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Gas-Phase Fragmentation of Peptides by MALDI in-Source Decay with Limited Amide Hydrogen ( 1 H/ 2 H) Scrambling
Abstract
To achieve a fundamental understanding of the function of proteins and protein complexes at the molecular level, it is crucial to obtain a detailed knowledge about their dynamic and structural properties. The kinetics of backbone amide hydrogen exchange is intimately linked to the structural dynamics of the protein, and in recent years, the monitoring of the isotopic exchange of these hydrogens by mass spectrometry has become a recognized method. At present, the resolution of this method is, however, limited and single-residue resolution is typically only obtained for a few residues in a protein. It would therefore be desirable if gas-phase fragmentation could be used to localize incorporated deuterons as this would ultimately lead to single-residue resolution. A central obstacle for this approach is, however, the occurrence of intramolecular migration of amide hydrogens upon activation of the gaseous protein (i.e., hydrogen scrambling). Here we investigate the occurrence of scrambling in selectively labeled peptides upon fragmentation by matrix-assisted laser desorption/ionization in-source decay (MALDI ISD). We have utilized peptides with a unique regioselective deuterium incorporation that allows us to accurately determine the extent of scrambling upon fragmentation. Our results show that the level of scrambling upon MALDI ISD is so low that the solution deuteration pattern is readily apparent in the gas-phase fragment ions. These results suggest that MALDI ISD may prove useful for hydrogen exchange studies of purified peptides and small proteins.
... 235,236 A few notable exceptions have been studies using very fast activation times (10 ns or less), such as in-source decay with MALDI mass spectrometry, to induce fragmentation before scrambling can take place. 237 Despite the inability to report sitespecific deuterium incorporation, HDX-MS with CID MS/MS has still proven useful. Percy et al. showed that one way to expand peak capacity for HDX-MS is to perform CID fragmentation and measure deuterium levels of fragment ions. ...
Solution-phase hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) is a widespread tool for structural analysis across academia and the biopharmaceutical industry. By monitoring the exchangeability of backbone amide protons, HDX-MS can reveal information about higher-order structure and dynamics throughout a protein, can track protein folding pathways, map interaction sites, and assess conformational states of protein samples. The combination of the versatility of the hydrogen/deuterium exchange reaction with the sensitivity of mass spectrometry has enabled the study of extremely challenging protein systems, some of which cannot be suitably studied using other techniques. Improvements over the past three decades have continually increased throughput, robustness, and expanded the limits of what is feasible for HDX-MS investigations. To provide an overview for researchers seeking to utilize and derive the most from HDX-MS for protein structural analysis, we summarize the fundamental principles, basic methodology, strengths and weaknesses, and the established applications of HDX-MS while highlighting new developments and applications.
... Currently, the size of peptic fragments generated by proteolysis control the resolution in a typical HDX-MS study. Four different approaches potentially further improve the resolution of HDX-MS data; (1) sublocalization of deuterium by subtracting the deuterium incorporations in two analogous peptic fragments utilizing non-specificity of acid proteases [6,[8][9][10], (2) development of new software that can utilize the isotope envelope shape of an peptide instead of the centroid value [7,11], (3) development of new proteases to cut the analyte protein at different sites [5,[12][13][14][15], and (4) sub-localization of deuterium within a peptide by gas-phase fragmentation [16][17][18][19][20][21][22][23]. ...
Protein backbone amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) typically utilizes enzymatic digestion after the exchange reaction and before MS analysis to improve data resolution. Gas-phase fragmentation of a peptic fragment prior to MS analysis is a promising technique to further increase the resolution. The biggest technical challenge for this method is elimination of intramolecular hydrogen/deuterium exchange (scrambling) in the gas phase. The scrambling obscures the location of deuterium. Jørgensen’s group pioneered a method to minimize the scrambling in gas-phase electron capture/transfer dissociation. Despite active investigation, the mechanism of hydrogen scrambling is not well-understood. The difficulty stems from the fact that the degree of hydrogen scrambling depends on instruments, various parameters of mass analysis, and peptide analyzed. In most hydrogen scrambling investigations, the hydrogen scrambling is measured by the percentage of scrambling in a whole molecule. This paper demonstrates that the degree of intramolecular hydrogen/deuterium exchange depends on the nature of exchangeable hydrogen sites. The deuterium on Tyr amide of neurotensin (9–13), Arg-Pro-Tyr-Ile-Leu, migrated significantly faster than that on Ile or Leu amides, indicating the loss of deuterium from the original sites is not mere randomization of hydrogen and deuterium but more site-specific phenomena. This more precise approach may help understand the mechanism of intramolecular hydrogen exchange and provide higher confidence for the parameter optimization to eliminate intramolecular hydrogen/deuterium exchange during gas-phase fragmentation.
... e subsequent MALDI-ISD to form c-ions is completed within several tens of nanoseconds or less without intramolecular hydrogen scrambling in the MALDI ion-source. 9,53) According to the mechanism of intermolecular hydrogen transfer for MALDI-ISD presented in Schemes 8 and 9, Demeure et al. have attempted to present a strategy for the rational selection of the optimum MALDI-ISD matrix materials, 18) and they determined the hydrogen-donating characteristics of several matrices and gave the order of the ability, i.e., picolinic acid (PA)>1,5-diaminonaphthalene (1,5-DAN)>2,5-DHB>sinapinic acid (SA)>α-cyano-4hydroxy-cinnamic acid (CHCA). We also reported on the hydrogen-donating properties of matrix materials as follows, 1,5-DAN>5-amino-salicylic acid (5-ASA)>2,5-DHB>SA≒CHCA. 54) Demeure et al. applied a mechanistic model (Scheme 9) to top-down proteomics using MALDI-ISD MS. 18,55) It should be emphasized from the experimental and rational results described above that the role of the X-ray crystallography and SPM analysis of matrix crystals is of great importance for developing a model construction of MALDI-ISD. ...
The in-source decay (ISD) phenomenon, the fragmentation at an N–Cα bond of a peptide backbone that occurs within several tens of nanoseconds in the ion-source in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), is discussed from the standpoints of the discovery and early publications dealing with MALDI-ISD, the formation of c-ions in energy-sudden desorption/ionization methods, the formation of radical species in a MALDI, model construction for ISD, and matrix materials that are suitable for use in MALDI-ISD. The formation of c-ions derived from peptides and proteins in MALDI-ISD can be rationalized by a mechanism involving intermolecular hydrogen transfer, denoted as the “Takayama’s model” by De Pauw’s group (Anal. Chem. 79: 8678–8685, 2007). It should be emphasized that the model for MALDI-ISD was constructed on the basis of X-ray crystallography and scanning probe microscopy (SPM) analyses of matrix crystals, as well as the use of isotopically-labelled peptides.
... It is also interesting that the ISD occurring within 53 ns in the MALDI ion source does not result in the loss of a phosphate group from either β-casein and its model peptide (Figures 1 and 3). This indicates that the ISD events that occur within 53 ns in the ion source are not a result originated from vibronic energy randomization resulting in an ergodic cleavage as pointed out by Lennon et al. [17], and that the ISD is mainly characterized by a prompt non-ergodic reaction without hydrogen scrambling [18, 19]. It is also of importance to recognize that the MALDI-ISD is essentially an RIF reaction of hydrogen-abundant neutral species [M + H]@BULLET and occurs independently of the ionization (protonation/deprotonation ) process [20]. ...
The fragment ions observed with time-of-flight (TOF) and quadrupole ion trap (QIT) TOF mass spectrometers (MS) combined with matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) experiments of phosphorylated analytes β-casein and its model peptide were compared from the standpoint of the residence timeframe of analyte and fragment ions in the MALDI ion source and QIT cell. The QIT-TOF MS gave fragment c-, z′-, z-ANL, y-, and b-ions, and further degraded fragments originating from the loss of neutrals such as H2O, NH3, CH2O (from serine), C2H4O (from threonine), and H3PO4, whereas the TOF MS merely showed MALDI source-generated fragment c-, z′-, z-ANL, y-, and w-ions. The fragment ions observed in the QIT-TOF MS could be explained by the injection of the source-generated ions into the QIT cell or a cooperative effect of a little internal energy deposition, a long residence timeframe (140 ms) in the QIT cell, and specific amino acid effects on low-energy CID, whereas the source-generated fragments (c-, z′-, z-ANL, y-, and w-ions) could be a result of prompt radical-initiated fragmentation of hydrogen-abundant radical ions [M + H + H]+ and [M + H – H]– within the 53 ns timeframe, which corresponds to the delayed extraction time. The further degraded fragment b/y-ions produced in the QIT cell were confirmed by positive- and negative-ion low-energy CID experiments performed on the source-generated ions (c-, z′-, and y-ions). The loss of phosphoric acid (98 u) from analyte and fragment ions can be explained by a slow ergodic fragmentation independent of positive and negative charges.
