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Optimization of parameters for coverage of low molecular weight proteins

Müller, Stephan A.; Kohajda, Tibor; Findeiß, Sven; Stadler, Peter F.; Washietl, Stefan; Kellis, Manolis; von Bergen, Martin; Kalkhof, Stefan
Fonte: Springer-Verlag Publicador: Springer-Verlag
Tipo: Artigo de Revista Científica
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Proteins with molecular weights of <25 kDa are involved in major biological processes such as ribosome formation, stress adaption (e.g., temperature reduction) and cell cycle control. Despite their importance, the coverage of smaller proteins in standard proteome studies is rather sparse. Here we investigated biochemical and mass spectrometric parameters that influence coverage and validity of identification. The underrepresentation of low molecular weight (LMW) proteins may be attributed to the low numbers of proteolytic peptides formed by tryptic digestion as well as their tendency to be lost in protein separation and concentration/desalting procedures. In a systematic investigation of the LMW proteome of Escherichia coli, a total of 455 LMW proteins (27% of the 1672 listed in the SwissProt protein database) were identified, corresponding to a coverage of 62% of the known cytosolic LMW proteins. Of these proteins, 93 had not yet been functionally classified, and five had not previously been confirmed at the protein level. In this study, the influences of protein extraction (either urea or TFA), proteolytic digestion (solely, and the combined usage of trypsin and AspN as endoproteases) and protein separation (gel- or non-gel-based) were investigated. Compared to the standard procedure based solely on the use of urea lysis buffer...

Real-time Hydrogen/Deuterium Exchange Kinetics via Supercharged Electrospray Ionization Tandem Mass Spectrometry

Sterling, Harry J.; Williams, Evan R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Amide hydrogen/deuterium exchange (HDX) rate constants of bovine ubiquitin in an ammonium acetate solution containing 1% of the electrospray ionization (ESI) “supercharging” reagent m-nitrobenzyl alcohol (m-NBA) were obtained using top-down, electron transfer dissociation (ETD) tandem mass spectrometry (MS). The supercharging reagent replaces the acid and temperature “quench” step in the conventional MS approach to HDX experiments by causing rapid protein denaturation to occur in the ESI droplet. The higher charge state ions that are produced with m-NBA are more unfolded, as measured by ion mobility, and result in higher fragmentation efficiency and higher sequence coverage with ETD. Single amino acid resolution was obtained for 44 of 72 exchangeable amide sites, and summed kinetic data were obtained for regions of the protein where adjacent fragment ions were not observed, resulting in an overall spatial resolution of 1.3 residues. Comparison of these results with previous values from NMR indicates that the supercharging reagent does not cause significant structural changes to the protein in the initial ESI solution, and that scrambling or back-exchange is minimal. This new method for top-down HDX-MS enables real-time kinetic data measurements under physiological conditions...

Characterization and Comparison of Disulfide Linkages and Scrambling Patterns in Therapeutic Monoclonal Antibodies - Using LC-MS with Electron Transfer Dissociation

Wang, Yi; Lu, Qiaozhen; Wu, Shiaw-Lin; Karger, Barry L.; Hancock, William S.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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The disulfides in three monoclonal antibodies (mAb), the anti-HER2, anti-CD11a, and GLP-1 with IgG4-Fc fusion protein, were completely mapped by LC-MS with the combination of ETD and CID fragmentation. In addition to mapping the 4 inter- and 12 intra-chain disulfides (total 16), the identification of scrambled disulfides in degraded samples (heat-stress) was achieved. The scrambling was likely attributed to an initial breakage between the light (Cys 214) and heavy (Cys 223) chains in anti-HER2, with the same observation found in a similar therapeutic mAb, anti-CD11a. On the other hand, the fusion antibody, with no light chain but containing only 2 heavy chains, generated much less scrambling under the same heat-stressed conditions. The preferred sites of scrambling were identified, such as the intra-chain disulfide for CxxC in the heavy chain, and the C194 of the heavy chain pairing with the terminal Cys residue (C214) in the light chain. The inter-chain disulfides between the light and heavy chains were weaker than the inter-chain disulfides between the two heavy chains. The relative high abundance ions observed in ETD provided strong evidence for the linked peptide information, which was particularly useful for the identification of the scrambled disulfides. The use of SDS-PAGE helped the separation of these misfolded proteins for the determination of scrambled disulfide linkages. This methodology is useful for comparison of disulfide stability generated from different structural designs...

