LETTER pubs.acs.org/ac
Spatially Resolved Protein Hydrogen Exchange Measured by Matrix-Assisted Laser Desorption Ionization In-Source Decay Kasper D. Rand,*,† Nicolai Bache, Morten M. Nedertoft, and Thomas J. D. Jørgensen* Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
bS Supporting Information ABSTRACT: Mass spectrometry has become a powerful tool for measuring protein hydrogen exchange and thereby reveal the structural dynamics of proteins in solution. Here we describe the successful application of a matrix-assisted laser desorption ionization (MALDI) mass spectrometry approach based on in-source decay (ISD) to measure spatially resolved amide backbone hydrogen exchange. By irradiating deuterated protein molecules in a crystalline matrix with a high laser fluence, they undergo prompt fragmentation. Spatially resolved deuteration levels are readily obtained by mass analysis of consecutive fragment ions. MALDI ISD analysis of deuterated cytochrome c yielded an extensive series of c-fragment ions which originate from cleavage of nearly all N Cα bonds (Cys17 to Glu104) allowing for a detailed analysis of the deuterium content of the backbone amides. While hydrogen scrambling can be major concern when using mass spectrometric fragmentation to obtain detailed information on protein hydrogen exchange, we show that the level of hydrogen scrambling in our MALDI ISD measurements is negligible and that the known dynamic behavior of cytochrome c in solution is accurately reflected in the deuterium contents of the fragment ions. The developed method combines several attractive features from a practical point of view as it is simple to perform and it readily provides a detailed mapping of the dynamic structure of a protein in solution.
P
rotein structures are dynamic by nature and they undergo structural changes to carry out their biological function. The conformational dynamics of enzymes during catalysis and the allosteric regulation of enzyme activity are well-know examples of the biological importance of the structural flexibility of proteins.1 Recently, amide hydrogen (1H/2H) exchange monitored by mass spectrometry (HX-MS) has become an important method for the characterization of protein structural dynamics.2,3 For example, it has been utilized to map structural responses upon allosteric activation of various enzymes.4,5 The basis of this method is that backbone amide hydrogens that are engaged in hydrogen bonds are protected against isotopic exchange with the solvent. Structural fluctuations and local unfolding events that transiently break these hydrogen bonds cause exposure of the amide hydrogens to the solvent and this leads to exchange.6 When a protein is incubated in D2O, its global deuterium uptake is readily monitored by mass spectrometry7 as each incorporated deuteron causes a mass increase of 1 Da. To obtain local information on the deuterium incorporation, the labeled protein is typically digested by pepsin at cold acidic conditions where the amide hydrogen exchange reaction is quenched (i.e., pH ∼2.5 and 0 °C).8 The resulting peptides are maintained at quench conditions and analyzed either directly by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry9 or separated by reversed-phase chromatography and analyzed by online electrospray ionization mass spectrometry. To minimize the unavoidable deuterium loss that occurs as a result of back-exchange with the solvents at quench conditions, the experiments should be performed as quickly as possible. r 2011 American Chemical Society
The spatial resolution of either the MALDI or the electrospray ionization (ESI) approach was until recently on the peptide level, although overlapping peptide sequences could increase the resolution to yield site-specific deuterium levels for typically a few residues in a protein. We have recently demonstrated the utility of gas-phase electron-based fragmentation to obtain sitespecific deuterium levels in model peptides10 12 and in a protein.13 The latter study employed pepsin digestion of the labeled protein followed by electron transfer dissociation of the deuterated peptic peptides (i.e, a bottom-up approach). Other investigators have utilized electron-based fragmentation of intact labeled proteins to obtain spatially resolved information on the deuterium incorporation (i.e., a top-down approach).14 20 Importantly, these mass spectrometry experiments are carried out at conditions where the deuterium labeling pattern of the backbone amides is unperturbed from solution into the gas-phase. In this regard, it is crucial to avoid excessive vibrational excitation of the precursor ions (peptides or proteins) in the mass spectrometer as this leads to hydrogen scrambling (i.e., a positional randomization of all hydrogen and deuterium atoms attached to nitrogen or oxygen).21 The use of MALDI-TOF-MS to measure protein hydrogen exchange has several attractive features from a practical point of view as it is very simple to perform and the analysis can be carried out in a very short time. However, no MALDI-MS method has been introduced with the potential to measure Received: September 17, 2011 Accepted: November 4, 2011 Published: November 04, 2011 8859
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Figure 2. Dissecting the solution structural dynamics of cytochrome c by MALDI-TOF in-source decay. Deuterium levels of the c-type fragment ions of cytochrome c after native state D-to-H exchange for 20 min measured by MALDI in-source decay (b) and NMR (red line). Error bars represent average ( s.d. for measurements made in triplicate. NMR deuterium levels were obtained from exchange rate constants determined by Milne et al.6 The theoretical deuterium content in the case of 100% gas phase H/D scrambling is indicated (green line). Figure 1. MALDI-TOF in-source decay (ISD) mass spectra of cytochrome c. The asterisked peaks in panel a correspond to c-type fragment ions obtained from nondeuterated cytochrome c, and the base peak corresponds to doubly protonated intact cytochrome c. Panel b shows the fragment ions c35, c36, c37, and c38 obtained from nondeuterated cytochrome c, while panel c shows the corresponding fragment ions obtained from labeled cytochrome c (D to H exchange for 20 min).
