Desorption Ionization Tandem Mass Spectrometry Research Articles

Protein Extraction, Enrichment and MALDI MS and MS/MS Analysis from Bitter Orange Leaves (Citrus aurantium).

Citrus aurantium is a widespread tree in the Mediterranean area, and it is mainly used as rootstock for other citrus. In the present study, a vacuum infiltration centrifugation procedure, followed by solid phase extraction matrix-assisted laser desorption ionization tandem mass spectrometry (SPE MALDI MS/MS) analysis, was adopted to isolate proteins from leaves. The results of mass spectrometry (MS) profiling, combined with the top-down proteomics approach, allowed the identification of 78 proteins. The bioinformatic databases TargetP, SignalP, ChloroP, WallProtDB, and mGOASVM-Loc were used to predict the subcellular localization of the identified proteins. Among 78 identified proteins, 20 were targeted as secretory pathway proteins and 36 were predicted to be in cellular compartments including cytoplasm, nucleus, and cell membrane. The largest subcellular fraction was the secretory pathway, accounting for 25% of total proteins. Gene Ontology (GO) of Citrus sinensis was used to simplify the functional annotation of the proteins that were identified in the leaves. The Kyoto Encyclopedia of Genes and Genomes (KEGG) showed the enrichment of metabolic pathways including glutathione metabolism and biosynthesis of secondary metabolites, suggesting that the response to a range of environmental factors is the key processes in citrus leaves. Finally, the Lipase GDSL domain-containing protein GDSL esterase/lipase, which is involved in plant development and defense response, was for the first time identified and characterized in Citrus aurantium.

Analysis of monodisperse, sequence-defined, and POSS-functionalized polyester copolymers by MALDI tandem mass spectrometry.

Monodisperse, sequence-defined polymers can be potentially used for digital data storage. This study reports the sequence analysis and differentiation of monodisperse polyester copolymers carrying side chains functionalized in a specific order by polyhedral oligomeric silsesquioxane (POSS) nanoparticles. Steglich esterification and succinic anhydride ring-opening chemistries were utilized iteratively to synthesize the intended sequences, which were characterized by matrix-assisted laser desorption ionization tandem mass spectrometry (MALDI-MS2). Isomeric oligomers were readily distinguished based on their different fragmentation patterns. The sequences embedded in the oligomers were decrypted by their specific backbone dissociation pathways. The robustness of using MALDI-MS2 as a sequencing method for monodisperse synthetic macromolecules was assessed and validated by the characterization of longer oligomers.

Scrambled Internal Standard Method for High-Throughput Protein Quantification by Matrix-Assisted Laser Desorption Ionization Tandem Mass Spectrometry.

Matrix-assisted laser desorption ionization (MALDI) could be advantageous for high-throughput MS acquisition but suffers from low signal reproducibility. The purpose of this study was to establish a reliable MALDI-tandem mass spectrometry (MS/MS)-based high-throughput quantification of tryptic peptides using our newly developed scrambled internal standard (sIS) method. The standard curves obtained with sIS peptides showed good linearity over a wide concentration range (5-1000 fmol/μL) compared to that with the IS-free method, and the coefficient of variation of data points at each concentration (5-1000 fmol/μL) was significantly reduced. Furthermore, the ion suppression effect of digested serum could be normalized with the sIS peptides. Differences of quantitative values obtained by MALDI-MS/MS and liquid chromatography-MS/MS with selected reaction monitoring were within 20% in the presence of 0.1-5 μL of immunoprecipitated model plasma. Furthermore, the effect of amino acid composition on peptide sensitivity was examined, and we found that sensitivity was significantly decreased if an aromatic amino acid was replaced with a nonaromatic amino acid. Thus, high sensitivity required the use of sIS peptides containing an aromatic amino acid. Finally, the sIS method enabled high-throughput quantification of tryptic peptides with high accuracy and a wide dynamic range.

Variable metastatic potentials correlate with differential plectin and vimentin expression in syngeneic androgen independent prostate cancer cells.