O-GlcNAcylation of the Plum pox virus capsid protein catalyzed by SECRET AGENT: characterization of O-GlcNAc sites by electron transfer dissociation mass spectrometry

Kim, Young-Cheon; Udeshi, Namrata D.; Balsbaugh, Jeremy L.; Shabanowitz, Jeffrey; Hunt, Donald F.; Olszewski, Neil E.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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The capsid protein of Plum pox virus (PPV-CP) is modified with O-linked β-N-acetylglucosamine (O-GlcNAc). In Arabidopsis thaliana this modification is made by an O-GlcNAc transferase named SECRET AGENT (SEC). Modification of PPV-CP by SEC is hypothesized to have a direct role in the infection process, because virus titer and rate of spread are reduced in SEC mutants. Previous studies used deletion mapping and site-directed mutagenesis to identify four O-GlcNAc sites on the capsid protein that are modified by Escherichia coli-expressed SEC. The infection process was not affected when two of these sites were mutated suggesting that O-GlcNAcylation of these sites does not have a significant role in the infection process or that a subset of the modifications is sufficient. Since it is possible that the mutational mapping approach missed or incorrectly identified O-GlcNAc sites, the modifications produced by E. coli-expressed SEC were characterized using mass spectrometry. O-GlcNAcylated peptides were enzymatically tagged with galactose, the products were enriched on immobilized Ricinus communis agglutinin I and sequenced by electron transfer dissociation (ETD) mass spectrometry. Five O-GlcNAc sites on PPV-CP were identified. Two of these sites were not identified in by the previous mutational mapping. In addition...

The Virus-Host Interface: Exploring Dynamic Protein Interactions via Targeted Proteomics

Cristea, I.
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /10/2011 Português
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Dynamic protein interactions carry out the majority of the processes within a cell, including cellular responses to environmental stimuli and pathogens. Isolation and characterization of protein complexes can provide invaluable insights into their biological functions. The development of approaches that can access stable and transient interactions is invaluable for numerous fields of study, including that of temporal and spatial virus-host protein interactions. Viruses have co-evolved with their hosts, developing remarkable mechanisms for subverting cellular processes for their own benefit. The study of virus host interactions has therefore emerged as a driving force in infectious disease research. Despite these efforts, the protein interactome remains in large part uncharted, and our knowledge of mechanisms controlling the outcome of an infection is limited. Modern proteomics techniques are currently emerging as powerful tools, bringing a new perspective to the field of virology. This presentation will describe the integration of targeted proteomics with genetic, molecular biology, and bioinformatics techniques for studying dynamic virus-host protein associations. Strategies for isolating protein complexes, quantifying infection-triggered changes in interactions...

Enhancing the Identification of Phosphopeptides from Putative Basophilic Kinase Substrates Using Ti (IV) Based IMAC Enrichment*

Zhou, Houjiang; Low, Teck Y.; Hennrich, Marco L.; van der Toorn, Henk; Schwend, Thomas; Zou, Hanfa; Mohammed, Shabaz; Heck, Albert J. R.
Fonte: The American Society for Biochemistry and Molecular Biology Publicador: The American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Metal and metal oxide chelating-based phosphopeptide enrichment technologies provide powerful tools for the in-depth profiling of phosphoproteomes. One weakness inherent to current enrichment strategies is poor binding of phosphopeptides containing multiple basic residues. The problem is exacerbated when strong cation exchange (SCX) is used for pre-fractionation, as under low pH SCX conditions phosphorylated peptides with multiple basic residues elute with the bulk of the tryptic digest and therefore require more stringent enrichment. Here, we report a systematic evaluation of the characteristics of a novel phosphopeptide enrichment approach based on a combination of low pH SCX and Ti4+-immobilized metal ion affinity chromatography (IMAC) comparing it one-to-one with the well established low pH SCX-TiO2 enrichment method. We also examined the effect of 1,1,1,3,3,3-hexafluoroisopropanol (HFP), trifluoroacetic acid (TFA), or 2,5-dihydroxybenzoic acid (DHB) in the loading buffer, as it has been hypothesized that high levels of TFA and the perfluorinated solvent HFP improve the enrichment of phosphopeptides containing multiple basic residues. We found that Ti4+-IMAC in combination with TFA in the loading buffer, outperformed all other methods tested...

