Hydrogen exchange studies on Alzheimer's amyloid-beta peptides by mass spectrometry using matrix-assisted laser desorption/ionization and electrospray ionization

The conformation and aggregation behavior of synthetic Alzheimer's amyloid peptides (Abeta) has been investigated using hydrogen-deuterium exchange measured by electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry. Mass spectrometric fragmentation of deuterated Abeta peptides was carried out by collision-induced dissociation, inlet fragmentation, and post-source decay. In contrast to the C-terminally truncated peptides Abeta(1-40) and Abeta(1-36) showing full hydrogen-deuterium exchange, Abeta(1-42) and the pyroglutamyl peptide Pyr(3)-Abeta(3-42) produced more complex signal patterns resulting from the formation of beta-sheet-structured oligomers having 18-20 strongly protected protons. Using mass spectrometric fragmentation the results show that the reduced isotope exchange of Abeta(1-42) can be attributed to the central part of the chain comprising residues 8-23. This confirms involvement of the hydrophobic binding domain LVFFA in the course of Abeta aggregation and demonstrates that hydrogen-deuterium exchange in combination with mass spectrometry is well suited for structural analysis of monomeric and reversibly associated amyloid peptides using picomole quantities of material.     

Site-specific amide hydrogen/deuterium exchange in E. coli thioredoxins measured by electrospray ionization mass spectrometry

Mass spectrometry as an analytical tool to study protein folding and structure by hydrogen/deuterium exchange is a relatively new approach. In this study, site-specific amide deuterium content was measured in oxidized and reduced E. coli thioredoxins by using the b(n) ions in electrospray ionization CID MS/MS experiments after 20-s incubation in D(2)O phosphate-buffered solution (pH 5.7). The deuterium levels correlated well with reported NMR-determined H/D exchange rate constants. The deuterium measured by y(n) ions, however, showed much less reliable correlation with rate exchange data. In general, residues in alpha helices and beta sheets, when measured by b(n) ions, showed low incorporation of deuterium while loops and turns had high deuterium levels. Most amide sites in the two protein forms showed similar deuterium levels consistent with the expected similarity of their structures, but there were some differences. The turn consisting of residues 18-22 in particular showed more variability in deuterium content consistent with reported structural differences in the two forms. The deuterium uptake by thioredoxins alkylated at Cys-32 by S-(2-chloroethyl)glutathione and S-(2-chloroethyl)cysteine, in peptides 1-24 and 45-58, was similar to that observed for oxidized and reduced thioredoxins, but several residues, particularly Leu-53 and Thr-54, showed slightly elevated deuterium levels, suggesting that structural changes had occurred from alkylation of the protein at Cys-32. It is concluded that b(n) ions are reliable for determining the extent of site-specific amide hydrogen isotope exchange and that mass spectrometry is useful as a complementary technique to NMR and other analytical methods for probing regional structural characteristics of proteins.     

The determination of high-affinity protein/inhibitor binding constants by electrospray ionization hydrogen/deuterium exchange mass spectrometry

Recently, a hydrogen/deuterium exchange method termed SUPREX (Stability of Unpurified Proteins from Rates of hydrogen/deuterium EXchange), capable of measuring protein/ligand binding constants, which utilizes matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), has been reported. Unlike more conventional approaches, SUPREX is inherently capable of measuring Kd values of tight binding ligands. Here we present a SUPREX-based method, incorporating automation and electrospray ionization (ESI)-MS, to measure Kd values for very potent inhibitors of the kinase PKCtheta. The use of ESI offers an alternative to MALDI, with the advantages of improved mass measurement precision for larger proteins, and amenability to automation. Kd values generated by this method are in good agreement with those generated by a molecular protein kinase assay.     

