Transfer Dissociation Tandem Mass Spectrometry Research Articles

A pain-causing and paralytic ant venom glycopeptide

SummaryAnts (Hymenoptera: Formicidae) are familiar inhabitants of most terrestrial environments. Although we are aware of the ability of many species to sting, knowledge of ant venom chemistry remains limited. Herein, we describe the discovery and characterization of an O-linked glycopeptide (Mg7a) as a major component of the venom of the ant Myrmecia gulosa. Electron transfer dissociation and higher-energy collisional dissociation tandem mass spectrometry were used to localize three α-N-acetylgalactosaminyl residues (α-GalNAc) present on the 63-residue peptide. To allow for functional studies, we synthesized the full-length glycosylated peptide via solid-phase peptide synthesis, combined with diselenide–selenoester ligation-deselenization chemistry. We show that Mg7a is paralytic and lethal to insects, and triggers pain behavior and inflammation in mammals, which it achieves through a membrane-targeting mode of action. Deglycosylation of Mg7a renders it insoluble in aqueous solution, suggesting a key solubilizing role of the O-glycans.

Evaluating the performance of an ETD-cleavable cross-linking strategy for elucidating protein structures

Chemical cross-linking is a powerful strategy for elucidating the structures of protein or protein complexes. The distance constraints obtained from cross-linked peptides represent the three-dimensional structures of the protein complexes. Unfortunately, structural analysis using cross-linking approach demands a significant amount of data to elucidate protein structures. This requires the development of several cleavable cross-linkers with different range of spacer chains. An Electron Transfer Dissociation (ETD) tandem mass spectrometry cleavable bond hydrazone was reported. Its fragmentation with conjugated peptides showed promise for the development of a new ETD cleavable cross-linker. However, no cross-linker was developed utilizing this ETD cleavable bond. For the first time, we attempted to develop an ETD cleavable cross-linker utilizing a hydrazone bond. We overcome the pitfall for the synthesis of this cross-linker and an easy synthesis scheme is reported. In this report, we evaluated the performance of this cross-linker called Hydrazone Incorporated ETD cleavable cross-linker (HI-ETD-XL) in model peptides and proteins. The characteristic fragmentation behavior of HI-ETD-XL during electron transfer dissociation and subsequent sequence identification of the peptide fragment ions by tandem mass spectrometry allowed the identification of cross-linked peptides unambiguously. We believe the availability of this ETD cleavable cross-linker will advance structural proteomics research significantly. SignificanceMany cellular processes rely on the structural dynamics of protein complexes. The detailed knowledge of the structure and dynamics of protein complexes is crucial for understanding their biological functions and regulations. However, most of the structure of these multiprotein entities remain uncharacterized and sometimes is very challenging to reveal with biophysical techniques alone. Chemical cross-linking combined with mass spectrometry (MS) has proven to be a dependable strategy in structural proteomics field. However, data complexity and false identifications are significant hindrances for unambiguous identification of cross-linked peptides. Confident identifications demand structural studies with cross-linkers with different properties and variable spacer chain lengths. This new ETD cleavable cross-linking workflow will provide additional confidence to overcome these drawbacks and allow us to pinpoint cross-linked peptides confidently.

Rapid Quantification of Peptide Oxidation Isomers From Complex Mixtures.

Hydroxyl radical protein footprinting (HRPF) is a powerful technique for probing changes in protein topography, based on quantifying the amount of oxidation of different regions of a protein. While quantification of HRPF oxidation at the peptide level is relatively common and straightforward, quantification at the residue level is challenging because of the influence of oxidation on MS/MS fragmentation and the large number of complex and only partially chromatographically resolved isomeric peptide oxidation products. HRPF quantification of isomeric peptide oxidation products (where the peptide sequence is the same but isomeric oxidation products are formed at different sites) at the residue level by electron transfer dissociation tandem mass spectrometry (ETD MS/MS) has been demonstrated in both model peptides and HRPF products, but the method is hampered by the partial separation of oxidation isomers by reversed phase chromatography. This requires custom MS/MS methods to equally sample all isomeric oxidation products across their elution window, greatly increasing method development time and reducing the oxidation products quantified in a single LC-MS/MS run. Here, we present a zwitterionic hydrophilic interaction capillary chromatography (ZIC-HILIC) method to ideally coelute all isomeric peptide oxidation products while separating different peptides. This allows us to relatively quantify peptide oxidation isomers using an ETD MS/MS spectrum acquired at any point across the single peptide oxidation isomer peak, greatly simplifying data acquisition and data analysis.