Graphical Abstract
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... The deuterium content of the gaseous c ions closely mimicked the known solution deuteration pattern of the model peptides. 40 Consequently, the level of scrambling in MALDI ISD of the peptides was low. However, it should be noted that the gas-phase data indicated the occurrence of an intraresidue H/D migration between the backbone amide of a lysine residue and its side-chain ε-amino group, but there were no indications of any inter-residue H/D migration, so the effect on the overall solution labeling pattern was negligible. ...
Conspectus Proteins are dynamic molecules that exhibit conformational flexibility to function properly. Well-known examples of this are allosteric regulation of protein activity and ligand-induced conformational changes in protein receptors. Detailed knowledge of the conformational properties of proteins is therefore pertinent to both basic and applied research, including drug development, since the majority of drugs target protein receptors and a growing number of drugs introduced to the market are therapeutic peptides or proteins. X-ray crystallography provides a static picture at atomic resolution of the lowest-energy structure of the native ensemble. There is a growing need for sensitive analytical tools to explore all of the significant molecular structures in the conformational landscape of proteins. Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has recently emerged as a powerful method for characterizing protein conformational dynamics. The basis of this method is the fact that backbone amides in stable hydrogen-bonded structures (e.g., α-helices and β-sheets) are protected against exchange with the aqueous solvent. All protein structures are dynamic, however, and eventually all of the protecting hydrogen bonds will transiently break as the protein-according to thermodynamic principles-cycles through partially unfolded states that correspond to excited free energy levels. As a result, all of the backbone amides will eventually become temporarily solvent-exposed and exchange-competent over time. Consequently, a folded protein in D2O will gradually incorporate deuterium into its backbone amides, and the kinetics of the process can be readily monitored by mass spectrometry. The deuterium uptake kinetics for the intact protein (global exchange kinetics) represents the sum of the exchange kinetics for the individual backbone amides. Local exchange kinetics is typically achieved by using pepsin digestion under quench conditions (i.e., under cold acidic conditions where the amide hydrogen exchange rate is slowed by many orders of magnitude). The ability to localize the individual deuterated residues (the spatial resolution) is determined by the size (typically ∼7-15 residues) and the number of peptic peptides. These peptides provide a relatively coarse-grained picture of the protein dynamics. A fundamental understanding of the relationship between protein function/dysfunction and conformational dynamics requires in many cases higher resolution and ultimately single-residue resolution. In this Account, we summarize our efforts to achieve single-residue deuterium levels in proteins by electron-based or laser-induced gas-phase fragmentation methods. A crucial analytical requirement for this approach is that the pattern of deuterium labeling from solution is retained in the gas-phase fragment ions. It is therefore essential to control and minimize any occurrence of gas-phase randomization of the solution deuterium label (H/D scrambling) during the MS experiment. For this purpose, we have developed model peptide probes to accurately measure the onset and extent of H/D scrambling. Our analytical procedures to control the occurrence of H/D scrambling are detailed along with the physical parameters that induce it during MS analysis. In light of the growing use of gas-phase dissociation experiments to measure the HDX of proteins in order to obtain a detailed characterization and understanding of the dynamic conformations and interactions of proteins at the molecular level, we discuss the perspectives and challenges of future high-resolution HDX-MS methodology.
... MALDI-ISD has provided unique strategies for molecular imaging, 16,17) collision-induced dissociation combined with ISD, 16,18,19) and conformation analysis of protein. 20,21) MALDI-ISD is a rapid fragmentation process which occurs within several tens of nanoseconds in the ion source, leading to speci c cleavage at the N-C α bond of the peptide backbone. e N-C α bond cleavage occurs due to hydrogen radicals released from the hydrogen-donating matrix. ...
The backbone flexibility of a protein has been studied from the standpoint of the susceptibility of amino acid residues to in-source decay (ISD) in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Residues more susceptible to MALDI-ISD, namely Xxx-Asp/Asn and Gly-Xxx, were identified from the discontinuous intense peak of c'-ions originating from specific cleavage at N-Cα bonds of the backbone of equine cytochrome c. The identity of the residues susceptible to ISD was consistent with the known flexible backbone amides as estimated by hydrogen/deuterium exchange (HDX) experiments. The identity of these flexible amino acid residues (Asp, Asn, and Gly) is consistent with the fact that these residues are preferred in flexible secondary structure free from intramolecular hydrogen-bonded structures such as α-helix and β-sheet. The MALDI-ISD spectrum of equine cytochrome c gave not only intense N-terminal side c'-ions originating from N-Cα bond cleavage at Xxx-Asp/Asn and Gly-Xxx residues, but also C-terminal side complement z'-ions originating from the same cleavage sites. The present study implies that MALDI-ISD can give information about backbone flexibility of proteins, comparable with the protection factors estimated by HDX.
... Gas-phase fragmentation of deuterium-labeled peptides or proteins by prompt dissociation techniques such as electron capture dissociation (ECD) [13,14], electron transfer dissociation (ETD) [15][16][17] or MALDI in-source decay (ISD) [18,19] provides an attractive alternate option for measuring the HDX of individual amides. For such high-resolution HDX-MS/MS type measurements, it is important to ensure that minimal vibrational excitation is experienced by the labeled peptide or protein ion prior to the prompt fragmentation event [13]. ...
This Feature describes the use of electron transfer dissociation (ETD) to analyze the hydrogen/deuterium exchange (HDX) of proteins at increased spatial resolution down to the level of individual residues. A practical overview of how to couple ETD to the classical bottom-up HDX-MS workflow is given and new options for method optimization are discussed and exemplified. In addition, the real-world applicability of the HDX-ETD method to pinpoint conformational changes in a large 75 kDa protein complex of therapeutic interest is demonstrated. This feature highlights how the conformation and interactions of complex protein systems of biological or pharmaceutical interest can now be analyzed at a hitherto unprecedented level of structural detail using an MS-based method.
... Also, the selective labeling with an N-terminal half devoid of deuterium ensures a maximal sensitivity for detecting and measuring the level of gas-phase hydrogen scrambling. Using such peptides, we have demonstrated that CID [9,[18][19][20] cause extensive hydrogen scrambling, while electron capture dissociation (ECD) [11], electron transfer dissociation (ETD) [10] and MALDI in-sourcedecay [21] proceed with a negligible level of hydrogen scrambling. These fragmentation techniques are thus available as experimental tools in either a "bottom-up" [22] or a "top-down" [23][24][25] approach to determine site-specific deuterium levels of proteins from solution H/D exchange experiments provided that any excessive vibrational excitation is avoided prior to the fragmentation event. ...
Hydrogen/deuterium exchange (HDX) monitored by mass spectrometry (HDX-MS) has become a valuable tool in studies of protein dynamics and protein interactions. Isotopic exchange is typically initiated by diluting a protein solution into deuterated buffer at physiological conditions. For MS analysis, the exchange reaction is quenched by acidification and cooling and the labeled protein (or a digest thereof) is analyzed by mass spectrometry. An inevitable deuterium loss occurs during quench conditions (i.e., back-exchange). The primary structure of the peptide influences the back-exchange rate due to steric hindrance by bulky side groups and by inductive and charge effects. Here we show that the back-exchange in histidine repeats (His6) at HDX-MS quench conditions is complete within a few seconds using either acetic acid or formic acid in the quench solution, while aspartic acid repeats (Asp6) retain deuterons for several minutes using formic acid. We employ electron transfer dissociation to obtain residue-specific deuterium levels of the Asp repeat in K2D6IIKIIK using a hybrid linear ion trap-Orbitrap mass spectrometer. Our results show an unexpected uneven distribution of deuterium in the Asp repeat. The implication of the rapid back-exchange of His repeats for HDX-MS protein hydrogen exchange studies is discussed. We also discuss the implications of retained deuterons in the Asp repeat of K2D6IIKIIK when this peptide is used as a probe for the occurrence of hydrogen scrambling.Graphical abstractView high quality image (151K)Research highlights▶ 100% back-exchange occurs in His repeats (His6) at HDX-MS quench conditions. ▶ Asp repeats (Asp6) retain deuterons for several minutes at HDX-MS quench conditions. ▶ Acetic acid accelerates the back-exchange rate at HDX-MS quench conditions. ▶ Residue-specific deuterium levels were obtained using a LTQ-Orbitrap with ETD.
... The application of collisional-induced dissociation to obtain site-specific information about the incorporation of deuterium into peptides and proteins is problematic due to "scrambling" of the deuterium position [213]. In contrast, in-source decay fragmentation induced less scrambling [214]. Although the higher salt tolerance and the simplified spectra are a strong advantage of MALDI and can often lead to abandonment of chromatographic purification, ESI is still the main ionization technique used for H/D exchange studies of biomolecular complexes. ...
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been demonstrated to be a valuable tool to investigate noncovalent interactions of biomolecules. The direct detection of noncovalent assemblies is often more troublesome than with electrospray ionization. Using dedicated sample preparation techniques and carefully optimized instrumental parameters, a number of biomolecule assemblies were successfully analyzed. For complexes dissociating under MALDI conditions, covalent stabilization with chemical cross-linking is a suitable alternative. Indirect methods allow the detection of noncovalent assemblies by monitoring the fading of binding partners or altered H/D exchange patterns.