Prostate cancer is a clinically heterogeneous disease, ranging from indolent asymptomatic disease to very aggressive metastatic and life threatening forms of the disease. Distant metastasis represents the major lethal cause of prostate cancer. The most critical clinical challenge in the management of the patients is identifying those individuals at risk of developing metastatic disease. To understand the molecular mechanisms of prostate cancer metastasis and identify markers with metastatic potential, we have analyzed protein expression in two syngeneic prostate cancer cells lines PC3-N2 and PC3-ML2 using isobaric tags for relative and absolute quantitation labeling and multi-dimensional protein identification technology liquid chromatography matrix assisted laser desorption ionization tandem mass spectrometry. PC3-N2 is lowly metastatic while PC3-ML2 highly metastatic. A total of 1,756 proteins were identified in the analyses with 130 proteins showing different expression levels (p<0.01) in the two cell lines. Out of these, 68 proteins were found to be significantly up-regulated while 62 are significantly down-regulated in PC3-ML2 cells compared with PC3-N2 cells. The upregulation of plectin and vimentin which were the most significantly differentially expressed were validated by Western blot and their functional relevance with respect to invasion and migration was determined by siRNA gene silencing. To our knowledge, this study is the first to demonstrate that up-regulation of vimentin and plectin expression positively correlates with the invasion and metastasis of androgen-independent PCA.

Pertussis toxoid structure: A collaboration and comparison of two-dimensional liquid chromatography–tandem mass spectrometry, ultraperformance liquid chromatography–mass spectrometryE, and capillary liquid chromatography–matrix-assisted laser desorption ionization–tandem mass spectrometry

The overall structure of pertussis toxoid has been established by analysis of its tryptic digest using two-dimensional liquid chromatography–tandem mass spectrometry (2D-LC–MS/MS), capillary liquid chromatography–matrix-assisted laser desorption ionization–tandem mass spectrometry (CapLC–MALDI–MS/MS), and ultraperformance liquid chromatography–mass spectrometryE (UPLC–MSE). In addition to oxidation and hydrolysis of amino acids losses of terminal peptides are observed. On-line UPLC–MSE generated a similar sequence coverage as the other two methods that involved off-line fraction collection. In light of recent favorable comparisons to data-dependent acquisition, UPLC–MSE should be the initial method of choice for analysis of a peptide mixture of moderate complexity.

Pdc1 Functions in the Assembly of P Bodies in Schizosaccharomyces pombe

P bodies are cytoplasmic RNA granules containing the Dcp1-Dcp2 decapping enzymes where mRNA decay can occur. Here, we describe the characterization of P bodies in the fission yeast Schizosaccharomyces pombe. Most information on the property and function of P bodies stems from studies in the distantly related budding yeast Saccharomyces cerevisiae, and Edc3 was identified as a scaffold protein required for P-body assembly. However, we found that, unlike in S. cerevisiae, fission yeast Edc3 was dispensable for P-body formation. Pdc1, a novel partner of the fission yeast decapping enzyme, with a limited similarity to plant Edc4/Varicose that is required for the assembly of P bodies, was identified (tandem affinity purification-matrix-assisted laser desorption ionization tandem mass spectrometry [TAP-MALDI MS/MS]). Pdc1 interacts with Dcp2 through its C terminus and contains a coiled-coil region for self-interaction to mediate P-body formation. In line with the model that Pdc1 cross-bridges different proteins, additional interactions can be demonstrated with components such as Edc3 and Ste13. Although Pdc1 is not required for the interaction between Dcp1 and Dcp2, our data suggest that Pdc1 acts as a functional homologue of Edc4, a third component of the decapping enzymes that is thought to be absent from fungi. Together, these results highlight the diverse P-body protein compositions between different species and might help to provide insight into their evolutionary paths.

