Gavin E. Reid

Identification of DIABLO, a Mammalian Protein that Promotes Apoptosis by Binding to and Antagonizing IAP Proteins

To identify proteins that bind mammalian IAP homolog A (MIHA, also known as XIAP), we used coimmuno-precipitation and 2D immobilized pH gradient/SDS PAGE, followed by electrospray ionization tandem mass spectrometry. DIABLO (direct IAP binding protein with low pI) is a novel protein that can bind MIHA and can also interact with MIHB and MIHC and the baculoviral IAP, OpIAP. The N-terminally processed, IAP-interacting form of DIABLO is concentrated in membrane fractions in healthy cells but released into the MIHA-containing cytosolic fractions upon UV irradiation. As transfection of cells with DIABLO was able to counter the protection afforded by MIHA against UV irradiation, DIABLO may promote apoptosis by binding to IAPs and preventing them from inhibiting caspases.

Vesiclepedia: A Compendium for Extracellular Vesicles with Continuous Community Annotation

Vesiclepedia is a community-annotated compendium of molecular data on extracellular vesicles.

ExoCarta 2012: database of exosomal proteins, RNA and lipids

Exosomes are membraneous nanovesicles of endocytic origin released by most cell types from diverse organisms; they play a critical role in cell–cell communication. ExoCarta (http://www.exocarta.org) is a manually curated database of exosomal proteins, RNA and lipids. The database catalogs information from both published and unpublished exosomal studies. The mode of exosomal purification and characterization, the biophysical and molecular properties are listed in the database aiding biomedical scientists in assessing the quality of the exosomal preparation and the corresponding data obtained. Currently, ExoCarta (Version 3.1) contains information on 11 261 protein entries, 2375 mRNA entries and 764 miRNA entries that were obtained from 134 exosomal studies. In addition to the data update, as a new feature, lipids identified in exosomes are added to ExoCarta. We believe that this free web-based community resource will aid researchers in identifying molecular signatures (proteins/RNA/lipids) that are specific to certain tissue/cell type derived exosomes and trigger new exosomal studies.

The Disulfide Bond Structure of Plasmodium Apical Membrane Antigen-1

Apical membrane antigen-1 (AMA-1) of Plasmodium falciparum is one of the leading asexual blood stage antigens being considered for inclusion in a malaria vaccine. The ability of this molecule to induce a protective immune response has been shown to be dependent upon a conformation stabilized by disulfide bonds. In this study we have utilized the reversed-phase high performance liquid chromatography of dithiothreitol-reduced and nonreduced tryptic digests of Plasmodium chabaudi AMA-1 secreted from baculovirus-infected insect cells, in conjunction with N-terminal sequencing and electrospray-ionization mass spectrometry, to identify and assign disulfide-linked peptides. All 16 cysteine residues that are conserved in all known sequences of AMA-1 are incorporated into intramolecular disulfide bonds. Six of the eight bonds have been assigned unequivocally, whereas the two unassigned disulfide bonds connect two Cys-Xaa-Cys sequences separated by 14 residues. The eight disulfide bonds fall into three nonoverlapping groups that define three possible subdomains within the AMA-1 ectodomain. Although the pattern of disulfide bonds within subdomain III has not been fully elucidated, one of only two possible linkage patterns closely resembles the cystine knot motif found in growth factors. Sites of amino acid substitutions in AMA-1 that are well separated in the primary sequence are clustered by the disulfide bonds in subdomains II and III. These findings are consistent with the conclusion that these amino acid substitutions are defining conformational disulfide bond-dependent epitopes that are recognized by protective immune responses.