Characterization and Quantification of Highly Sulfated Glycosaminoglycan Isomers by Gated-Trapped Ion Mobility Spectrometry Negative Electron Transfer Dissociation MS/MS.

Glycosaminoglycans (GAGs) play vital roles in many biological processes and are naturally present as complex mixtures of polysaccharides with tremendous structural heterogeneity, including many structural isomers. Mass spectrometric analysis of GAG isomers, in particular highly sulfated heparin (Hep) and heparan sulfate (HS), is challenging because of their structural similarity and facile sulfo losses during analysis. Herein, we show that highly sulfated Hep/HS isomers may be resolved by gated-trapped ion mobility spectrometry (gated-TIMS) with negligible sulfo losses. Subsequent negative electron transfer dissociation (NETD) tandem mass spectrometry (MS/MS) analysis of TIMS-separated Hep/HS isomers generated extensive glycosidic and cross-ring fragments for confident isomer differentiation and structure elucidation. The high mobility resolution and preservation of labile sulfo modifications afforded by gated-TIMS MS analysis also allowed relative quantification of highly sulfated heparin isomers. These results show that the gated-TIMS-NETD MS/MS approach is useful for both qualitative and quantitative analysis of highly sulfated Hep/HS compounds in a manner not possible with other techniques.

Roles of Macrophage Exosomes in Immune Response to Calcium Oxalate Monohydrate Crystals.

In kidney stone disease, macrophages secrete various mediators via classical secretory pathway and cause renal interstitial inflammation. However, whether their extracellular vesicles, particularly exosomes, are involved in kidney stone pathogenesis remained unknown. This study investigated alterations in exosomal proteome of U937-derived macrophages (by phorbol-12-myristate-13-acetate activation) after exposure to calcium oxalate monohydrate (COM) crystals for 16-h using 2-DE-based proteomics approach. Six significantly altered proteins in COM-treated exosomes were successfully identified by nanoscale liquid chromatography–electrospray ionization–electron transfer dissociation tandem mass spectrometry as proteins involved mainly in immune processes, including T-cell activation and homeostasis, Fcγ receptor-mediated phagocytosis, interferon-γ (IFN-γ) regulation, and cell migration/movement. The decreased heat shock protein 90-beta (HSP90β) and increased vimentin were confirmed by Western blotting. ELISA showed that the COM-treated macrophages produced greater level of interleukin-1β (IL-1β), one of the markers for inflammasome activation. Functional studies demonstrated that COM-treated exosomes enhanced monocyte and T-cell migration, monocyte activation and macrophage phagocytic activity, but on the other hand, reduced T-cell activation. In addition, COM-treated exosomes enhanced production of proinflammatory cytokine IL-8 by monocytes that could be restored to its basal level by small-interfering RNA targeting on vimentin (si-Vimentin). Moreover, si-Vimentin could also abolish effects of COM-treated exosomes on monocyte and T-cell migration as well as macrophage phagocytic activity. These findings provided some implications to the immune response during kidney stone pathogenesis via exosomal pathway of macrophages after exposure to COM crystals.

Negative Polarity Helium Charge Transfer Dissociation Tandem Mass Spectrometry: Radical-Initiated Fragmentation of Complex Polysulfated Anions.

This work provides the first use of helium charge transfer dissociation (He-CTD) tandem mass spectrometry (MS/MS) in negative polarity mode. Three sulfated oligosaccharides of natural origin were chosen as representative structures that are difficult to solve by conventional MS/MS approaches. Negative polarity He-CTD provided a full set of structurally informative fragments, which permitted the unambiguous determination of the complete structures of these molecules, including the characterization of labile sulfated functional groups. Despite close structural features, the three molecules led to distinct fragmentation patterns depending on the position of the sulfate group in the heterocycle. The observed fragments showed a consistent radical-initiated mechanism of dissociation, which shares similarities with fragment types produced in electron detachment dissociation (EDD), negative electron transfer dissociation (NETD), or extreme UV photodissociation (XUV-PD). Short times of data collection and the fact that the technique can be affordably implementable in any standard laboratory and with a classical ion trap mass spectrometer were other remarkable characteristics of negative polarity He-CTD.