Alterations in Glycopeptides Associated with Herceptin Treatment of Human Breast Carcinoma MCF-7 and T-Lymphoblastoid Cells

The therapeutic humanized monoclonal antibody IgG1 known as Herceptin® has shown remarkable antitumor effects. Although this type of therapy has increased the cancer-free survival of patients, not all tumors respond to this treatment and cancers often develop resistance to the antibody. Despite the fact that Herceptin function has been extensively studied, the precise mechanism underlying its antitumor activity still remains incompletely defined. We previously demonstrated on human breast MCF-7 carcinoma and T-lymphoblastoid CEM cells that monoclonal antibody in combination with Lipoplex consisting of Lipofectamine mixed with plasmid DNA showed a more profound effect on cancer cell viability than antibody alone. The analyses of N-glycans isolated from cancer cells showed dramatic differences in profiles when cells were exposed to Herceptin. Moreover, the investigation of glycosylated peptides from the same cancer cell models after treatment revealed further alterations in the post-translational modifications. Tandem mass spectra obtained from the samples treated confirmed the presence of a series of glycopeptides bearing characteristic oligosaccharides as described in IgG1. However some of them differed by mass differences that corresponded to peptide backbones not described previously and more of them were detected from Herceptin treated samples than from cells transfected with Heceptin/Lipoplex. The results indicate that the presence of Lipoplex prevents antibody transformation and elongates its proper function. The better understanding of the multipart changes described in the glycoconjugates could provide new insights into the mechanism by which antibody induces regression in cancers.

Glycation sites in neoglycoglycoconjugates from the terminal monosaccharide antigen of the O‐PS of Vibrio cholerae O1, serotype Ogawa, and BSA revealed by matrix‐assisted laser desorption–ionization tandem mass spectrometry

We present the MALDI-TOF/TOF-MS analyses of various hapten-bovine serum albumin (BSA) neoglycoconjugates obtained by squaric acid chemistry coupling of the spacer-equipped, terminal monosaccharide of the O-specific polysaccharide of Vibrio cholerae O1, serotype Ogawa, to BSA. These analyses allowed not only to calculate the molecular masses of the hapten-BSA neoglycoconjugates with different hapten-BSA ratios (4.3, 6.6 and 13.2) but, more importantly, also to localize the covalent linkages (conjugation sites) between the hapten and the carrier protein. Determination of the site of glycation was based on comparison of the MALDI-TOF/TOF-MS analysis of the peptides resulting from the digestion of BSA with similar data resulting from the digestion of BSA glycoconjugates, followed by sequencing by MALDI-TOF/TOF-MS/MS of the glycated peptides. The product-ion scans of the protonated molecules were carried out with a MALDI-TOF/TOF-MS/MS tandem mass spectrometer equipped with a high-collision energy cell. The high-energy collision-induced dissociation (CID) spectra afforded product ions formed by fragmentation of the carbohydrate hapten and amino acid sequences conjugated with fragments of the carbohydrate hapten. We were able to identify three conjugation sites on lysine residues (Lys235, Lys437 and Lys455). It was shown that these lysine residues are very reactive and bind lysine specific reagents. We presume that these Lys residues belong to those that are considered to be sterically more accessible on the surface of the tridimensional structure. The identification of the y-series product ions was very useful for the sequencing of various peptides. The series of a- and b-product ions confirmed the sequence of the conjugated peptides.

Protein adducts as prospective biomarkers of nevirapine toxicity.

Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor used against human immunodeficiency virus type-1 (HIV-1), mostly to prevent mother-to-child HIV-1 transmission in developing countries. Despite its clinical efficacy, NVP administration is associated with a variety of toxic responses that include hepatotoxicity and skin rash. Although the reasons for the adverse effects of NVP administration are still unclear, increasing evidence supports the involvement of metabolic activation to reactive electrophiles. In particular, Phase II activation of the NVP metabolite 12-hydroxy-NVP is thought to mediate NVP binding to bionucleophiles, which may be at the onset of toxicity. In the present study, we investigated the nature and specific locations of the covalent adducts produced in human serum albumin and human hemoglobin by reaction in vitro with the synthetic model electrophile 12-mesyloxy-NVP, used as a surrogate for the Phase II metabolite 12-sulfoxy-NVP. Multiple sites of modification were identified by two different mass spectrometry-based methodologies, liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and matrix-assisted laser desorption ionization tandem mass spectrometry (MALDI-TOF-TOF-MS). These two distinct methodologies, which in some instances afforded complementary information, allowed the identification of multiple adducts involving cysteine, lysine, tryptophan, histidine, serine, and the N-terminal valine of hemoglobin. Tryptophan, which is not a common site of covalent protein modification, was the NVP-modified amino acid residue detected in the two proteins and consistently identified by both LC-ESI-MS/MS and MALDI-TOF-TOF-MS. The propensity of tryptophan to react with the NVP-derived electrophile is further emphasized by the fact that human serum albumin possesses a single tryptophan residue, which suggests a remarkable selectivity that may be useful for biomonitoring purposes. Likewise, the NVP adduct with the terminal valine of hemoglobin, detected by LC-ESI-MS/MS after N-alkyl Edman degradation, appears as an easily assessed marker of NVP binding to proteins. Our results demonstrate the merits and complementarity of the two MS-based methodologies for the characterization of protein binding by NVP and suggest a series of plausible biomarkers of NVP toxicity that should be useful in the monitoring of toxicity effects in patients administered NVP.

The Haemophilus influenzae HMW1 Adhesin Is a Glycoprotein with an Unusual N-Linked Carbohydrate Modification

The Haemophilus influenzae HMW1 adhesin mediates adherence to respiratory epithelial cells, a critical early step in the pathogenesis of H. influenzae disease. In recent work, we demonstrated that HMW1 undergoes glycosylation. In addition, we observed that glycosylation of HMW1 is essential for HMW1 tethering to the bacterial surface, a prerequisite for HMW1-mediated adherence to host epithelium. In this study, we examined HMW1 proteolytic fragments by mass spectrometry, achieved 89% amino acid sequence coverage, and identified 31 novel modification sites. All of the modified sites were asparagine residues, in all but one case in the conventional consensus sequence of N-linked glycans, viz. NX(S/T). Liquid chromatography-tandem mass spectrometry analysis using a hybrid linear quadrupole ion trap Fourier transform ion cyclotron mass spectrometer, accurate mass measurements, and deuterium exchange studies established that the modifying glycan structures were mono- or dihexoses rather than the N-acetylated chitobiosyl core that is characteristic of N-glycosylation. This unusual carbohydrate modification suggests that HMW1 glycosylation requires a glycosyltransferase with a novel activity.

Probing peptide libraries from Conus achatinus using mass spectrometry and cDNA sequencing: identification of δ and ω‐conotoxins

The peptide library present in the venom of the piscivorous marine snail Conus achatinus has been probed using a combination of mass spectrometry and cDNA sequencing methods. Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) analysis, before and following global reduction/alkylation of peptide mixtures, permits the rapid classification of individual components on the basis of the number of disulfide bonds. Mass fingerprinting and the reverse phase HPLC retention times permit a further deconvolution of the library in terms of peptide size and hydrophobicity. Sequencing of cDNA derived using O-superfamily specific primers yielded five complete conotoxin precursor sequences, ranging in polypeptide length from 75-87 residues containing six Cys residues at the C-terminus. Sequence analysis permits classification of the five putative mature peptides (Ac 6.1 to Ac 6.5) as delta, omega, and omega-like conotoxins. The presence of these predicted peptides in crude venom was established by direct matrix assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS) sequencing following trypsin digestion of the peptide mixture after global reduction/alkylation. The determination of partial peptide sequences and comparison with the predicted sequences resulted in the identification of four of the five predicted conotoxins. The characterization of posttranslationally modified analogs, which are hydroxylated at proline or amidated at the C-terminus is also demonstrated. Crude venom analysis should prove powerful in studying both inter- and intra-species variation in peptide libraries.