Hydrogen/deuterium exchange on aromatic rings during atmospheric pressure chemical ionization mass spectrometry

It has been demonstrated that substituted indoles fully labelled with deuterium on the aromatic ring can undergo substantial exchange back to partial and even fully protonated forms during atmospheric pressure chemical ionisation (APCI) liquid chromatography/mass spectrometry (LC/MS). The degree of this exchange was strongly dependent on the absolute quantity of analyte, the APCI desolvation temperature, the nature of the mobile phase, the mobile phase flow rate and the instrument used. Hydrogen/deuterium (H/D) exchange on several other aromatic ring systems during APCI LC/MS was either undetectable (nitrobenzene, aniline) or extremely small (acetanilide) compared to the effect observed for substituted indoles. This observation has major implications for quantitative assays using deuterium‐labelled internal standards and for the detection of deuterium‐labelled products from isotopically labelled feeding experiments where there is a risk of back exchange to the protonated form during the analysis. Copyright © 2010 John Wiley & Sons, Ltd.     

Structural characterization of methionine and leucine enkephalins by hydrogen/deuterium exchange and electrospray ionization tandem mass spectrometry

Conformational changes in two endogenous opioid active pentapeptides methionine enkephalin (Met-enk) and leucine enkephalin (Leu-enk) induced by trifluoroethanol (TFE) were identified using hydrogen/deuterium exchange (HDX), coupled with electrospray ionization (ESI) mass spectrometry. The exchange features in individual amino acid residues were characterized by acquiring tandem mass spectra of the deuterated peptides. The exact identity of the labile hydrogens involved in HDX reveals that the monomer forms of both peptides adopt an unfolded conformation in aqueous solvent, but prefer the 5-->2 beta-turn secondary structure under the membrane-mimetic environment. The ESI mass spectra of Met-enk and Leu-enk also reveal that the dimer structure of these peptides coexists with the monomer conformation. The extent of the dimer structure is dependent on the peptide concentration and nature of the solvent. The non-polar solvents facilitate the dimer formation.     

Gas phase hydrogen / deuterium exchange in electrospray ionization mass spectrometry as a practical tool for structure elucidation

Two methods for gas phase hydrogen/deuterium exchange have been developed for the analysis of small molecules. Hydrogen/deuterium exchange has been implemented by making simple modifications to the plumbing for the nebulizer and curtain gases on a nebulization-assisted electrospray ion source. The nebulizer gas exchange method has demonstrated deuterium exchange levels of 84–97% for a variety of molecules representing a wide range of structural classes containing up to 51 potentially exchangeable hydrogens; this allowed determination of the number of exchangeable hydrogens for all of the molecules studied containing ≤ 25 labile hydrogens (M _r ≤ 3000). ND₃ gas consumption is minimized in the nebulizer method by toggling the nebulizer from air to ND₃ for only a few scans of the total sample elution period. The curtain gas exchange method is more variable, yielding exchange levels of 32–98% for the same set of molecules; this was still sufficient to allow determination of > 70% of the molecules studied containing ≤ 25 labile hydrogens. Gas consumption is minimized in the curtain method by replacing ≤ 10% of the curtain gas flow with ND₃. Neither the nebulizer nor curtain exchange method requires the use of deuterated or aprotic solvents at typical 2 μL/min flow rates.     

Conformational changes in Akt1 activation probed by amide hydrogen/deuterium exchange and nano‐electrospray ionization mass spectrometry

Amide hydrogen exchange coupled to nano‐electrospray ionization mass spectrometry (nano‐ESI‐MS) has been used to identify and characterize localized conformational changes of Akt upon activation. Active or inactive Akt was incubated in D₂O buffer, digested with pepsin, and analyzed by nano‐ESI‐MS to determine the deuterium incorporation. The hydrogen/deuterium (H/D) exchange profiles revealed that Akt undergoes considerable conformational changes in the core structures of all three individual domains after activation. In the PH domain, four β‐strand (β1, β2 β5 and β6) regions containing membrane‐binding residues displayed higher solvent accessibility in the inactive state, suggesting that the PH domain is readily available for the binding to the plasma membrane for activation. In contrast, these β‐strands became less exposed or more folded in the active form, which is favored for the dissociation of Akt from the membrane. The beginning α‐helix J region and the C‐terminal locus (T450‐470P) of the regulatory domain showed less folded structures that probably enable substrate entry. Our data also revealed detailed conformational changes of Akt in the kinase domain due to activation, some of which may be attributed to the interaction of the basic residues with phosphorylation sites. Our H/D exchange results indicating the conformational status of Akt at different activation states provided new insight for the regulation of this critical protein involved in cell survival. Published in 2009 by John Wiley & Sons, Ltd.     