Toward a suitable structural analysis of gene delivery carrier based on polycationic carbohydrates by electron transfer dissociation tandem mass spectrometry

Polycationic carbohydrates represent an attractive class of biomolecules for several applications and particularly as non viral gene delivery vectors. In this case, the establishment of structure-biological activity relationship requires sensitive and accurate characterization tools to both control and achieve fine structural deciphering. Electrospray-tandem mass spectrometry (ESI-MS/MS) appears as a suitable approach to address these questions. In the study herein, we have investigated the usefulness of electron transfer dissociation (ETD) to get structural data about five polycationic carbohydrates demonstrated as promising gene delivery agents. A particular attention was paid to determine the influence of charge states as well as both fluoranthene reaction time and supplementary activation (SA) on production of charge reduced species, fragmentation yield, varying from 2 to 62%, as well as to obtain the most higher both diversity and intensity of fragments, according to charge states and targeted compounds. ETD fragmentation appeared to be mainly directed toward pending group rather than carbohydrate cyclic scaffold leading to a partial sequencing for building blocks when amino groups are close to carbohydrate core, but allowing to complete structural deciphering of some of them, such as those including dithioureidocysteaminyl group which was not possible with CID only. Such findings clearly highlight the potential to help the rational choice of the suitable analytical conditions, according to the nature of the gene delivery molecules exhibiting polycationic features. Moreover, our ETD-MS/MS approach open the way to a fine sequencing/identification of grafted groups carried on various sets of oligo-/polysaccharides in various fields such as glycobiology or nanomaterials, even with unknown or questionable extraction, synthesis or modification steps.

High-Efficiency Recognition and Identification of Disulfide Bonded Peptides in Rat Neuropeptidome Using Targeted Electron Transfer Dissociation Tandem Mass Spectrometry.

The main goal of the present study is to develop a method to recognize and identify endogenous intrachain disulfide bonded peptide, which are rarely sequenced in current peptidomics studies. In order to achieve highly efficient detection of these peptides in a neuropeptidome analysis, we alkylated the peptides, mined the raw mass spectrometry data, and then recognized the candidates of untreated disulfide bonded peptides from unalkylated peptide extracts. After removing more than 90% features, targeted electron transfer dissociation fragmentation was performed for detecting and fragmenting disulfide bonded peptides, and even most of them were present in low abundance in the original sample. Diverse endogenous disulfide bonded peptides were then detected and sequenced, opening up new perspectives for comprehensively understanding the response of a neuropeptidome.

O-GlcNAc Modification of the runt-Related Transcription Factor 2 (Runx2) Links Osteogenesis and Nutrient Metabolism in Bone Marrow Mesenchymal Stem Cells

Runx2 is the master switch controlling osteoblast differentiation and formation of the mineralized skeleton. The post-translational modification of Runx2 by phosphorylation, ubiquitinylation, and acetylation modulates its activity, stability, and interactions with transcriptional co-regulators and chromatin remodeling proteins downstream of osteogenic signals. Characterization of Runx2 by electron transfer dissociation tandem mass spectrometry revealed sites of O-linked N-acetylglucosamine (O-GlcNAc) modification, a nutrient-responsive post-translational modification that modulates the action of numerous transcriptional effectors. O-GlcNAc modification occurs in close proximity to phosphorylated residues and novel sites of arginine methylation within regions known to regulate Runx2 transactivation. An interaction between Runx2 and the O-GlcNAcylated, O-GlcNAc transferase enzyme was also detected. Pharmacological inhibition of O-GlcNAcase (OGA), the enzyme responsible for the removal of O-GlcNAc from Ser/Thr residues, enhanced basal (39.9%) and BMP2/7-induced (43.3%) Runx2 transcriptional activity in MC3T3-E1 pre-osteoblasts. In bone marrow-derived mesenchymal stem cells differentiated for 6 days in osteogenic media, inhibition of OGA resulted in elevated expression (24.3%) and activity (65.8%) of alkaline phosphatase (ALP) an early marker of bone formation and a transcriptional target of Runx2. Osteogenic differentiation of bone marrow-derived mesenchymal stem cells in the presence of BMP2/7 for 8 days culminated in decreased OGA activity (39.0%) and an increase in the abundance of O-GlcNAcylated Runx2, as compared with unstimulated cells. Furthermore, BMP2/7-induced ALP activity was enhanced by 35.6% in bone marrow-derived mesenchymal stem cells differentiated in the presence of the OGA inhibitor, demonstrating that direct or BMP2/7-induced inhibition of OGA is associated with increased ALP activity. Altogether, these findings link O-GlcNAc cycling to the Runx2-dependent regulation of the early ALP marker under osteoblast differentiation conditions.