... Thus, EDD may be used for deducing the solution deuteration pattern for deprotonated peptides, in a manner similar to electron capture dissociation (ECD) for protonated peptides [39]. Alternatively, MALDI in-source decay of deprotonated peptides is likely to preserve their solution deuteration pattern, as fragmentation by this technique occurs with a very low degree of scrambling for positive ions [40]. ...
We have previously shown that peptide amide hydrogens undergo extensive intramolecular migration (i.e., complete hydrogen scrambling) upon collisional activation of protonated peptides (Jørgensen et al. J. Am. Chem. Soc. 2005, 127, 2785-2793). The occurrence of hydrogen scrambling enforces severe limitations on the application of gas-phase fragmentation as a convenient method to obtain information about the site-specific deuterium uptake for proteins and peptides in solution. To investigate whether deprotonated peptides exhibit a lower level of scrambling relative to their protonated counterparts, we have now measured the level of hydrogen scrambling in a deprotonated, selectively labeled peptide using MALDI tandem time-of-flight mass spectrometry. Our results conclusively show that hydrogen scrambling is prevalent in the deprotonated peptide upon collisional activation. The amide hydrogens ((1)H/(2)H) have migrated extensively in the anionic peptide, thereby erasing the original regioselective deuteration pattern obtained in solution.
... This can also help to assess the use of tandem mass spectrometry to localize the incorporated deuteriums following hydrogen/deuterium exchange experiments. The deuterium migration during ion activation and fragmentation has been actively discussed for peptides [1][2][3][4][5], but not for oligonucleotides. To contribute to the understanding of the fragmentation mechanism, we examined, in this paper, the fragmentation of a totally deuterated dinucleotide, dAT Ϫ , in labile positions (heteroatom-bound hydrogens). ...
The fragmentation of the totally deuterated dinucleotide dAT(-) in labile positions (heteroatom-bound hydrogens) was compared for different MS/MS methods: CID, IRMPD, and EID. These experiments allowed us to affirm the coexistence of several fragmentation channels. They can be classified according to the involvement of nonlabile or labile protons in the fragmentation process. Moreover, double resonance experiments were performed in IRMPD and EID. They demonstrated the existence of consecutive fragmentation processes. The probability with which each channel is taken depends on the fragmentation technique used, i.e., the energy and the time scale of the method. The fragmentation channels that involve labile protons requiring peculiar three-dimensional structures are entropically unfavorable and enthalpically favorable. They are more observed in IRMPD and EID. The involvement of labile and, therefore, exchangeable protons in the fragmentation mechanism casts doubt on the use of tandem mass spectrometry to localize incorporated deuteriums in oligonucleotides.
Peptide therapeutics are a growing modality in the pharmaceutical industry and expanding these therapeutics to hit intracellular targets would require establishing cell permeability. Rapid measurement target-agnostic cell permeability of peptides is still analytically challenging. In this study, we demonstrate the development of a rapid high-throughput label-free methodology based on a MALDI-hydrogen-deuterium exchange mass spectrometry (MALDI-HDX-MS) approach to rank-order peptide cell membrane permeability using live THP-1 and AsPc-1 cells. Peptides were incubated in the presence of live cells and their permeability into the cells over time was measured by MALDI-HDX-MS. A differential hydrogen-deuterium exchange approach was used to distinguish the peptides outside of the cells from those inside. The peptides on the outside of the cells were labeled using sufficiently short exposure to deuterium oxide, while the peptides inside of the cells were protected from labeling as a result of permeation into the cells. The deuterium labeled and peak area ratios of unlabeled peptides were compared and plotted over time. The developed methodology, referred to as Cell-based Approach Membrane Permeability Assay (CAMPA), was applied to study an array of 24 diverse peptides including cell-penetrating peptides, stapled and macrocyclic peptides. The cell membrane permeability results observed by CAMPA were corroborated by previously reported in literature data. The CAMPA MALDI-MS analysis was fully automated including MS data processing using internally developed Python scripts. Moreover, CAMPA was demonstrated to be useful for differentiating passive and active cell transportation by using an endocytosis inhibitor in cell incubation media for selected peptides.
Matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) with a reducing matrix is believed to be initiated by hydrogen transfer from the matrix to the peptide. Several new matrices have recently been developed to achieve more efficient MALDI-ISD. In particular, the use of matrices containing aniline groups facilitates MALDI-ISD to a greater extent than that of matrices containing phenol groups, although the N-H bond in aniline is stronger than the O-H bond in phenol. In this study, photoelectron yield spectroscopy of matrix solids revealed that conversion of the phenol group to the aniline group decreased the ionization energy of the matrix solids. Crucially, the use of a matrix with lower ionization energy has been found to result in efficient cleavage at N-Cα and disulfide bonds by MALDI-ISD. Therefore, electron association with the peptide rather than the fragmentation mechanism involving hydrogen atom attachment is proposed as the initial step of the MALDI-ISD process. In this mechanism, electron transfer from the reducing matrix to the peptide produces a peptide anion radical, which provides either a [cn + H]/[zm]• or [an]•/[ym + H] fragment pair. Fragmentation of the peptide anion radical strongly depends on the gas-phase acidity of the matrix used. Subsequently, the resultant fragments/radicals underwent a reaction in the MALDI plume, producing observable even-electron ions. Consequently, MALDI-ISD fragments are observed as both positive and negative ions, even though MALDI-ISD with a reducing matrix involves fragmentation of peptide anion radicals. The proposed mechanism is suitable for obtaining a better understanding of the MALDI-ISD process.
Ultraviolet photodissociation (UVPD) has recently been introduced as an ion activation method for the determination of single-residue deuterium levels in H/D exchange tandem mass spectrometry experiments. In this regard, it is crucial to know which fragment ion types can be utilized for this purpose. UVPD yields rich product ion spectra where all possible backbone fragment ion types (a/x, b/y, and c/z) are typically observed. Here we provide a detailed investigation of the level of H/D scrambling for all fragment ion types upon UVPD of the peptide scrambling probe P1 (HHHHHHIIKIIK) using an Orbitrap tribrid mass spectrometer equipped with a solid-state 213 nm UV laser. The most abundant UVPD-generated fragment ions (i.e., b/y ions) exhibit extensive H/D scrambling. Similarly, a/x and c/z ions have also undergone H/D scrambling due to UV-induced heating of the precursor ion population. Therefore, dominant b/y ions upon UVPD of protonated peptides are a strong indicator for the occurrence of extensive H/D scrambling of the precursor ion population. In contrast to peptide P1, UV-irradiation of ubiquitin did not induce H/D scrambling in the nonfragmented precursor ion population. However, the UVPD-generated b2 and a4 ions from ubiquitin exhibit extensive H/D scrambling. To minimize H/D scrambling, short UV-irradiation time and high gas pressures are recommended.
Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) enables the study of protein dynamics by measuring the time-resolved deuterium incorporation into a protein incubated in D2O. Using electron-based fragmentation in the gas phase it is possible to measure deuterium uptake at single-residue resolution. However, a prerequisite for this approach is that the solution-phase labeling is conserved in the gas phase prior to precursor fragmentation. It is therefore essential to reduce or even avoid intramolecular hydrogen/deuterium migration, which causes randomization of the deuterium labels along the peptide (hydrogen scrambling). Here, we describe an optimization strategy for reducing scrambling to a negligible level while minimizing the impact on sensitivity on a high-resolution Q-TOF equipped with ETD and an electrospray ionization interface consisting of a glass transfer capillary followed by a dual ion funnel. In our strategy we narrowed down the optimization to two accelerating potentials, and we defined the optimization of these in a simple rule by accounting for their interdependency in relation to scrambling and transmission efficiency. Using this rule, we were able to reduce scrambling from 75% to below 5% on average using the highly scrambling-sensitive quadruply charged P1 peptide scrambling probe resulting in a minor 33% transmission loss. To demonstrate the applicability of this approach, we probe the dynamics of certain regions in cytochrome c.
The pharmaceutical industry's focus has expanded to include peptide and protein-based therapeutics; however, some analytical challenges have arisen along the way, including the urgent need for fast and robust measurement of the membrane permeability of peptides and small proteins. In this study, a simple and efficient approach that utilizes MALDI-TOF-MS to study peptide and protein permeability through an artificial liposome membrane in conjunction with a differential hydrogen-deuterium exchange (HDX) methodology is described. A non-aqueous (aprotic) matrix was evaluated for use with MALDI sample preparation in order to eliminate undesirable hydrogen-deuterium back-exchange. Peptides and proteins were incubated with liposomes and their penetration into the liposome membrane over time was measured by MALDI-MS. A differential HDX approach was used to distinguish the peptides outside of the liposome from those inside. In this regard, the peptides on the outside of the liposomes were labeled using short exposure to deuterium oxide, while the peptides inside of the liposomes were protected from labeling. Subsequently, the unlabeled versus labeled peak area ratios for peptide and protein samples were compared using MALDI-TOF-MS. In this proof-of-concept study, we developed the Liposome Artificial Membrane Permeability Assay (LAMPA) workflow to study three well-known membrane-active model peptides (melittin, alamethicin, and gramicidin) and two model proteins (aprotinin and ubiquitin). The permeability results obtained from this were corroborated by previously reported data for studied peptides and proteins. The proposed LAMPA by MALDI-HDX-MS can be applied in an ultra-high-throughput manner for studying and rank-ordering membrane permeability of peptides and small proteins.