hydrogen exchange of individual residues in a protein. Herein we present a novel top-down MALDI fragmentation approach for investigating protein hydrogen exchange at a spatial resolution approaching single-residue resolution. In MALDI, a pulsed UV laser irradiates a crystalline layer of matrix molecules in which analyte molecules are embedded.22 Laser irradiation of the matrix crystals doped with peptide or protein causes a rapid phase transition and forms an expanding plume of neutrals and ions. Intact protein or peptide ions are produced by this process. However, by increasing the laser fluence somewhat above the threshold value for ion formation, protein ions fragment by specific cleavage of the N Cα bond in the polypeptide backbone yielding primarily c and z fragment ions.23,24 This fragmentation process is known as MALDI in source decay (ISD). This type of fragmentation is distinct from the commonly employed post source decay (PSD), which is based on unimolecular decomposition of vibrationally excited ions fragmentation of the peptide bond yielding b and y fragment ions. The backbone dissociation mechanism of ISD is thought to involve a hydrogen radical transfer from the matrix molecule to the protein ion.25 27 Fragmentation occurs on a nanosecond time scale, and such a rapid dissociation causes the protein fragment ions to obtain the same kinetic energy as the intact protein ions after acceleration in the ion source (the mass calibration of the precursor ions is therefore also valid for the fragment ions).28,29 We have previously
utilized model peptides to show that MALDI ISD proceeds with very limited hydrogen scrambling.30 Here we demonstrate the potential of MALDI ISD as a straightforward experimental tool for obtaining local information on the hydrogen exchange kinetics of proteins. We have chosen cytochrome c as a reference protein for the present study as its site-specific amide hydrogen exchange rate constants are known.6 Native-state D-to-H exchange of oxidized cytochrome c was carried out by incubating fully deuterated protein in 50-fold (v/v) excess of 1H2O ammonium acetate buffer at neutral pH for 20 min at 20 °C. Under these conditions, the most stable regions in cytochrome c are protected against isotopic exchange with the solvent and they will retain their deuterium, while the more dynamic and flexible regions undergo exchange. After 20 min, the isotopic exchange reaction of backbone amides was quenched by acidification and the sample was kept frozen until MALDI ISD analysis. Mass spectra were acquired in positive linear mode using a high-vacuum UV MALDI TOF mass spectrometer and sinapinic acid as the MALDI matrix. A detailed experimental description is given in the Supporting Information. MALDI ISD analysis of nonlabeled cytochrome c yields an extensive series of c fragment ions which originate from cleavage of nearly all N Cα bonds from Cys17 to the C-terminal residue Glu104 (Figure 1a). This allows for a detailed analysis of the deuterium content of backbone amides in the protein. The retained deuterium can be measured directly by the mass increase of the c-ions obtained from MALDI ISD analysis of D-to-H exchanged cytochrome c (compare parts b and c of Figure 1). The deuterium content of the c-ion series is plotted in Figure 2. An excellent correlation is observed between the deuterium content of the c-ion series and the graph showing the cumulative deuterium content obtained from known NMR exchange rates (compare filled circles with red graph in Figure 2). The cumulative deuterium graph exhibits a distinctive profile, where the 8860
dx.doi.org/10.1021/ac202468v |Anal. Chem. 2011, 83, 8859–8862
Analytical Chemistry nearly horizontal portions of the graph correspond to highly dynamic regions in cytochrome c that are nearly devoid of deuterium, while the protected regions retain deuterium and exhibit a nearly linear increase in their D-content. The various regions with different dynamics correspond to recognizable secondary structural elements of the protein. For example, the flat portion from c39 to c56 encompasses an omega loop (highlighted in yellow in Figure 2) which is one of the most dynamic regions in cytochrome c.31 In contrast, the 60s helix (highlighted in blue in Figure 2) belongs to the second-most stable structural elements in cytochrome c and hence deuterium is retained in this region indicated by the increase in cumulative D-content from c62 to c69. A similar behavior is observed for the N- and C-terminal helices which are the most