On the Viability of Heterolytic Peptide N–Cα Bond Cleavage in Electron Capture and Transfer Dissociation Mass Spectrometry

While frequently employed as an experimental technique, the mechanistic picture surrounding the gas-phase dissociation of peptides carrying multiple positive charges during electron capture and electron transfer dissociation tandem mass spectrometry remains incomplete. Despite this mechanistic uncertainty, most proposals agree that the peptide backbone N-Cα bond located to the C-terminal (right) side of an aminoketyl radical formed in a peptide backbone during the electron capture process is homolytically cleaved. Recently, we introduced the "enol" mechanism, which proposes that a backbone N-Cα bond located to the N-terminal (left) side of an aminoketyl radical is cleaved heterolytically. Here, we further validate this mechanism using replica-exchange molecular dynamics to create unbiased representative sets of low-energy conformers for several model tryptic peptide systems (H-Alax-Lys-OH(2+), x = 3-5). Transition state barrier enthalpies for the cleavage of N-Cα bonds proceeding via the homolytic (right-side) and heterolytic (left-side) pathways, determined by density functional computations, identify the preferred cleavage route for each conformer. These findings support our original hypothesis that heterolytic N-Cα cleavage can exist in a competitive balance with homolytic cleavages, independent of the relative energy of the precursor dication species. Smaller peptide systems see decreased heterolytic N-Cα cleavage probabilities, likely resulting from an insufficient hydrogen-bonding network needed to stabilize and ultimately annihilate the transition state zwitterion. This observation may explain the early dismissal of left-side cleavage pathways based on computational studies employing small model systems.

Ping-Pong Protons: How Hydrogen-Bonding Networks Facilitate Heterolytic Bond Cleavage in Peptide Radical Cations

Electron capture and electron transfer dissociation (ECD/ETD) tandem mass spectrometry (MS/MS) are commonly employed techniques for biomolecular analysis. The ECD/ETD process predominately cleaves N-Cα peptide backbone bonds, leading to primary sequence information complementary to other mass spectrometry techniques. Despite frequent laboratory use, the mechanistic underpinnings surrounding N-Cα bond cleavage remain debated. While the majority of mechanisms assume a homolytic bond rupture, we recently showed that heterolytic cleavage is also thermodynamically viable. For a cleavage of this type to be feasible, the charge separation created upon breaking of the N-Cα backbone bond must be quickly annihilated. In this work, we show, using density functional computations, that specific hydrogen-bonding motifs and structural rearrangements involving proton transfers stabilize the transition state associated with heterolytic cleavage and eliminate the ensuing charge separation from the final product fragments. The movement of protons can occur either directly from the z- to c-fragment or in a more complex manner including a ping-pong-type mechanism. The nature of these diverse hydrogen-bonding motifs reveals that not only those functional groups proximate to the bond rupture site, but also the entire global chemical environment, play important roles in backbone cleavage characteristic of ECD/ETD MS/MS. For doubly charged systems, both conformation and electron localization site dictate which of the two fragments retains the final positive charge.

Discovery and Confirmation of O-GlcNAcylated Proteins in Rat Liver Mitochondria by Combination of Mass Spectrometry and Immunological Methods