A Tandem Affinity Purification-based Technology Platform to Study the Cell Cycle Interactome in Arabidopsis thaliana

Defining protein complexes is critical to virtually all aspects of cell biology because many cellular processes are regulated by stable protein complexes, and their identification often provides insights into their function. We describe the development and application of a high throughput tandem affinity purification/mass spectrometry platform for cell suspension cultures to analyze cell cycle-related protein complexes in Arabidopsis thaliana. Elucidation of this protein-protein interaction network is essential to fully understand the functional differences between the highly redundant cyclin-dependent kinase/cyclin modules, which are generally accepted to play a central role in cell cycle control, in all eukaryotes. Cell suspension cultures were chosen because they provide an unlimited supply of protein extracts of actively dividing and undifferentiated cells, which is crucial for a systematic study of the cell cycle interactome in the absence of plant development. Here we report the mapping of a protein interaction network around six known core cell cycle proteins by an integrated approach comprising generic Gateway-based vectors with high cloning flexibility, the fast generation of transgenic suspension cultures, tandem affinity purification adapted for plant cells, matrix-assisted laser desorption ionization tandem mass spectrometry, data analysis, and functional assays. We identified 28 new molecular associations and confirmed 14 previously described interactions. This systemic approach provides new insights into the basic cell cycle control mechanisms and is generally applicable to other pathways in plants.

Design, Synthesis, and Application of a Hydrazide-Functionalized Isotope-Coded Affinity Tag for the Quantification of Oxylipid−Protein Conjugates

An isotopically coded affinity probe was developed and evaluated for the characterization and quantification of proteins adducted by 2-alkenals derived from lipid peroxidation (LPO) processes. Lipid-derived 2-alkenals, such as acrolein and 4-hydroxy-2-nonenal (HNE), have the ability to react with cysteine, histidine, and lysine residues in proteins, thus causing protein damage and loss of protein function. Such modifications of proteins are difficult to characterize in biological samples by mass spectrometry due to the complexity of protein extracts and the low abundance of adducted proteins. The novel aldehyde-reactive, hydrazide-functionalized, isotope-coded affinity tag (HICAT) described in this study was found effective for the selective isolation, detection, and quantification of Michael-type adducts of 2-alkenals with proteins using a combination of affinity isolation, nanoLC, and matrix-assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS). The chemical and mass spectrometric properties of the new probe are demonstrated on a model protein treated with HNE. The efficacy of HICAT for the analysis of complex samples was tested using preparations of mitochondrial proteins that were modified in vitro with HNE. The potential of the HICAT strategy for the identification, characterization, and quantification of in vivo oxylipid-protein conjugates is demonstrated on cardiac mitochondrial protein preparations, in which, for example, the ADP/ATP translocase 1 was found adducted to the 2-alkenals, acrolein and 4-hydroxy-2-hexenal, at Cys-256.

Determination of the Disulfide Bond Arrangement of Dengue Virus NS1 Protein

The 12 half-cystines of NS1 proteins are absolutely conserved among flaviviruses, suggesting their importance to the structure and function of these proteins. In the present study, peptides from recombinant Dengue-2 virus NS1 were produced by tryptic digestion in 100% H(2)(16)O, peptic digestion in 50% H(2)(18)O, thermolytic digestion in 50% H(2)(18)O, or combinations of these digestion conditions. Peptides were separated by size exclusion and/or reverse phase high performance liquid chromatography and examined by matrix-assisted laser desorption ionization-time of flight mass spectrometry, matrix-assisted laser desorption ionization post-source decay, and matrix-assisted laser desorption ionization tandem mass spectrometry. Where digests were performed in 50% H(2)(18)O, isotope profiles of peptide ions aided in the identification and characterization of disulfide-linked peptides. It was possible to produce two-chain peptides containing C1/C2, C3/C4, C5/C6, and C7/C12 linkages as revealed by comparison of the peptide masses before and after reduction and by post-source decay analysis. However, the remaining four half-cystines (C8, C9, C10, and C11) were located in a three-chain peptide of which one chain contained adjacent half-cystines (C9 and C10). The linkages of C8/C10 and C9/C11 were determined by tandem mass spectrometry of an in-source decay fragment ion containing C9, C10, and C11. This disulfide bond arrangement provides the basis for further refinement of flavivirus NS1 protein structural models.