stable structural elements in cytochrome c.31 The known dynamic behavior of cytochrome c in solution is thus readily apparent in our HX-ISD measurements. Hydrogen scrambling is a major concern when using mass spectrometric fragmentation as an experimental tool to obtain detailed information on deuterium levels in proteins and peptides.21 However, the level of hydrogen scrambling in our MALDI measurements is negligible, as our experimental data closely mimic the solution deuterium pattern and not the theoretical graph for 100% scrambling (Figure 2). Cleavage of the backbone between Cys14 and Cys17 does not yield any fragment ions as the heme-group of cytochrome c covalently bridges these cysteine residues. Similarly, cleavage of the N Cα bond at any proline residues does not result in the formation of c-ions due to the cyclic nature of the proline side chain (c29, c43, c70, and c75 are therefore not observed). A few c-ions were omitted from the data analysis due to the presence of interfering ions. For example, the fragment ion c32 (theoretical m/z 4124.6) was not included in the data analysis as the peak corresponding to c32 overlaps with the peak corresponding to triply protonated cytochrome c (theoretical m/z 4121.0). MALDI ISD experiments require a somewhat higher laser fluence which increases the mass measurement uncertainty for the type of MALDI TOF mass spectrometer employed in the present study. The uncertainty increases with the c-ion size. In our case, the upper m/z limit for useable ions is approximately m/z 8200 (corresponding to c68). It should be noted, however, that the increased laser fluence in MALDI ISD experiments should not affect the mass measurement uncertainty for other types of MALDI mass spectrometers where the mass analysis is uninfluenced by the initial kinetic energy distribution (e.g., MALDI quadrupole-TOF, MALDI-Orbitrap, MALDI-ion trap mass spectrometers). In HX-MS experiments, an unavoidable loss of deuterium occurs when the sample is handled at quench conditions (i.e., back exchange). To measure the level of back exchange occurring during the described HX-ISD experiments on cytochrome c, a fully deuterated sample (100% D control) was prepared and analyzed by MALDI ISD. The deuterium content of c-type fragment ions obtained from MALDI ISD analysis of the 100% D control sample is shown in Figure 3. The measured deuterium level exhibits a nearly linear increase with c-ion size as expected for a uniform labeling with an average deuterium loss of 30%. This level of back exchange is due to the combined effect of the experimental quench conditions (i.e., temperature, pH, solvent composition, the presence of sinapinic acid, crystallization time, and total analysis time). A 30% loss of deuterium is comparable to protocols in which traditional ESI or MALDI MS approaches are used to measure protein hydrogen exchange.
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Figure 3. Determining the inadvertent loss of deuterium during MALDI-TOF in-source decay analysis. Deuterium levels of the c-type fragment ions obtained from fully deuterated cytochrome c (100% D control) is plotted with the red graph indicating the cumulative deuterium content of backbone amides assuming an average deuterium loss of 30% per residue. Error bars represent average ( s.d. for measurements made in triplicate.
In the present study, we have demonstrated that the fragmentation of proteins by MALDI ISD proceeds with a negligible level of gas phase hydrogen scrambling. Furthermore, we have shown a novel application of MALDI mass spectrometry for providing a detailed map of protein dynamics. We envision that the use of other recently introduced matrixes capable of producing alternate cleavages of the polypeptide backbone (such as the Cα C bond) during ISD32 could further improve the ability to extract spatially resolved hydrogen exchange information for proteins in solution using MALDI ISD.
’ ASSOCIATED CONTENT
bS
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] (K.D.R.);
[email protected] (T.J.D.J.). Present Addresses †
Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, DK-2100 Copenhagen Ø, Denmark.
’ ACKNOWLEDGMENT We thank Prof. Ole N. Jensen for supporting and encouraging this work. We gratefully acknowledge financial support by The Danish Council for Independent Research in Natural Sciences (FNU Grant No. 09-063876, K.D.R. and FNU Grant No. 09-070686, T.J.D.J.) and the Human Frontiers Science Program (K.D.R). 8861
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