Matrix-assisted laser desorption/ionization (MALDI) coupled with a time-of-flight (TOF) mass-spectrometry (MS) detector is acknowledged to be very useful for analysis of biological molecules. At the same time, hydrogen-deuterium exchange (HDX) is a well-known technique for studying protein higher-order structure. However, coupling MALDI with HDX may be challenging due to undesired back-exchange reactions during analysis. In this report, we survey an approach that utilizes MALDI coupled with automated sample preparation to compare global conformational changes of proteins under different solution conditions using differential HDX. A non-aqueous matrix was proposed for MALDI sample preparation to minimize undesirable back-exchange. An automated experimental setup based on the use of a liquid-handling robot and automated data acquisition allowed for tracking protein conformational changes as a difference in the number of protons exchanged to deuterons at specified solution conditions. Experimental time points to study the deuteration-labeling kinetics were obtained in a fully automated manner. The use of a non-aqueous matrix solution allowed experimental error to be minimized to within 1% RSD. We applied a newly developed MALDI-HDX workflow to study the effect of several common excipients on insulin folding stability. The observed results were corroborated by literature data and were obtained in a high-throughput and automated manner. The proposed MALDI-HDX approach can also be applied in a high-throughput manner for batch-to-batch higher order structure comparison, as well as for the optimization of protein chemical modification reactions.
Generation of overlapping peptides in solution via multiple proteases requires a very high peak capacity for the LC-MS analysis to minimize signal overlap. An inherent advantage of a gas-phase fragmentation step is that the additional gas-phase fragment ions used to sublocalize deuterium are produced after precursor ion selection and thus do not add complexity to the LC-MS analysis. The key to obtaining optimal spatial resolution in a hydrogen exchange mass spectrometry (HX-MS) experiment is the fragmentation efficiency. This chapter discusses common fragmentation techniques like collision-induced dissociation (CID) occur with complete Hydrogen-deuterium (H/D) scrambling, while other techniques that induce dissociation on a faster timescale through radical-based fragmentation channels, like electron-capture dissociation (ECD) and electron-transfer dissociation (ETD), occur inherently without H/D scrambling, thus making them suitable for HX applications. By combining the classic bottom-up HX-MS workflow with gas-phase fragmentation by ETD, detailed information on protein HX can be obtained.
Matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) induces N-Cα bond cleavage via hydrogen transfer from the matrix to the peptide backbone, which produces a c′/z• fragment pair. Subsequently, the z• generates z′ and [z + matrix] fragments via further radical reactions because of the low stability of the z•. In the present study, we investigated MALDI-ISD of a cyclic peptide. The N-Cα bond cleavage in the cyclic peptide by MALDI-ISD produced the hydrogen-abundant peptide radical [M + 2H]+• with a radical site on the α-carbon atom, which then reacted with the matrix to give [M + 3H]+ and [M + H + matrix]+. For 1,5-diaminonaphthalene (1,5-DAN) adducts with z fragments, post-source decay of [M + H + 1,5-DAN]+ generated from the cyclic peptide showed predominant loss of an amino acid with 1,5-DAN. Additionally, MALDI-ISD with Fourier transform-ion cyclotron resonance mass spectrometry allowed for the detection of both [M + 3H]+ and [M + H]+ with two 13C atoms. These results strongly suggested that [M + 3H]+ and [M + H + 1,5-DAN]+ were formed by N-Cα bond cleavage with further radical reactions. As a consequence, the cleavage efficiency of the N-Cα bond during MALDI-ISD could be estimated by the ratio of the intensity of [M + H]+ and [M + 3H]+ in the Fourier transform-ion cyclotron resonance spectrum. Because the reduction efficiency of a matrix for the cyclic peptide cyclo(Arg-Gly-Asp-D-Phe-Val) was correlated to its tendency to cleave the N-Cα bond in linear peptides, the present method could allow the evaluation of the efficiency of N-Cα bond cleavage for MALDI matrix development.
Electron transport phosphorylation is the central mechanism for most prokaryotic species to harvest energy released in the respiration of their substrates as ATP. Microorganisms have evolved incredible variations on this principle, most of these we perhaps do not know, considering that only a fraction of the microbial richness is known. Besides these variations, microbial species may show substantial versatility in using respiratory systems. In connection herewith, regulatory mechanisms control the expression of these respiratory enzyme systems and their assembly at the translational and posttranslational levels, to optimally accommodate changes in the supply of their energy substrates. Here, we present an overview of methods and techniques from the field of proteomics to explore bacterial electron transfer chains and their regulation at levels ranging from the whole organism down to the Ångstrom scales of protein structures. From the survey of the literature on this subject, it is concluded that proteomics, indeed, has substantially contributed to our comprehending of bacterial respiratory mechanisms, often in elegant combinations with genetic and biochemical approaches. However, we also note that advanced proteomics offers a wealth of opportunities, which have not been exploited at all, or at best underexploited in hypothesis-driving and hypothesis-driven research on bacterial bioenergetics. Examples obtained from the related area of mitochondrial oxidative phosphorylation research, where the application of advanced proteomics is more common, may illustrate these opportunities.
This chapter discusses the application of mass spectrometry to functional and structural characterization of proteins. Mass spectrometry techniques commonly applied to both the functional proteomics (identity and expression level of the proteins) and the structural characterization of proteins (post-translational modifications, tertiary structure and stability, subunit composition, and molecular shape) are discussed. The chapter reviews the current and historical methodologies for these applications, provides an in-depth description of the instrumentation requirements, and highlights some of examples of the techniques used. Overall, the chapter reviews biophysical, biochemical, analytical, and biological aspects of the structural and functional protein mass spectrometry.
The association of hydrogen/deuterium exchange and mass spectrometry (HDX/MS) has been emerged as a powerful analytical tool for probing structural and dynamic features of proteins. In classical bottom-up approach, the deuterated protein is digested by pepsin and the proteolytic digest is analyzed by mass spectrometry. Unfortunately, this approach suffers from two main drawbacks: (1) the resolution, which is limited by the size of the peptides after digestion and (2) the back-exchange that can take place in solution before the analysis by mass spectrometry. Classical tandem mass spectrometry cannot be used to improve resolution since Collision Induced Dissociation (CID) experiments on deuterated peptides have been shown for intramolecular deuterium migration before backbone cleavage ("scrambling"). Therefore, there is a great interest in the development of alternative HDX/MS approaches in order to improve back-exchange, scrambling and resolution problems. In this work, a complete optimized workflow for robust top-down HDX/MS has been set up. New system has been developed for introduction of the deuterated samples in the mass spectrometer, named "cryosource" and electron-based activation techniques, Electron Capture/Transfer Dissociation, were used. ECD and ETD have been shown recently to fragment, in proper experimental conditions, entire deuterated proteins with minimized scrambling and single residue resolution. The cryosource leads to negligible back-exchange during long periods of time and allows low flow rates. Optimization of all parameters was done using model peptides and protein. Our final goal was to use this methodology for the structural analysis of the complex aIF2.
Matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) is a very easy way to obtain large sequence tags and, thereby, reliable identification of peptides and proteins. Recently discovered new matrices have enhanced the MALDI-ISD yield and opened new research avenues. The use of reducing and oxidizing matrices for MALDI-ISD of peptides and proteins favors the production of fragmentation pathways involving “hydrogen-abundant” and “hydrogen-deficient” radical precursors, respectively. Since an oxidizing matrix provides information on peptide/protein sequences complementary to that obtained with a reducing matrix, MALDI-ISD employing both reducing and oxidizing matrices is a potentially useful strategy for de novo peptide sequencing. Moreover, a pseudo-MS3 method provides sequence information about N- and C-terminus extremities in proteins and allows N- and C-terminal side fragments to be discriminated within the complex MALDI-ISD mass spectrum. The combination of high mass resolution of a Fourier transform-ion cyclotron resonance (FTICR) analyzer and the software suitable for MALDI-ISD facilitates the interpretation of MALDI-ISD mass spectra. A deeper understanding of the MALDI-ISD process is necessary to fully exploit this method. Thus, this review focuses first on the mechanisms underlying MALDI-ISD processes, followed by a discussion of MALDI-ISD applications in the field of proteomics. © 2014 Wiley Periodicals, Inc., Mass Spec Rev
Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become an important method to study protein dynamics in solution. Recently, electron-based fragmentation methods (ECD and ETD) have been utilized in HDX-MS/MS experiments as experimental tools to increase the spatial resolution (the ability to obtain deuterium levels of individual residues). An essential prerequisite for this approach is that the level of hydrogen scrambling is negligible. The occurrence of hydrogen scrambling depends critically on the extent of vibrational excitation in the mass spectrometer. In particular, the desolvation process in the electrospray ion source is likely to induce scrambling at standard operating conditions. Consequently, finding experimental conditions that minimize hydrogen scrambling to a negligible level is thus pivotal for the application of electron-based fragmentation in HDX-MS/MS experiments. In the present work, we investigate the occurrence of scrambling in the Apollo I electrospray ion source using ECD of selectively deuterium labeled peptides. The electrospray ion source settings leading to minimal scrambling were identified. Furthermore, an energy dependent loss of deuterium occurring in the ion source was also observed. This loss was critically dependent on the occurrence of scrambling. (c) 2014 Elsevier B.V. All rights reserved.
