS analysis, as in addition to the significant structural heterogeneity (which manifests itself via broad, poorly resolved peaks) there are two ionic populations with partially overlapping charge state distributions (Figure 2B). While it is clear that the ion peaks in the lower m/z region correspond to free Hp molecules in solution, the ionic species populating the higher m/z region of the mass spectrum may represent a number of different Hp/Hb complexes. While Hp is known to be capable of binding either one or two Hb dimers (Rβ),14 a complex with three globin chains bound to a single Hp molecule was observed alongside

“canonical” complexes Rβ · Hp and (Rβ)2 · Hp in a recent study using a combination of chemical cross-linking and high-mass MALDI MS.9 However, this Hb/Hp complex with unusual stoichiometry may be an artifact caused by incomplete chemical cross-linking of the proteins followed by dissociation of noncovalent complexes in the MALDI source. In order to verify the stoichiometry of Hb/Hp noncovalent complexes observed in ESI MS (Figure 2B), a narrow population of ionic species around m/z 5000 were mass-selected followed by ETD-induced charge reduction. The m/z value of the progeny ions (representing the most abundant species in the original ESI MS) provides unequivocal evidence that their charge state is +24. The mass of the Hp/Hb complex, calculated based on this charge assignment, is 124.7 kDa. This number is only slightly higher than the calculated mass of the Rβ · Hp complex (123 kDa), and the difference between the two values is clearly within the range expected for nonspecific adduct formation during the ESI process. It is important to note that interaction of protein ions with fluoranthene does not lead to their dissociation. Indeed, it has been noted that even though electron-based dissociation methods are very effective when applied to peptides and unfolded proteins, fragmentation yields of low charge density protein ions (produced by ESI under native conditions) are extremely low. This is most likely due to the stabilizing effect of multiple hydrogen bonds, which prevent physical separation of fragments in the gas phase even if the backbone cleavages do occur.15 It is not therefore surprising that neither monomeric proteins nor their complexes fragment under the conditions employed in our work. Another very interesting question that arises here is the reason for the failure of the standard curve-fitting methods in obtaining the charge state assignment of extensively glycosylated protein ions in ESI MS. We note that these methods implicitly assume constancy of the mass profile of all ions irrespective of their charge.2 While this is a very reasonable assumption when it comes to relatively homogeneous proteins, the situation is much more complicated in the case of highly heterogeneous systems. The extent of multiple charging of macromolecules in ESI MS strongly depends on their physical size in solution.5-7 Carbohydrate chains in glycoproteins provide additional sites that can accommodate extra charges during the ESI process. Therefore, more extensive glycosylation favors a higher number of charges, giving rise to the apparent dependence of the ionic mass on its charge. Indeed, the masses of Hp ions based on the apexes of their peaks in mass spectra shown in Figure 2 increase monotonically with the increase of charge (see the Supporting Information for more detail). Interestingly, a similar trend is observed in the negative ion ESI mass spectrum of Hp, which is better resolved due to the lower number of charges (see the Supporting Information). In this spectrum, the relative intensity of the ion signal corresponding to higher mass glycoforms clearly shows a monotonic increase as a function of the protein ion charge state. The new method of charge state assignment presented in this work is insensitive to this problem, since only a narrow distribution of protein ions is mass-selected for consequent charge reduction.

(14) Ascenzi, P.; Bocedi, A.; Visca, P.; Altruda, F.; Tolosano, E.; Beringhelli, T.; Fasano, M. IUBMB Life 2005, 57, 749–759.

(15) Breuker, K.; Jin, M.; Han, X.; Jiang, H.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 2008, 19, 1045–1053.

Figure 2. ESI MS of Hp (A) and Hp/Hb mixture at 2:1 molar ratio (B) acquired with a hybrid quadrupole-TOF MS (gray trace). The results of incomplete charge reduction of mass-selected narrow populations of protein ions are shown in black.

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CONCLUSIONS Straightforward application of deconvolution strategies to interpret ESI MS data for large extensively glycosylated proteins and their complexes may fail due to apparent dependence of the extent of multiple charging of macromolecular ions in ESI MS on their physical size. Our approach to deconvoluting the ESI MS data for such heterogeneous systems combines complexity reduction (by mass-selecting narrow distribution of ionic species from a heterogeneous mixture) and gas-phase chemistry (electron transfer reactions) to reduce the ionic charge states. The resulting spectra are devoid of complexity and are easy to interpret, leading to correct ionic charge assignment and protein mass calculation. In addition to glycoproteins, this method should become a useful tool aiding interpretation of the results of ESI MS studies for a large variety of other highly heterogeneous systems, such as functionalized nanoparticles and protein-polymer conjugates. Native ESI MS and hydrogen exchange (HDX) are arguably the two most popular techniques to study protein higher order structure and dynamics in solution. Methods of gas-phase chem(16) Kaltashov, I. A.; Bobst, C. E.; Abzalimov, R. R. Anal. Chem. 2009, 81, 7892– 7899.

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istry, particularly electron-based dissociation techniques, such as ETD, have already become a part of the experimental arsenal of hydrogen deuterium exchange (HDX) MS.16 This work demonstrates that they also have a great potential as a tool aiding interpretation of the results of native ESI MS experiments. ACKNOWLEDGMENT This work was supported by NSF Grant CHE-0750389. The FTICR MS was acquired with support of the NSF Major Research Instrumentation Grant CHE-0923329. We are grateful to Drs. Philip Savickas and John Thomas (Shire Human Genetic Therapies) for providing human R-galactosidase. 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 12, 2010. Accepted August 17, 2010. AC101848Z