Fragmentation of mycosporine-like amino acids by hydrogen/deuterium exchange and electrospray ionisation tandem mass spectrometry

The determination and identification of mycosporine-like amino acids (MAAs) from algae remain a major challenge due to the low concentration. Mass spectrometry (MS) can make an invaluable contribution in the search and identification of MAAs because of its high sensitivity, possibility of coupling with liquid chromatography, and the availability of powerful tandem mass spectrometric techniques. However, the unequivocal determination of the presence and location of important functional groups present on the basic skeleton of the MAAs is often elusive due to their inherent instability under MS conditions. In this study, the use of hydrogen/deuterium (H/D) exchange and electrospray ionisation tandem mass spectrometry (ESI-MS/MS) for characterisation of four MAAs (palythine, asterina, palythinol and shinorine) isolated from the macroalgae Gracilaria tenuistipitata Chang et Xia was investigated. The accurate-mass confirmation of the protonated molecules was performed on a Q-TOF instrument. We demonstrate that employing deuterium labelling in ESI-MS/MS analysis provides a convenient tool for the determination of new MAAs. Although the fragmentation patterns of MAAs were discussed earlier, to our knowledge, this is the first time that mechanisms are proposed.     

C-terminal sequencing of peptides using electrospray ionization mass spectrometry.

A low-flow reactor is described for the on-line monitoring of peptides digested with carboxypeptidase P by electrospray ionization. Two peptides were analyzed using this technique: glucagon (average MW 3482.8 Da), and apomyoglobin (average MW 16,951.5). Both peptides gave interpretable results. The first 19 amino acids of glucagon were successfully sequenced. Apomyoglobin yielded sequence information to the 30th amino acid with some gaps. At 300 nL/min, 50% of the first 30 amino acids were sequenced and at 1 microL/min, 67% of the first 30 amino acids were observed.     

Diastereomeric differentiation of norbornene amino acid peptides by electrospray ionization tandem mass spectrometry

A new class of diastereomeric pairs of non‐natural amino acid peptides derived from butyloxycarbonyl (Boc‐)protected cis‐(2S,3R)‐ and trans‐(2S,3S)‐β‐norbornene amino acids including a monomeric pair have been investigated by electrospray ionization (ESI) tandem mass spectrometry using quadrupole time‐of‐flight (Q‐TOF) and ion‐trap mass spectrometers. The protonated cis‐BocN‐β‐nbaa (2S,3R) (1) (βnbaa = β‐norbornene amino acid) eliminates the Boc group to form [M+H–Boc+H]⁺, whereas an additional ion [M+H–C₄H₈]⁺ is formed from trans‐BocN‐β‐nbaa (2S,3S) (2). Similarly, it is observed that the peptide diastereomers (di‐, tri‐ and tetra‐), with cis‐BocN‐β‐nbaa (2S,3R)‐ at the N‐terminus, initially eliminate the Boc group to form [M+H–Boc+H]⁺ which undergo further fragmentation to give a set of product ions that are different for the peptides with trans‐BocN‐β‐nbaa (2S,3S)‐ at the N‐terminus. Thus the Boc group fragments differently depending on the configuration of the amino acid present at the N‐terminus. It is also observed that the peptide bond cleavage in these peptides is less favoured and most of the product ions are formed due to retro‐Diels‐Alder fragmentation. Interestingly, sodium‐cationized peptide diastereomers mainly yield a series of retro‐Diels‐Alder fragment ions which are different for each diastereomer as they are formed starting from [M+Na–Boc+H]⁺ in peptides with cis‐BocN‐β‐nbaa (2S,3R)‐ at the N‐terminus, and [M+Na–C₄H₈]⁺ in peptides with trans‐BocN‐β‐nbaa (2S,3S)‐ at the N‐terminus. All these results clearly indicate that these diastereomeric pairs of peptides yield characteristic product ions which help distinguish the isomers. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of micelle mass by electrospray ionization mass spectrometry