In-Source Decay (ISD) in Matrix-Assisted Laser Desorption/Ionization (MALDI) mass spectrometry is a fast and easy top-down activation method. Our objective is to find a suitable matrix to locate the deuterons following in-solution hydrogen/deuterium exchange (HDX). This matrix must circumvent the commonly encountered undesired back-exchange reactions, in order to preserve the regioselective deuteration pattern.
The 1,5-diaminonaphthalene (1,5-DAN) matrix is known to be suitable for MALDI-ISD fragmentation. MALDI Mass Spectrometry Imaging (MSI) was employed to compare 1,5-DAN and other commonly used MALDI matrices with respect to the extent of back-exchange and the uniformity of the H/D exchange profiles within the MALDI spots. We tested the back-exchange on the highly sensitive amyloid-beta peptide (1-40), and proved the regioselectivity on ubiquitin and β-endorphin.
MALDI-MSI results show that 1,5-DAN leads to the least back-exchange over all the spot. MALDI-ISD fragmentation combined with H/D exchange using 1,5-DAN matrix was validated by localizing deuterons in native ubiquitin. Results agree with previous data obtained by Nuclear Magnetic Resonance (NMR) and Electron Transfer Dissociation (ETD). Moreover, 1,5-DAN matrix was used to study the H/D exchange profile of the methanol-induced helical structure of β-endorphin, and the relative protection can be explained by the polarity of residues involved in hydrogen bond formation.
We found that controlling crystallization is the most important parameter when combining H/D exchange with MALDI. The 1,5-DAN matrix is characterized by a fast crystallization kinetics, and therefore gives robust and reliable H/D exchange profiles using MALDI-ISD. Copyright © 2013 John Wiley & Sons, Ltd.
Hydrogen/deuterium exchange (HDX) methods generate useful information on protein structure and dynamics, ideally at the individual residue level. Most MS-based HDX methods involve a rapid proteolytic digestion followed by LC/MS analysis, with exchange kinetics monitored at the peptide level. Localizing specific sites of HDX is usually restricted to a resolution the size of the host peptide because gas-phase processes can scramble deuterium throughout the peptide. Subtractive methods may improve resolution, where deuterium levels of overlapping and nested peptides are used in a subtractive manner to localize exchange to smaller segments. In this study, we explore the underlying assumption of the subtractive method, namely, that the measured back exchange kinetics of a given residue is independent of its host peptide. Using a series of deuterated peptides, we show that secondary structure can be partially retained under quenched conditions, and that interactions between peptides and reversed-phase LC columns may both accelerate and decelerate residue HDX, depending upon peptide sequence and length. Secondary structure is induced through column interactions in peptides with a solution-phase propensity for structure, which has the effect of slowing HDX rates relative to predicted random coil values. Conversely, column interactions can orient random-coil peptide conformers to accelerate HDX, the degree to which correlates with peptide charge in solution, and which can be reversed by using stronger ion pairing reagents. The dependency of these effects on sequence and length suggest that subtractive methods for improving structural resolution in HDX-MS will not offer a straightforward solution for increasing exchange site resolution.
Figure
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Matrix-assisted laser desorption/ionization (MALDI) is now a mature method allowing the identification and, more challenging, the quantification of biopolymers (proteins, nucleic acids, glycans, etc). MALDI spectra show mostly intact singly charged ions. To obtain fragments, the activation of singly charged precursors is necessary, but not efficient above 3.5 kDa, thus making MALDI MS/MS difficult for large species. In-source decay (ISD) is a prompt fragmentation reaction that can be induced thermally or by radicals. As fragments are formed in the source, precursor ions cannot be selected; however, the technique is not limited by the mass of the analyzed compounds and pseudo MS3 can be performed on intense fragments. The discovery of new matrices that enhance the ISD yield, combined with the high sensitivity of MALDI mass spectrometers, and software development, opens new perspectives. We first review the mechanisms involved in the ISD processes, then discuss ISD applications like top-down sequencing and post-translational modifications (PTMs) studies, and finally review MALDI-ISD tissue imaging applications.
Mass spectrometry has become a valuable method for studying structural dynamics of proteins in solution by measuring their backbone amide hydrogen/deuterium exchange (HDX) kinetics. In a typical exchange experiment one or more proteins are incubated in deuterated buffer at physiological conditions. After a given period of deuteration, the exchange reaction is quenched by acidification (pH 2.5) and cooling (0 °C) and the deuterated protein (or a digest thereof) is analyzed by mass spectrometry. The unavoidable loss of deuterium (back-exchange) that occurs under quench conditions is undesired as it leads to loss of information. Here we describe the successful application of a chip-based nanoelectrospray ionization mass spectrometry top-down fragmentation approach based on cooling to subzero temperature (-15 °C) which reduces the back-exchange at quench conditions to very low levels. For example, only 4% and 6% deuterium loss for fully deuterated ubiquitin and β(2)-microglobulin were observed after 10 min of back-exchange. The practical value of our subzero-cooled setup for top-down fragmentation HDX analyses is demonstrated by electron-transfer dissociation of ubiquitin ions under carefully optimized mass spectrometric conditions where gas-phase hydrogen scrambling is negligible. Our results show that the known dynamic behavior of ubiquitin in solution is accurately reflected in the deuterium contents of the fragment ions.
To interpret the wealth of information contained in the hydrogen/deuterium exchange (HDX) behavior of peptides and proteins in the gas-phase, analytical tools are needed to resolve the HDX of individual exchanging sites. Here we show that ETD can be combined with fast gas-phase HDX in ND(3) gas and used to monitor the exchange of side-chain hydrogens of individual residues in both small peptide ions and larger protein ions a few milliseconds after electrospray. By employing consecutive traveling wave ion guides in a mass spectrometer, peptide and protein ions were labeled on-the-fly (0.1-10 ms) in ND(3) gas and subsequently fragmented by ETD. Fragment ions were separated using ion mobility and mass analysis enabled the determination of the gas-phase deuterium uptake of individual side-chain sites in a range of model peptides of different size and sequence as well as two proteins; cytochrome C and ubiquitin. Gas-phase HDX-ETD experiments on ubiquitin ions ionized from both denaturing and native solution conditions suggest that residue-specific HDX of side-chain hydrogens is sensitive to secondary and tertiary structural features occurring in both near-native and unfolded gas-phase conformers present shortly after electrospray. The described approach for online gas-phase HDX and ETD paves the way for making mass spectrometry techniques based on gas-phase HDX more applicable in bioanalytical research.
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-C(α) 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.
The application of electron-transfer dissociation (ETD) to obtain single-residue resolution in hydrogen exchange-mass spectrometry (HX-MS) experiments has recently been demonstrated. For such measurements, it is critical to ensure that the level of gas-phase hydrogen scrambling is negligible. Here we utilize the abundant loss of ammonia upon ETD of peptide ions as a universal reporter of positional randomization of the exchangeable hydrogens (hydrogen scrambling) during HX-ETD experiments. We show that the loss of ammonia from peptide ions proceeds without depletion of deuterium when employing optimized mild electrospray ion source settings for the HX-ETD analysis of a selectively labeled model peptide and peptides derived from fully labeled β(2)-microglobulin. Hydrogen scrambling, as induced by excessive vibrational excitation of peptide ions during harsh declustering conditions, is easily detected by a depletion of deuterium when deuterated ammonia is lost from peptides during ETD. This straightforward method requires no modifications to the experimental workflow and has the great advantage that the occurrence of hydrogen scrambling can be directly detected in the actual peptides analyzed in the HX-ETD experiment.