O-linked β-N-acetylglucosamine (O-GlcNAc) is an important post-translational modification (PTM) consisting of a single N-acetylglucosamine moiety attached via an O-β-glycosidic linkage to serine and threonine residues. Glycosylation with O-GlcNAc occurs on myriad nuclear and cytosolic proteins from almost all functional classes. However, with respect to O-GlcNAcylated proteins special in mitochondria, little attention has been paid. In this study, we combined mass spectrometry and immunological methods to perform global exploration of O-GlcNAcylated proteins specific in mitochondria of rat liver. First, highly purified mitochondrial proteins were obviously shown to be O-GlcNAcylated by immunoblot profiling. Then, β-elimination followed by Michael Addition with Dithiothreitol (BEMAD) treatment and LC-MS/MS were performed to enrich and identify O-GlcNAcylated mitochondrial proteins, resulting in an unambiguous assignment of 14 O-GlcNAcylation sites, mapping to 11 O-GlcNAcylated proteins. Furthermore, the identified O-GlcNAcylated mitochondrial proteins were fully validated by both electron transfer dissociation tandem mass spectrometry (ETD/MS/MS) and western blot. Thus, for the first time, our study definitely not only identified but also validated that some mitochondrial proteins in rat liver are O-GlcNAcylated. Interestingly, all of these O-GlcNAcylated mitochondrial proteins are enzymes, the majority of which are involved in a wide variety of biological processes, such as urea cycle, tricarboxylic acid cycle and lipid metabolism, indicating a role for protein O-GlcNAcylation in mitochondrial function.

Precision Mapping of Coexisting Modifications in Histone H3 Tails from Embryonic Stem Cells by ETD-MS/MS

Post-translational modifications (PTMs) of histones play a major role in regulating chromatin dynamics and influence processes such as transcription and DNA replication. Here, we report 114 distinct combinations of coexisting PTMs of histone H3 obtained from mouse embryonic stem (ES) cells. Histone H3 N-terminal tail peptides (amino acids 1-50, 5-6 kDa) were separated by optimized weak cation exchange/hydrophilic interaction liquid chromatography (WCX/HILIC) and sequenced online by electron transfer dissociation (ETD) tandem mass spectrometry (MS/MS). High mass accuracy and near complete sequence coverage allowed unambiguous mapping of the major histone marks and discrimination between isobaric and nearly isobaric PTMs such as trimethylation and acetylation. Hierarchical data analysis identified H3K27me2-H3K36me2 as the most frequently observed PTMs in H3. Modifications at H3 residues K27 and K36 often coexist with the abundant mark K23ac, and we identified two frequently occurring quadruplet marks 'K9me1K23acK27me2K36me2' and 'K9me3K23acK27me2K36me', which might indicate a role in crosstalk. Co-occurrence frequency analysis revealed also an interplay between methylations of K9, K27, and K36, suggesting interdependence between histone methylation marks. We hypothesize that the most abundant coexisting PTMs may provide a signature for the permissive state of mouse ES cells.

Identification of O-Linked N-Acetylglucosamine (O-GlcNAc)-modified Osteoblast Proteins by Electron Transfer Dissociation Tandem Mass Spectrometry Reveals Proteins Critical for Bone Formation

The nutrient-responsive β-O-linked N-acetylglucosamine (O-GlcNAc) modification of critical effector proteins modulates signaling and transcriptional pathways contributing to cellular development and survival. An elevation in global protein O-GlcNAc modification occurs during the early stages of osteoblast differentiation and correlates with enhanced transcriptional activity of RUNX2, a key regulator of osteogenesis. To identify other substrates of O-GlcNAc transferase in differentiating MC3T3E1 osteoblasts, O-GlcNAc-modified peptides were enriched by wheat germ agglutinin lectin weak affinity chromatography and identified by tandem mass spectrometry using electron transfer dissociation. This peptide fragmentation approach leaves the labile O-linkage intact permitting direct identification of O-GlcNAc-modified peptides. O-GlcNAc modification was observed on enzymes involved in post-translational regulation, including MAST4 and WNK1 kinases, a ubiquitin-associated protein (UBAP2l), and the histone acetyltransferase CREB-binding protein. CREB-binding protein, a transcriptional co-activator that associates with CREB and RUNX2, is O-GlcNAcylated at Ser-147 and Ser-2360, the latter of which is a known site of phosphorylation. Additionally, O-GlcNAcylation of components of the TGFβ-activated kinase 1 (TAK1) signaling complex, TAB1 and TAB2, occurred in close proximity to known sites of Ser/Thr phosphorylation and a putative nuclear localization sequence within TAB2. These findings demonstrate the presence of O-GlcNAc modification on proteins critical to bone formation, remodeling, and fracture healing and will enable evaluation of this modification on protein function and regulation.