Proteomic analysis of the human colon carcinoma cell line (LIM 1215): Development of a membrane protein database

The proteomic definition of plasma membrane proteins is an important initial step in searching for novel tumor marker proteins expressed during the different stages of cancer progression. However, due to the charge heterogeneity and poor solubility of membrane‐associated proteins this subsection of the cells proteome is often refractory to two‐dimensional electrophoresis (2‐DE), the current paradigm technology for studying protein expression profiles. Here, we describe a non‐2‐DE method for identifying membrane proteins. Proteins from an enriched membrane preparation of the human colorectal carcinoma cell line LIM1215 were initially fractionated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE, 4—20%). The unstained gel was cut into 16 × 3 mm slices, and peptide mixtures resulting from in‐gel tryptic digestion of each slice were individually subjected to capillary‐column reversed phase‐high performance liquid chromatography (RP‐HPLC) coupled with electrospray ionization‐ ion trap‐ mass spectrometry (ESI‐IT‐MS). Interrogation of genomic databases with the resulting collision‐induced dissociation (CID) generated peptide ion fragment data was used to identify the proteins in each gel slice. Over 284 proteins (including 92 membrane proteins) were identified, including many integral membrane proteins not previously identified by 2‐DE, many proteins seen at the genomic level only, as well as several proteins identified by expressed sequence tags (ESTs) only. Additionally, a number of peptides, identified by de novo MS sequence analysis, have not been described in the databases. Further, a “targeted” ion approach was used to unambiguously identify known low‐abundance plasma membrane proteins, using the membrane‐associated A33 antigen, a gastrointestinal‐specific epithelial cell protein, as an example. Following localization of the A33 antigen in the gel by immunoblotting, ions corresponding to the theoretical A33 antigen tryptic peptide masses were selected using an “inclusion” mass list for automated sequence analysis. Six peptides corresponding to the A33 antigen, present at levels well below those accessible using the standard automated “nontargeted” approach, were identified. The membrane protein database may be accessed via the World Wide Web (WWW) at http://www.ludwig. edu.au/jpsl/jpslhome.html.

Techniques for phosphopeptide enrichment prior to analysis by mass spectrometry

Mass spectrometry is the tool of choice to investigate protein phosphorylation, which plays a vital role in cell regulation and diseases such as cancer. However, low abundances of phosphopeptides and low degrees of phosphorylation typically necessitate isolation and concentration of phosphopeptides prior to MS analysis. This review discusses the enrichment of phosphopeptides with immobilized metal affinity chromatography, reversible covalent binding, and metal oxide affinity chromatography. Capture of phosphopeptides on TiO(2) seems especially promising in terms of selectivity and recovery, but the success of all methods depends on careful selection of binding, washing, and elution solutions. Enrichment techniques are complementary, such that a combination of methods greatly enhances the number of phosphopeptides isolated from complex samples. Development of a standard series of phosphopeptides in a highly complex mixture of digested proteins would greatly aid the comparison of different enrichment methods. Phosphopeptide binding to magnetic beads and on-plate isolation prior to MALDI-MS are emerging as convenient methods for purification of small (microL) samples. On-plate enrichment can yield >70% recoveries of phosphopeptides in mixtures of a few digested proteins and can avoid sample-handling steps, but this technique is likely limited to relatively simple samples such as immunoprecipitates. With recent advances in enrichment techniques in hand, MS analysis should provide important insights into phosphorylation pathways.

Tandem mass spectrometry strategies for phosphoproteome analysis

Protein phosphorylation is involved in nearly all essential biochemical pathways and the deregulation of phosphorylation events has been associated with the onset of numerous diseases. A multitude of tandem mass spectrometry (MS/MS) and multistage MS/MS (i.e., MS(n) ) strategies have been developed in recent years and have been applied toward comprehensive phosphoproteomic analysis, based on the interrogation of proteolytically derived phosphopeptides. However, the utility of each of these MS/MS and MS(n) approaches for phosphopeptide identification and characterization, including phosphorylation site localization, is critically dependant on the properties of the precursor ion (e.g., polarity and charge state), the specific ion activation method that is employed, and the underlying gas-phase ion chemistries, mechanisms and other factors that influence the gas-phase fragmentation behavior of phosphopeptide ions. This review therefore provides an overview of recent studies aimed at developing an improved understanding of these issues, and highlights the advantages and limitations of both established (e.g., CID) and newly maturing (e.g., ECD, ETD, photodissociation, etc.) yet complementary, ion activation techniques. This understanding is expected to facilitate the continued refinement of existing MS/MS strategies, and the development of novel MS/MS techniques for phosphopeptide analysis, with great promise in providing new insights into the role of protein phosphorylation on normal biological function, and in the onset and progression of disease.