A new low-temperature plasma (LTP), based on dielectric barrier discharge (DBD), has been developed as an alternative ionization source for ambient mass spectrometry. For organic samples, the source is able to produce two different fragmentation patterns which are selectable by an electrical switch. The two source modes are different only in the second electrodes: in configuration (A), bar-plate and in configuration (B), coaxial bar-cylinder shapes are used. A disposable graphite probe is used as the first electrode, the same in both configurations, and a copper foil is used as the second electrode. The ionization source is applicable to gas and liquid samples, without any change being necessary in its design. Under optimal conditions, to take ethylbenzene as an example, a detection limit of less than 25 ng was obtained and a relative standard deviation (RSD) of 13.36% has been demonstrated for 50 ng of ethylbenzene (n = 11). We have found several interesting differences in the mass spectra of the tested volatile organic compounds (VOCs) in the two modes, which might be applicable in identification studies. We have investigated the effect of variation of the first electrode material and the second electrode length in mode B. Moreover, in this design the graphite electrode is capable of acting as a sample adsorbent, which is a new sampling method for LTP mass spectrometry (MS). This capability was investigated by adsorption of the selected VOCs onto the surface of the graphite electrode in a headspace solid-phase microextraction (SPME) system, and direct desorption and ionization of the samples by LTPMS.
The rate of exchange of peptide group NH hydrogens with the hydrogens of aqueous solvent is sensitive to neighboring side chains. To evaluate the effects of protein side chains, all 20 naturally occurring amino acids were studied using dipeptide models. Both inductive and steric blocking effects are apparent. The additivity of nearest-neighbor blocking and inductive effects was tested in oligo- and polypeptides and, surprisingly, confirmed. Reference rates for alanine-containing peptides were determined and effects of temperature considered. These results provide the information necessary to evaluate measured protein NH to ND exchange rates by comparing them with rates to be expected for the same amino acid sequence is unstructured oligo- and polypeptides. The application of this approach to protein studies is discussed.
In this report, we evaluate the validity of using hydrogen/deuterium exchange in combination with collision-induced dissociation mass spectrometry (CID MS) for the detailed structural and conformational investigation of peptides and proteins. This methodology, in which partly deuterated peptide ions are subjected to collision-induced dissociation in the vacuum of a mass spectrometer, has emerged as a useful tool in structural biology. It may potentially provide quantitatively the extent of deuterium incorporation per individual amino acid in peptides and proteins, thus providing detailed structural information related to protein structure and folding. We report that this general methodology has limitations caused by the fact that the incorporated deuterium atoms migrate prior or during the CID MS analysis. Our data are focused on a variety of transmembrane peptides, incorporated in a lipid bilayer, in which the near-terminal amino acids that anchor at the lipid-water interface are systematically varied. Our findings suggest that, under the experimental conditions we use, the extent of intramolecular hydrogen scrambling is strongly influenced by experimental factors, such as the exact amino acid sequence of the peptide, the nature of the charge carrier, and therefore most likely by the gas-phase structure of the peptide ion. Moreover, the observed scrambling seems to be independent of the nature of the peptide fragment ions (i.e., protonated B and Y' ' ions, and sodiated A and Y' ions). Our results strongly suggest that scrambling may be reduced by using alkali metal cationization instead of protonation in the ionization process.
Considerable controversy exists in the literature as to the occurrence of intramolecular migration of amide hydrogens upon collisional activation of protonated peptides and proteins. This phenomenon has important implications for the application of CID as an experimental tool to obtain site-specific information about the incorporation of deuterium into peptides and proteins in solution. Using a unique set of peptides with their carboxyl-terminal half labeled with deuterium we have shown unambiguously that hydrogen (1H/2H) scrambling is such a dominating factor during low energy collisional activation of doubly protonated peptides that the original regioselective deuterium pattern of these peptides is completely erased (Jørgensen, T. J. D., Gårdsvoll, H., Ploug, M., and Roepstorff, P. (2005) Intramolecular migration of amide hydrogens in protonated peptides upon collisional activation. J. Am. Chem. Soc.127, 2785-2793). Taking further advantage of this unique test system we have now investigated the influence of the charge state and collision energy on the occurrence of scrambling in protonated peptides. Our MALDI tandem time-of-flight experiments clearly demonstrate that complete positional randomization among all exchangeable sites (i.e. all N- and O-linked hydrogens) also occurs upon high energy collisional activation of singly protonated peptides. This intense proton/deuteron traffic precludes the use of MALDI tandem time-of-flight mass spectrometry to obtain reliable information on the specific incorporation pattern of deuterons obtained during exchange experiments in solution.
Coagulation factor VIIa (FVIIa) is a serine protease that, after binding to tissue factor (TF), plays a pivotal role in the initiation of blood coagulation. We used hydrogen exchange monitored by mass spectrometry to visualize the details of FVIIa activation by comparing the exchange kinetics of distinct molecular states, namely zymogen FVII, endoproteolytically cleaved FVIIa, TF-bound zymogen FVII, TF-bound FVIIa, and FVIIa in complex with an active site inhibitor. The hydrogen exchange kinetics of zymogen FVII and FVIIa are identical indicating highly similar solution structures. However, upon tissue factor binding, FVIIa undergoes dramatic structural stabilization as indicated by decreased exchange rates localized throughout the protease domain and in distant parts of the light chain, spanning across 50A and revealing a concerted interplay between functional sites in FVIIa. The results provide novel insights into the cofactor-induced activation of this important protease and reveal the potential for allosteric regulation in the trypsin family of proteases.
Because proteins are central to cellular function, researchers have sought to uncover the secrets of how these complex macromolecules execute such a fascinating variety of functions. Although static structures are known for many proteins, the functions of proteins are governed ultimately by their dynamic character (or 'personality'). The dream is to 'watch' proteins in action in real time at atomic resolution. This requires addition of a fourth dimension, time, to structural biology so that the positions in space and time of all atoms in a protein can be described in detail.
Disulfide bonds in gaseous multiply-protonated proteins are preferentially cleaved in the mass spectrometer by low-energy electrons, in sharp contrast to excitation of the ions by photons or low-energy collisions. For S−S cyclized proteins, capture of one electron can break both an S−S bond and a backbone bond in the same ring, or even both disulfide bonds holding two peptide chains together (e.g., insulin), enhancing the sequence information obtainable by tandem mass spectrometry on proteins in trace amounts. Electron capture at uncharged S−S is unlikely; cleavage appears to be due to the high S−S affinity for H• atoms, consistent with a similar favorability found for tryptophan residues. RRKM calculations indicate that H• capture dissociation of backbone bonds in multiply-charged proteins represents nonergodic behavior, as proposed for the original direct mechanism of electron capture dissociation.
Solution folding of a protein removes major sections of it from their aqueous environment. Complete removal, by forming water-free gaseous protein ions with electrospray ionization/mass spectrometry, profoundly changes the conformation of cytochrome c. Of these ions' exchangeable hydrogen atoms, gaseous D2O replaces 30% to 70% in distinct values indicative of at least six conformational states. Although this is increased to >95% by colliding ions with D2O, colliding instead with N2 and subsequent D2O exposure gives the same H/D exchange values, although in different proportions; on solvent removal, denatured ions spontaneously refold. Deuterated State I, II, and V ions of a range of charge values up to 17+ when charge stripped to 9+ ions do not fold appreciably, even though their cross section decreases by 20%, confirming that each has a characteristic conformational structure insensitive to electrostatic repulsion; the charge solvation of an added protonated side chain also protects additional exchangeable sites. Dramatic temperature effects on H/D exchange also support unique State I, II, IV, and V conformers with a variety of charge values. Despite extensive H/D scrambling, dissociation to locate D sites of State I, II, IV, and V ions indicates that four small α-helical regions are maintained even in the most open ionic conformations; these regions are consistent with salt bridge stabilization. In the more open conformers the α-helical regions could be partially converted to either β-sheet or denatured structures. No close similarities were found between the gaseous conformer structures and those in solution, a cautionary note for the use of ESI/MS gas-phase data to characterize noncovalent interactions in solution.
In matrix assisted laser desorption ionisation (MALDI) a large fraction of analyte ions undergo postsource decay (PSD) during flight in the field free drift path. By means of a modified two-state reflectron, daughter ion time-of-flight spectra of medium sized linear peptides (up to 2800 u) were recorded containing full sequence information. Precision, accuracy and mass resolution of fragment ions were almost as good as obtained in high energy CAD studies performed in four-sector instruments. Instrumental sensitivity was better at least one order of magnitude. In reflectron time-of-flight mass spectrometry (RETOF-MS) the cleavage pattern of PSD products is different from that obtained by high energy and low energy CAD. In our instrument, conditions which were energetically comparable to high energy and low energy CAD could easily and comparatively be studied in the same experiment by varying instrumental parameters. Activation mechanisms of PSD were found to be largely determined by collisional events (ion/neutral) induced by the acceleration field during early plume expansion. Future potentials of PSD analysis after MALDI are discussed.
The specific cleavage of NCα bonds on the peptide backbone to form the so-called ‘c’ and ‘z + 2’ products, which can be used for the rapid determination of protein amino-acid sequences, has been examined to clarify the mechanism(s) that occur during hydrogen abstraction induced by bombardment with 337-nm laser photons in matrix-assisted laser desorption/ionization (MALDI) method. Intramolecular hydrogen abstraction, which results from the hydrogen(s) on the Cα or Cβ carbon, did not occur with a deuterium-labeled dodecapeptide. To confirm a proposition that intermolecular hydrogen abstraction occurs between the peptide and the MALDI matrix, a deuterium dodecapeptide embedded in a deuterium 2,5-dihydroxybenzoic acid matrix at a molar ratio of 1:7000 was analyzed. The resulting deuterium c product ions suggested that c ions form via intermolecular hydrogen abstraction, although the results obtained did not deny any other possibilities such as intramolecular transfer of labile hydrogen. A mechanism for the NCα bond cleavage has been proposed that the formation of hypervalent radical species and subsequent prompt bond cleavages occur. The proposed mechanism successfully rationalizes the formation of both the z + 2 and the c product ions.