Large-scale Identification of Endogenous Secretory Peptides Using Electron Transfer Dissociation Mass Spectrometry

Mass spectrometry-based unbiased analysis of the full complement of secretory peptides is expected to facilitate the identification of unknown biologically active peptides. However, tandem MS sequencing of endogenous peptides in their native form has proven difficult because they show size heterogeneity and contain multiple internal basic residues, the characteristics not found in peptide fragments produced by in vitro digestion. Endogenous peptides remain largely unexplored by electron transfer dissociation (ETD), despite its widespread use in bottom-up proteomics. We used ETD, in comparison to collision induced dissociation (CID), to identify endogenous peptides derived from secretory granules of a human endocrine cell line. For mass accuracy, both MS and tandem MS were analyzed on an Orbitrap. CID and ETD, performed in different LC-MS runs, resulted in the identification of 795 and 569 unique peptides (ranging from 1000 to 15000 Da), respectively, with an overlap of 397. Peptides larger than 3000 Da accounted for 54% in CID and 46% in ETD identifications. Although numerically outperformed by CID, ETD provided more extensive fragmentation, leading to the identification of peptides that are not reached by CID. This advantage was demonstrated in identifying a new antimicrobial peptide from neurosecretory protein VGF (non-acronymic), VGF[554-577]-NH2, or in differentiating nearly isobaric peptides (mass difference less than 2 ppm) that arise from alternatively spliced exons of the gastrin-releasing peptide gene. CID and ETD complemented each other to add to our knowledge of the proteolytic processing sites of proteins implicated in the regulated secretory pathway. An advantage of the use of both fragmentation methods was also noted in localization of phosphorylation sites. These findings point to the utility of ETD mass spectrometry in the global study of endogenous peptides, or peptidomics.

Automated and High Confidence Protein Phosphorylation Site Localization Using Complementary Collision-Activated Dissociation and Electron Transfer Dissociation Tandem Mass Spectrometry

Reversible protein phosphorylation plays a critical role in cell signaling and is responsible for the regulation of many biological processes in most living organisms. The low stoichiometry of protein phosphorylation requires sensitive analysis by tandem mass spectrometry. However, incomplete peptide fragmentation and the loss of labile phosphate groups complicate identification of the site of the phosphate motif. Here, we have implemented and evaluated a novel approach for phospho-site localization by the combined use of peptide tandem mass spectrometry data obtained using both collision-activated dissociation and electron transfer dissociation, an approach termed the Cscore. The scoring algorithm used in the Cscore was adapted from the widely used Ascore method. The analytical benefit of integrating the product ion information of both ETD and CAD data are evident by increased confidence in phospho-site localization and the number of assigned phospho-sites at a fixed false-localization rate. The average calculated Cscore from a large data set (>7000 phosphopeptide MS/MS spectra) was ∼32 compared to ∼23 and ∼17 for the Ascore using collision-activated dissociation (CAD) or electron transfer dissociation (ETD), respectively. Compared with the Ascore using either CAD or ETD, the Cscore identified up to 88% more phosphorylation sites. Using a phosphopeptide library revealed that the score threshold for obtaining a false-localization rate of 0.5% was lower for the Cscore than either the Ascore (CAD) or the Ascore (ETD).

Limited Proteolysis via Millisecond Digestions in Protease-Modified Membranes

Sequential adsorption of poly(styrene sulfonate) (PSS) and proteases in porous nylon yields enzymatic membrane reactors for limited protein digestion. Although a high local enzyme density (~30 mg/cm(3)) and small pore diameters in the membrane lead to digestion in <1 s, the low membrane thickness (170 μm) affords control over residence times at the millisecond level to limit digestion. Apomyoglobin digestion demonstrates that peptide lengths increase as the residence time in the membrane decreases. Moreover, electron transfer dissociation (ETD) tandem mass spectrometry (MS/MS) on a large myoglobin proteolytic peptide (8 kDa) provides a resolution of 1-2 amino acids. Under denaturing conditions, limited membrane digestion of bovine serum albumin (BSA) and subsequent ESI-Orbitrap MS analysis reveal large peptides (3-10 kDa) that increase the sequence coverage from 53% (2 s digestion) to 82% (0.05 s digestion). With this approach, we also performed membrane-based limited proteolysis of a large Arabidopsis GTPase, Root Hair Defective 3 (RHD3) and showed suitable probing for labile regions near the C-terminus to suggest what protein reconstruction might make RHD3 more suitable for crystallization.