By electrospray ionization (ESI) mass spectrometry, micelle solutions of sodium cholate were investigated in detail in the presence and absence of ethanol. The average aggregation number could be evaluated from the spectra acquired under conditions where soft collisions adequate to measure the micelle solution were induced, and the value agreed well with that obtained previously by other methods. From the dependence on ethanol content, it was also found that the average aggregation number in aqueous solution without organic solvent could be reliably estimated. The ESI method proved to be a useful tool for determining the micelle mass in the original aqueous phase.     

Cluster chemical ionization and deuterium exchange mass spectrometry in supersonic molecular Beams

A cluster-based chemical ionization method has been developed that produces protonated molecular ions from molecules introduced through a supersonic molecular beam interface. Mixed clusters of the analyte and a clustering agent (water or methanol) are produced in the expansion region of the beam, and are subsequently ionized by “fly through” electron impact (EI) ionization, which results in a mass spectrum that is a combination of protonated molecular ion peaks together with the conventional EI fragmentation pattern. The technique is presented and discussed as a tool complementary to electron impact ionization in supersonic molecular beams. Surface-induced dissociation on a rhenium oxide surface is also applied to simplify the mass spectra of clusters and reveal the analyte spectrum. The high gas flow rates involved with the supersonic molecular beam interface that enable the easy introduction of the clustering agents also have been used to introduce deuterating agents. An easy-to-use, fast, and routine on-line deuterium exchange method was developed to exchange active hydrogens (NH, OH). This method, combined with electron impact ionization, is demonstrated and discussed in terms of the unique information available through the EI fragmentation patterns, its ability to help in isomer identification, and possible applications with fast gas chromatography-mass spectrometry in supersonic molecular beams.     

Fragmentation mechanisms of oligodeoxynucleotides studied by H/D exchange and electrospray ionization tandem mass spectrometry

We used solution-phase hydrogen/deuterium (H/D) exchange and multistage tandem mass spectrometry (MS/MS) experiments in an electrospray ion-trap mass spectrometer operating in the negative-ion mode to investigate the consequences of the loss of a high proton-affinity (PA) base from T-rich tetra and hexadeoxynucleotides. The T-rich oligodeoxynucleotides containing one or two other nucleobases take advantage of the mass spectral inertness of T because fragmentation of a T-rich oligomer is simple, allowing a tight focus on those processes of interest. Furthermore, determination of T-rich oligodeoxynucleotides may be a starting point in the development of a mass spectrometric scheme to understand the mutagenicity of various types of DNA damage by UV radiation. For nine oligodeoxynucleotides, the nucleobases were charged by nearly exclusive D transfer and then expelled as neutral bases. Loss of the base located at the 3' end is preferred over that from the 5' terminus when the two bases are identical. The observation of partially exchanged fragments from a completely exchanged precursor ion proves intramolecular H/D exchange between hydrogen atoms that can exchange in water and those that cannot. The multiplicity of the product-ion peaks provides information on decomposition pathways and origins of the product ions and shows that the loss of base is the first step in all fragmentation of hexanucleotides, but is a competitive process for tetranucleotide fragmentation.     

New example of proline-induced fragmentation in electrospray ionization mass spectrometry of peptides

The positive ion electrospray ionization (ESI+) mass spectra of peptides usually display only protonated molecules provided that soft ionization conditions are applied (low cone voltage to prevent in-source dissociations). Such ions can be multiply charged depending on the molecular weight of the studied compounds. We have experienced an unexpected behavior during the ESI analysis of a modified peptide of relatively high mass (3079 Da). A specific fragmentation occurred even under soft energetic conditions, leading to a mass spectrum containing multiply charged molecular and fragment ions. The selective rupture involved the amide bond between the glutamic acid and proline residues (E-P sequence). The successive replacement of each amino acid by an alanine residue (positional scanning study) was undertaken to assess which part of the sequence induced such selective and abundant fragmentation on multiply charged species. The succession P-P was evidenced as the minimum unit giving rise to the first peptide bond rupture in the sequence X-P-P. Any acidic amino acid at the X position (X = D, E) favored the fragmentation by an intramolecular interaction. Such proline-induced fragmentation occurring readily in the source differed from the literature data on the specific behavior of proline-containing peptides where bond ruptures occur solely in dissociation conditions.     