The MD+ ions of a variety of amino acids and small peptides have been prepared using CD4 and (CD3)2CO as chemical ionization reagents. Using tandem mass spectrometry the fragmentation reactions of these MD+ ions have been studied, both those occurring unimolecularly on the metastable ion time scale (CD4 CI) and those occurring following collisional activation ((CD3)2CO CI). The results show that the added D+ has undergone extensive interchange (leading to H/D scrambling) with all labile hydrogens including carboxylic hydrogens, hydroxylic hydrogens, amidic hydrogens and amino hydrogens. The results indicate that the proton added to amino acids and simple peptides is very mobile and samples all positions bearing labile hydrogens prior to fragmentation of the protonated species.
In the following, the first results on ultraviolet laser desorption (UVLD) of bioorganic compounds in the mass range above 10000 daltons are reported. Strong molecular ion signals were registered by use of an organic matrix with strong absorption at the wavelength used for controlled energy deposition and soft desorption (7)
By utilizing delayed pulsed ion extraction of ions generated via the matrix-assisted laser desorption/ionization (MALDI) technique, fast (< 320 ns) metastable ion fragmentation is observed for both peptide and protein analytes in the ion source of a linear time-of-flight mass spectrometer. Small peptides such as the oxidized B chain of bovine insulin exhibit fragmentation at the amide linking bond between peptide residues. Overlapping sequence information is provided by fragmentation from both the C- and N-terminal ends of the peptide (cn-, yn-, and z*n-type fragment ions). Larger proteins can also exhibit a wealth of sequence specific fragment ions in favorable cases. One example is cytochrome c, which undergoes substantial (approximately 80%) fast fragmentation at the amide bonds along the amino acid backbone of the protein. Only amide bond cleavages initiating from the C-terminal end (cn fragments) are observed. The observed fragmentation pattern provides a significant amount of potential sequence information for these molecules. External mass calibration of the intact protonated molecular ions is demonstrated with mass accuracies typically around 100 ppm. Mass accuracies for the observed fragment ions ranged from +/- 0.20 Da for the smaller peptides studied (i.e., oxidized B chain of bovine insulin) to +/- 0.38 Da for the largest protein studied (cytochrome c), based upon the known sequences.
Continuous segments of amino acid sequence information as long as 41 residues have been deduced by interpretation of matrix-assisted laser desorption/ionization-generated ion signals dominated by Cn fragmentation within the ion source of a linear time-of-flight mass spectrometer utilizing delayed ion extraction. The technique has been applied successively to five proteins of mass 12.2 kDa to 18.3 kDa, yielding segments of continuous sequence as long as 41 residues without the need for prior proteolytic fragmentation. Intact crosslinks such as disulfides or heme linkages interrupt the generation of these data.
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) was used to determine amide proton/deuteron (H/D) exchange rates. The method has broad application to the study of protein conformation and folding and to the study of protein-ligand interactions and requires no modifications of the instrument. Amide protons were allowed to exchange with deuterons in buffered D2O at room temperature, pD 7.25. Exchanged deuterons were "frozen" in the exchanged state by quenching at pH 2.5, 0 degree C and analyzed by MALDI-TOF MS. The matrix mixture consisted of 5 mg/mL alpha-cyano-4-hydroxycinnamic acid, acetonitrile, ethanol, and 0.1% TFA. The matrix was adjusted to pH 2.5, and the chilled MALDI target was rapidly dried. Deuteration of amide protons on cyclic AMP-dependent protein kinase was measured after short times of incubation in deuterium by pepsin protein digestion and MALDI-TOF MS analysis. The unseparated peptic digest was analyzed in a single spectrum of the mixture. From five spectra, H/D exchange rates were determined for some 40 peptides covering 65% of the protein sequence.
A technique is described for identifying and locating posttranslational modifications (PTMs) in peptides and proteins of known sequence by interpretation of c(n) ion signals generated by in-source decay during delayed ion extraction in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Sites of phosphorylation in seven synthetic peptides were determined, as was the location of both the heme group and N,N,N-trimethyllysine in yeast cytochrome c. A semi-automated data analysis process facilitates the identification of segments of the sequence on each side of the PTM, permitting its placement at the junction of the segments and definition of the added mass. A graphical display facilitates illustration of both the location and mass of the PTM.
It has been apparent for some years that the structures of proteins are dynamic rather than static. For some proteins, dynamics
is essential to function (e.g., refs. 1–7). These structural changes have been detected for more than 30 yr by observing hydrogen exchange between peptide amide hydrogens
and solvent containing the hydrogen isotopes tritium or deuterium (8–10). Although tritium is no longer used extensively for this purpose, deuterium is widely used in hydrogen exchange studies,
especially in multidimensional nuclear magnetic resonance (NMR), in which amide hydrogen signals disappear on deuteration.
Since deuterium weighs 1 Dalton more than protium, hydrogen exchange in proteins can also be detected by mass spectrometry.
This approach is complementary to NMR in some respects and clearly advantageous in others.
Understanding the molecular mechanisms of enzyme catalysis and allosteric regulation has been a primary goal of biochemistry for many years. The dynamics of these processes, approached through a variety of kinetic methods, are discussed. The results obtained for many different enzymes suggest that multiple intermediates and conformations are general characteristics of the catalytic process and allosteric regulation. Ribonuclease, dihydrofolate reductase, chymotrypsin, aspartate aminotransferase, and aspartate transcarbamoylase are considered as specific examples. Typical and maximum rates of conformational changes and catalysis are also discussed, based on results obtained from model systems. The nature and rates of interconversion of the intermediates, along with structural information, can be used as the bases for understanding the incredible catalytic efficiency of enzymes. Potential roles of conformational changes in the catalytic process are discussed in terms of static and environmental effects, and in terms of dynamic coupling within the enzyme-substrate complex.
The extent of internal hydrogen exchange (scrambling) within multiply charged solvent-free protein ions was investigated using a small model protein. The site-specific backbone amide protection data were obtained using protein ion fragmentation in the gas phase and compared with the available NMR data. Only minimal scrambling was detected when relatively high-energy collisional activation was used to fragment intact protein ions, while low-energy fragmentation resulted in more significant but not random internal exchange. Increased conformational flexibility of protein ions in the gas phase did not have any effect on the extent of hydrogen scrambling under the conditions of higher-energy collisional activation but resulted in totally random redistribution of labile hydrogen atoms when the protein ion fragmentation was induced by multiple low-energy collisions.
This paper reports detailed studies on the internal energy of ions formed in matrix-assisted laser desorption/ionization (MALDI) using delayed extraction MALDI-time-of-flight (TOF) and atmospheric pressure (AP) MALDI mass spectrometric (MS) methods. We use benzylpyridinium cations as internal energy probes. Our study reveals three distinct contributions to internal energy build-up in vacuum-MALDI (classical MALDI-TOF), each having different effects on ion fragmentation. Some fragments are formed before ion extraction (i.e. no more than 100 ns after the laser impact), and they are therefore well resolved and recorded as sharp signals in the MALDI-TOFMS scan. This prompt fragmentation can have two origins: (i) in-plume thermal activation, presumably always present, and (ii) in-plume chemical activation, in the course of reactions with hydrogen radicals. In addition to early internal energy build-up associated with these well-resolved promptly formed fragments, a broad peak slightly offset to higher masses could be detected corresponding to fragments formed after the extraction has started. This second signal corresponds to a third source of internal energy in MALDI ions, (iii) the extraction-induced collisional activation of the ions with the neutral components of the plume. These three contributions are difficult to quantify in vacuum-MALDI, because of the combined influence of several parameters (nature of the matrix, spot-to-spot variability, total laser exposure, delay time, acceleration voltage) on extraction-induced fragmentation. AP-MALDI, on the other hand, has two advantages for comparative studies of analyte fragmentation. First, extraction-induced fragmentation is absent, and only the contributions of early plume activation remain. Second, the reproducibility is far better than in vacuum-MALDI. AP-MALDI is therefore expected to shed new light on the early steps of the MALDI process.