Perturbing Peptide Cation-Radical Electronic States by Thioxoamide Groups: Formation, Dissociations, and Energetics of Thioxopeptide Cation-Radicals

Thioxodipeptides Gly-thio-Lys (GtK), Ala-thio-Lys (AtK), and Ala-thio-Arg (AtR) in which the amide group has been modified to a thioxoamide were made into dications by electrospray ionization and converted to cation-radicals, (GtK + 2H)(+•), (AtK + 2H)(+•), and (AtR + 2H)(+•), by electron transfer dissociation (ETD) tandem mass spectrometry using fluoranthene anion-radical as an electron donor. The common and dominant dissociation of these cation-radicals was the loss of a hydrogen atom. The dissociation products were characterized by collision-induced dissociation (CID) multistage tandem mass spectrometry up to CID-MS(5). The ground electronic states of several (GtK + 2H)(+•), (AtK + 2H)(+•), and (AtR + 2H)(+•) conformers were explored by extensive ab initio and density functional theory calculations of the potential energy surface. In silico electron transfer to the precursor dications, (GtK + 2H)(2+), (AtK + 2H)(2+), and (AtR + 2H)(2+), formed zwitterionic intermediates containing thioenol anion-radical and ammonium cation groups that were local energy minima on the potential energy surface of the ground electronic state. The zwitterions underwent facile isomerization by N-terminal ammonium proton migration to the thioenol anion-radical group forming aminothioketyl intermediates. Combined potential energy mapping and RRKM calculations of dissociation rate constants identified N-C(α) bond cleavages as the most favorable dissociation pathways, in a stark contrast to the experimental results. This discrepancy is interpreted as being due to the population upon electron transfer of low-lying excited electronic states that promote loss of hydrogen atoms. For (GtK + 2H)(+•), these excited states were characterized by time-dependent density functional theory as A-C states that had large components of Rydberg-like 3s molecular orbitals at the N-terminal and lysine ammonium groups that are conducive to hydrogen atom loss.

Reduction in Database Search Space by Utilization of Amino Acid Composition Information from Electron Transfer Dissociation and Higher-Energy Collisional Dissociation Mass Spectra

With high-mass accuracy and consecutively obtained electron transfer dissociation (ETD) and higher-energy collisional dissociation (HCD) tandem mass spectrometry (MS/MS), reliable (≥97%) and sensitive fragment ions have been extracted for identification of specific amino acid residues in peptide sequences. The analytical benefit of these specific amino acid composition (AAC) ions is to restrict the database search space and provide identification of peptides with higher confidence and reduced false negative rates. The 6706 uniquely identified peptide sequences determined with a conservative Mascot score of >30 were used to characterize the AAC ions. The loss of amino acid side chains (small neutral losses, SNLs) from the charge reduced peptide radical cations was studied using ETD. Complementary AAC information from HCD spectra was provided by immonium ions. From the ETD/HCD mass spectra, 5162 and 6720 reliable SNLs and immonium ions were successfully extracted, respectively. Automated application of the AAC information during database searching resulted in an average 3.5-fold higher confidence level of peptide identification. In addition, 4% and 28% more peptides were identified above the significance level in a standard and extended search space, respectively.

Heme Binding in Gas-Phase Holo-Myoglobin Cations: Distal Becomes Proximal?

His64 and His93 are the two well-known sites of heme binding in water-dissolved holo-myoglobin, with His93 being a proximal, strongly binding partner, while the distal His64 weakly coordinates to the heme through a small-molecule ligand, e.g., water or O(2). The heme bonding scheme in a water-free environment is as yet unclear. Here we employed electron transfer dissociation tandem mass spectrometry to study the preferential attachment site of the ferri-heme (Fe(3+)) in electrospray-produced 12+, 14+, and 16+ holo-myoglobin ions. Contrary to expectations, in lower-charge complexes that should have a structure resembling that in solution, the heme seems to be preferentially attached to the "distal" histidine. In contrast, in the highest studied charge state, the "proximal" histidine is the site of preferential attachment; the 14+ charge state is an intermediate case. This surprising finding raises a question of heme coordination in proteins transferred to water-free environment, as well as the effect of the protonation sites on heme bonding.