Determination of mu-oxo exchange rates in di-mu-oxo dimanganese complexes by electrospray ionization mass spectrometry

A time-resolved mass spectrometric technique has been used for the determination of rates of exchange of mu-O atoms with water for the complexes [(mes-terpy)2Mn2(III/IV)(mu-O)2(H2O)2](NO3)3 (1, mes-terpy = 4'-mesityl-2,2':6',2' '-terpyridine), [(bpy)4Mn2(III/IV)(mu-O)2](ClO4)3 (2, bpy = 2,2'-bipyridine), [(phen)4Mn2(III/IV)(mu-O)2](ClO4)3 (3, phen = 1,10-phenanthroline), [(bpea)2Mn2(III/IV)(mu-O)2(mu-OAc)](ClO4)2 (4, bpea = bis(2-pyridyl)ethylamine), [(bpea)2Mn2(IV/IV)(mu-O)2(mu-OAc)](ClO4)3 (4ox), [(terpy)4Mn4(IV/IV/IV/IV)(mu-O)5(H2O)2](ClO4)6 (5, terpy = 2,2':6',2'-terpyridine), and [(tacn)4Mn4(IV/IV/IV/IV)(mu-O)6]Br(3.5)(OH)0.5.6H2O (6, tacn = 1,4,7-triazacyclononane). The rate of exchange of mu-OAc bridges with free acetate in solution has been measured for complexes 4 and 4ox. These are the first measurements of rates of ligand exchange on biologically relevant high-valent Mn complexes. The data analysis method developed here is of general utility in the quantitation of isotope exchange processes by mass spectrometry. We find that the presence of labile coordination sites on Mn increases mu-O exchange rates, and that all-Mn(IV) states are more inert toward exchange than mixed Mn(III)-Mn(IV) states. The rates of mu-O exchange obtained in this work for a di-mu-oxo Mn2(III/IV) dimer with labile coordination sites are compared with the oxygen isotope incorporation rates from substrate water to evolved dioxygen measured in different S states of the oxygen evolving complex (OEC) of photosystem II (PSII). On the basis of this comparison, we propose that both substrate waters are not bound as mu-O bridges between Mn atoms in the S2 and S3 states of the OEC.     

Study of non-covalent complexation between catechin derivatives and peptides by electrospray ionization mass spectrometry

The recent development of electrospray ionization mass spectrometry (ESI-MS) has allowed its use to study molecular interactions driven by non-covalent forces. ESI-MS has been used to detect non-covalent complexes between proteins and metals, ligands and peptides and interactions involving DNA, RNA, oligonucleotides and drugs. Surprisingly, the study of the interaction between polyphenolic molecules and peptides/proteins is still an area where ESI-MS has not benefited. With regard to the important influence of these interactions in the biological and food domains, ESI-MS was applied to the detection and the characterization of soluble polyphenol-peptide complexes formed in model solution. The ability to observe and monitor the weak interactions involved in such macromolecular complexation phenomena was demonstrated for monomeric and dimeric flavonoid molecules (catechin-derived compounds) largely encountered in plants and plant derived products. Intact non-covalent polyphenol-peptide complexes were observed by ESI-MS using different experimental conditions. Utilizing mild ESI interface conditions allowed the detection of 1 : 1 polyphenol-peptide complexes in all tested solutions and 2 : 1 complexes for the dimers and galloylated polyphenols (flavanols). These results show that there is a preferential interaction between polymerized and/or galloylated polyphenols and peptide compared with that between monomeric polyphenols and peptides. Thus, ESI-MS shows potential for the study of small polyphenolic molecule-peptide interactions and determination of stoichiometry.     