A mass spectrometric and ab initio study of the pathways for dehydration of simple glycine and cysteine-containing peptide [M+H]+ ions

The gas phase fragmentation reactions of the [M+H]+ and [M+H−H2O]+ ions of glycylglycine, glycylcysteine, N-acetylglycine, N-acetylcysteine, their corresponding methyl esters, as well as several other related model systems have been examined by electrospray ionization (ESI) tandem mass spectrometry (MSn) using triple quadrupole and quadrupole ion trap mass spectrometers. Two discrete gas phase fragmentation pathways for the loss of water from glycine-containing peptides, corresponding to retro-Koch and retro-Ritter type reactions were observed. Two pathways were also observed for the loss of water from C-terminal cysteine-containing peptides: a retro-Koch type reaction and an intramolecular nucleophilic attack at the carbonyl of the amide bond by the cysteinyl side chain thiol. Various intermediates involved in these reactions, derived from the [M+H−H2O]+ ions of N-formylglycine and N-formylcysteine, were modeled using ab initio calculations at the MP2(FC)/6-31G*//HF/6-31G* level of theory. These calculations indicate that: (i) the retro-Koch reaction product is predicted to be more stable than the product from the retro-Ritter reaction for N-formylglycine, and (ii) the intramolecular nucleophilic attack product is preferred over the retro-Koch and retro-Ritter reaction products for N-formylcysteine. The results from these ab initio calculations are in good agreement with the experimentally determined ion abundances for these processes.

Do all b2 ions have oxazolone structures? Multistage mass spectrometry and ab initio studies on protonated N-acyl amino acid methyl ester model systems

Abstract The tandem mass spectrometry fragmentation reactions of 21 protonated N-acyl amino acid methyl esters are examined as models for more complicated peptides. Four main types of reactions are observed: loss of CH2CO from the N-terminal acetyl group; loss of CH3OH from the C-terminal ester group to yield a model system for a b2 ion structure; loss of water from amino acids without an OH side chain group; fragmentation of the side chain by way of small molecule loss (e.g. H2O, NH3, and CH3SH). CH3OH loss is the only common reaction observed for all systems. The resultant [M+H−CH3OH]+ ions were examined in further detail by way of MS3 experiments because previous studies have shown that the oxozolone structures liberate CO. Only lysine and arginine do not fragment by way of CO, which is suggestive of alternative cyclic structures involving the side chain. Ab initio calculations (at the MP2/6-31G∗//HF/6-31G∗) were carried out on isomeric b2 ions of both types (oxazolone and that involving side-chain interaction) derived from arginine, histidine, lysine, methionine, asparagine, glutamine, and serine. For arginine, histidine, and lysine the cyclic structures involving the side chain are more stable than the oxozolone structures. Finally, solution phase data relevant to the gas phase processes are highlighted.

GAS PHASE ION-MOLECULE REACTIONS IN A MODIFIED ION TRAP: H/D EXCHANGE OF NON-COVALENT COMPLEXES AND COORDINATIVELY UNSATURATED PLATINUM COMPLEXES

A commercially available electrospray ionization ion trap mass spectrometer has been modified to carry out gas phase ion–molecule reactions. The ability to study gas phase ion–molecule reactions in conjunction with collision induced dissociation (CID) based methods and the multistage trapping capabilities of the ion trap have been exploited in two ways: (i) gas phase H/D exchange reactions inside the ion trap, coupled with CID tandem mass spectrometry have been used to provide insights into the reactivity of non covalent complexes of amino acids and simple peptides, and (ii) CID prior to performing ion–molecule reactions has been used to synthesize and examine the reactivity of coordinatively unsaturated platinum complexes.