The fragmentation pathways of protonated peptides are reviewed in the present paper paying special attention to classification of the known fragmentation channels into a simple hierarchy defined according to the chemistry involved. It is shown that the 'mobile proton' model of peptide fragmentation can be used to understand the MS/MS spectra of protonated peptides only in a qualitative manner rationalizing differences observed for low-energy collision induced dissociation of peptide ions having or lacking a mobile proton. To overcome this limitation, a deeper understanding of the dissociation chemistry of protonated peptides is needed. To this end use of the 'pathways in competition' (PIC) model that involves a detailed energetic and kinetic characterization of the major peptide fragmentation pathways (PFPs) is proposed. The known PFPs are described in detail including all the pre-dissociation, dissociation, and post-dissociation events. It is our hope that studies to further extend PIC will lead to semi-quantative understanding of the MS/MS spectra of protonated peptides which could be used to develop refined bioinformatics algorithms for MS/MS based proteomics. Experimental and computational data on the fragmentation of protonated peptides are reevaluated from the point of view of the PIC model considering the mechanism, energetics, and kinetics of the major PFPs. Evidence proving semi-quantitative predictability of some of the ion intensity relationships (IIRs) of the MS/MS spectra of protonated peptides is presented.
In-source decay (ISD) in matrix-assisted laser desorption/ionization (MALDI) shares some similarities with the novel fragmentation technique electron capture dissociation (ECD). In both reactions, the otherwise strong N-C(alpha) bond is cleaved, forming fragment ions of the c and z types, while labile posttranslational modifications are preserved. Therefore, it is tempting to assume that ISD and ECD have some mechanistic aspects in common. Because electrons are present in the MALDI plume, we investigated the previously suggested possibility that ISD is a variation of ECD. However, experiments with peptides with only one site for efficient protonation revealed that ISD is not caused by electron capture. Instead, ICD seems to be induced by hydrogen atoms generated by a photochemical reaction of the matrix. We provide evidence for this reaction by hydrogen/deuterium exchange experiments with peptides containing a minimal number of exchangeable hydrogen atoms. The hydrogen atom model in ECD is indirectly supported by the proposed fragmentation mechanism for ISD, because our data suggest that hydrogen radicals can induce fragmentation by cleavage of the N-C(alpha) bond, independent from their origin.
Presently different opinions exist as to the degree of scrambling of amide hydrogens in gaseous protonated peptides and proteins upon collisional activation in tandem mass spectrometry experiments. This unsettled controversy is not trivial, since only a very low degree of scrambling is tolerable if collision-induced dissociation (CID) should provide reliable site-specific information from (1)H/(2)H exchange experiments. We have explored a series of unique, regioselectively deuterium-labeled peptides as model systems to probe for intramolecular amide hydrogen migration under low-energy collisional activation in an orthogonal quadrupole time-of-flight electrospray ionization (Q-TOF ESI) mass spectrometer. These peptides contain a C-terminal receptor-binding sequence and an N-terminal nonbinding region. When the peptides form a receptor complex, the amide hydrogens of the interacting sequences are protected against exchange with the solvent, while the amide hydrogens of the nonbinding sequences exchange rapidly with the solvent. We have utilized such long-lived complexes to generate peptides labeled with deuterium in either the binding or nonbinding region, and the expected regioselectivity of this labeling was confirmed after pepsin proteolysis. CID of such deuterated peptides, [M + 2H](2+), yielded fragment ions (b- and y-ions) having a deuterium content that resemble the theoretical values calculated for 100% scrambling. Thus, complete randomization of all hydrogen atoms attached to nitrogen and oxygen occurs in the gaseous peptide ion prior to its dissociation.
Hydrogen exchange coupled to mass spectrometry (MS) has become a valuable analytical tool for the study of protein dynamics. By combining information about protein dynamics with more classical functional data, a more thorough understanding of protein function can be obtained. In many cases, protein dynamics are directly related to specific protein functions such as conformational changes during enzyme activation or protein movements during binding. The method is made possible because labile backbone hydrogens in a protein will exchange with deuterium atoms when the protein is placed in a D2O solution. The subsequent increase in protein mass over time is measured with high-resolution MS. The location of the deuterium incorporation is determined by monitoring deuterium incorporation in peptic fragments that are produced after the labeling reaction. In this review, we will summarize the general principles of the method, discuss the latest variations on the experimental protocol that probe different types of protein movements, and review other recent work and improvements in the field.
It remains an open question as to whether experiments involving collision-induced dissociation (CID) can provide a viable approach for monitoring spatially resolved deuteration levels in electrosprayed polypeptide ions. A number of laboratories reported the successful application of CID following solution-phase H/D exchange (HDX), whereas others found that H/D scrambling precluded site-specific measurements. The aim of the current work is to help clarify the general feasibility of HDX-CID methods, using a 22-residue zinc-bound protein domain (Zn-ZBD) as model system. Metal binding in Zn-ZBD should confer structural rigidity, and the presence of several basic residues should sequester mobile charge carriers in the gas phase. Both of these factors were expected to suppress the extent of scrambling. HDX was carried out by employing rapid on-line mixing, thereby mimicking conditions typically encountered in kinetic pulse-labeling studies. Quadrupole time-of-flight MS/MS of pulse-labeled Zn-ZBD provides high sequence coverage. However, the measured fragment deuteration levels do not correlate with the known H-bonding pattern of Zn-ZBD, suggesting the occurrence of extensive scrambling. Instead of showing a uniform distribution, the fragment ions reveal a distinct nonrandom pattern of deuteration levels. In the absence of prior information, these data could erroneously be ascribed to the presence of protected sites. However, the observed patterns clearly originate from other factors; possibly they are caused by modulations of the amide CID efficiency by kinetic isotope effects. It is concluded that scrambling does not represent the only conceptual problem in HDX-CID studies and that control experiments on uniformly labeled samples are essential for ruling out interpretation artifacts.
Proteins from biological samples are often identified by mass spectrometry (MS) with the two following "bottom-up" approaches: peptide mass fingerprinting or peptide sequence tag. Nevertheless, these strategies are time-consuming (digestion, liquid chromatography step, desalting step), the N- (or C-) terminal information often lacks and post-translational modifications (PTMs) are hardly observed. The in-source decay (ISD) occurring in a matrix assisted laser desorption/ionization (MALDI) source appears an interesting analytical tool to obtain N-terminal sequence, to identify proteins and to characterize PTMs by a "top-down" strategy. The goal of this review deals with the usefulness of the ISD technique in MALDI source in proteomics fields. In the first part, the ISD principle is explained and in the second part, the use of ISD in proteomic studies is discussed for protein identification and sequence characterization.
Hydrogen (1H/2H) exchange combined with mass spectrometry (HX-MS) has become a valuable method for the analysis of protein structural dynamics. Currently, localization of the incorporated deuterons is made by enzymatic cleavage of the labeled proteins, and single-residue resolution is typically only achieved for a few residues. Determination of site-specific deuterium levels by gas-phase fragmentation would greatly increase the applicability of the HX-MS method. It is, however, mandatory for this gas-phase approach that hydrogen (1H/2H) scrambling in the gaseous peptide is negligible. Thus, it is important to have a simple reference system where the onset of scrambling processes is readily detected. Here we describe a simple well-characterized set of peptides with a unique regioselective labeling that ensures an inherent high sensitivity for the detection of scrambling. This selective labeling is achieved by utilizing differences in the intrinsic exchange rates between various amino acid residues. We demonstrate that our peptides can be infused directly into an electrospray ion source by means of a cooled glass syringe, while maintaining their selective labeling in solution. We further show that the selective labeling is completely erased upon low-energy collisional activation in a tandem mass spectrometry experiment as a result of extensive hydrogen (1H/2H) scrambling.
Hydrogen (1H/2H) exchange combined with mass spectrometry (HX-MS) has become a recognized method for the analysis of protein structural dynamics. Presently, the incorporated deuterons are typically localized by enzymatic cleavage of the labeled proteins and single residue resolution is normally only obtained for a few residues. Determination of site-specific deuterium levels by gas-phase fragmentation in tandem mass spectrometers would greatly increase the applicability of the HX-MS method. The biggest obstacle in achieving this goal is the intramolecular hydrogen migration (i.e., hydrogen scrambling) that occurs during vibrational excitation of gas-phase ions. Unlike traditional collisional ion activation, electron capture dissociation (ECD) is not associated with substantial vibrational excitation. We investigated the extent of intramolecular backbone amide hydrogen (1H/2H) migration upon ECD using peptides with a unique selective deuterium incorporation. Our results show that only limited amide hydrogen migration occurs upon ECD, provided that vibrational excitation prior to the electron capture event is minimized. Peptide ions that are excessively vibrationally excited in the electrospray ion source by, e.g., high declustering potentials or during precursor ion selection (via sideband excitation) in the external linear quadrupole ion trap undergo nearly complete hydrogen (1H/2H) scrambling. Similarly, collision-induced dissociation (CID) in the external linear quadrupole ion trap results in complete or extensive hydrogen (1H/2H) scrambling. This precludes the use of CID as a method to obtain site-specific information from proteins that are labeled in solution-phase 1H/2H exchange experiments. In contrast, the deuteration levels of the c- and z-fragment ions generated from ECD closely mimic the known solution deuteration pattern of the selectively labeled peptides. This excellent correlation between the results obtained from gas phase and solution suggests that ECD holds great promise as a general method to obtain single residue resolution in proteins from solution 1H/2H exchange experiments.