Hydrogen/deuterium exchange using a coaxial sheath-flow interface for capillary electrophoresis/mass spectrometry

The interfacing of capillary electrophoresis (CE) with mass spectrometry (MS) is well established and may be accomplished by use of either a coaxial arrangement or by employing a liquid T-junction. In both these interfaces a make-up flow is introduced. This is required because of the mismatch in flow rates for capillary electrophoresis approximately nL/min and 'true' electrospray approximately 2-10 microL/min. Electrical connectivity may also be established where the liquid flows meet (the introduction of nanospray renders the use of make-up flow unnecessary). Hydrogen/deuterium (H/D) exchange occurs in solution when there are labile hydrogen atoms present in a molecule. The establishment of the presence and the number of such exchangeable hydrogen atoms may be of importance in the identification and differentiation of compounds. It may also be an aid in the structural elucidation of unknown materials. We have investigated the feasibility of carrying out H/D exchange via a CE/MS interface. This involved the addition of D2O to the sheath flow and our preliminary results showing the separations of drug substances, subsequently undergoing exchange, are presented.     

Interaction of angiotensin peptides and zinc metal ions probed by electrospray ionization mass spectrometry

Electrospray ionization-tandem mass spectrometry experiments were used to provide evidence regarding the sites of interactions between zinc metal ions and angiotensin peptides. The electrospray ionization mass spectra of histidine-containing human angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) and angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) in the presence of zinc show abundant multiply charged ions for the zinc-attached peptide [M + aZn²⁺ +(c ? 2a)H⁺]^c+, where a = 1, 2 and c is charge. From collisionally activated dissociation experiments, with both low energy (triple quadrupole mass spectrometry) and high energy collisions (linked scan at constant B/E with a double focusing instrument) of the [M + Zn]²⁺ and [M + Zn + H]³⁺ ions for angiotensin II, a [b ₆ + Zn]²⁺ species is produced as the most abundant product ion, suggesting that the zinc interaction site is in the vicinity of the His⁶ residue. Additionally, tandem mass spectra from the zinc-attached ions for angiotensin I show abundant [b ₆ + Zn]²⁺ and [b ₉ + Zn]²⁺ products, providing evidence that both His⁶ and His⁹ are involved in zinc coordination.     

Analysis of sulfated peptides using positive electrospray ionization tandem mass spectrometry.

Presented is a method for analyzing sulfated peptides, and differentiating the post-translational modification (PTM) from its isobaric counterpart phosphorylation, using quadrupole time-of-flight (Qq/TOF) mass spectrometry (MS) and positive ion nanoelectrospray MS/MS. A set of commercially available sulfo- and phosphopeptide standards was analyzed via in-source dissociation and MS/MS to generate fragmentation signatures that were used to characterize and differentiate the two modifications. All of the phosphorylated peptides retained their +80 Da modifications under collision-induced decomposition (CID) conditions and peptide backbone fragmentation allowed for the site-specific identification of the modification. In sharp contrast, sulfated peptides lost SO3 from the precursor as the collision energy (CE) was increased until only the non-sulfated form of the peptide was observed. The number of 80 Da losses indicated the number of sulfated sites. By continuing to ramp the CE further, it was possible to fragment the non-sulfated peptides and obtain detailed sequence information. It was not possible to obtain site-specific information on the location of the sulfate moieties using positive ion MS/MS as none of the original precursor ions were present at the time of peptide backbone fragmentation. This method was applied to the analysis of recombinant human B-domain deleted factor VIII (BDDrFVIII), which has six well-documented sulfation sites and several potential phosphorylation sites located in two of the sulfated regions of the protein. Seven peptides with single and multiple +80 Da modifications were isolated and analyzed for their respective PTMs. The fragmentation patterns obtained from the BDDrFVIII peptides were compared with those obtained for the standard peptides; and in all cases the peptides were sulfated. None of the potential phosphorylation sites were found to be occupied, and these results are consistent with the literature.