Eye Tracking Dysfunction in Schizophrenia: Characterization and Pathophysiology

Levy, Deborah L.; Sereno, Anne B.; Gooding, Diane C.; O’Driscoll, Gilllian A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em //2010 Português
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Eye tracking dysfunction (ETD) is one of the most widely replicated behavioral deficits in schizophrenia and is over-represented in clinically unaffected first-degree relatives of schizophrenia patients. Here, we provide an overview of research relevant to the characterization and pathophysiology of this impairment. Deficits are most robust in the maintenance phase of pursuit, particularly during the tracking of predictable target movement. Impairments are also found in pursuit initiation and correlate with performance on tests of motion processing, implicating early sensory processing of motion signals. Taken together, the evidence suggests that ETD involves higher-order structures, including the frontal eye fields, which adjust the gain of the pursuit response to visual and anticipated target movement, as well as early parts of the pursuit pathway, including motion areas (the middle temporal area and the adjacent medial superior temporal area). Broader application of localizing behavioral paradigms in patient and family studies would be advantageous for refining the eye tracking phenotype for genetic studies.

Designer Reagents for Mass Spectrometry-Based Proteomics: Clickable Cross-Linkers for Elucidation of Protein Structures and Interactions

Sohn, Chang Ho; Agnew, Heather D.; Lee, J. Eugene; Sweredoski, Michael J.; Graham, Robert L.J.; Smith, Geoffrey T.; Hess, Sonja; Czerwieniec, Gregg; Loo, Joseph A.; Heath, James R.; Deshaies, Raymond J.; Beauchamp, J. L.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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We present novel homobifunctional amine-reactive clickable cross-linkers (CXLs) for investigation of three-dimensional protein structures and protein-protein interactions (PPIs). CXLs afford consolidated advantages not previously available in a simple cross-linker, including (1) their small size and cationic nature at physiological pH, resulting in good water solubility and cell permeability, (2) an alkyne group for bio-orthogonal conjugation to affinity tags via the click reaction for enrichment of cross-linked peptides, (3) a nucleophilic displacement reaction involving the 1,2,3-triazole ring formed in the click reaction, yielding a lock-mass reporter ion for only clicked peptides, and (4) higher charge states of cross-linked peptides in the gas-phase for augmented electron transfer dissociation (ETD) yields. Ubiquitin, a lysine-abundant protein, is used as a model system to demonstrate structural studies using CXLs. To validate the sensitivity of our approach, biotin-azide labeling and subsequent enrichment of cross-linked peptides are performed for cross-linked ubiquitin digests mixed with yeast cell lysates. Cross-linked peptides are detected and identified by collision induced dissociation (CID) and ETD with linear quadrupole ion trap (LTQ)-Fourier transform ion cyclotron resonance (FTICR) and LTQ-Orbitrap mass spectrometers. The application of CXLs to more complex systems (e.g....

An N-glycosylation Analysis of Human Alpha-2-Macroglobulin Using an Integrated Approach

Lin, Zhenxin; Lo, Andy; Simeone, Diane M.; Ruffin, Mack T.; Lubman, David M.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 31/05/2012 Português
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Assignment of glycosylation sites and site microheterogeneity is of both biological and clinical significance. Herein, the detailed N-glycosylation pattern of human serum alpha-2-macroglobulin was studied using an integrative approach, including permethylation of N-glycans, collision induced dissociation (CID) and electron transfer dissociation (ETD) of chymotryptic N-glycopeptides, and partial deglycosylation of chymotryptic N-glycopeptides with endo-β-N-acetylglucosaminidase F3 (Endo F3). Three N-glycosylation sites were found to be occupied by four biantennary complex type N-glycans using N-glycan analysis and the ETD/CID method. Endo F3 assisted mass spectrometric analysis yielded five N-glycosylation sites with and without core fucosylation. In total, six out of eight potential N-glycosylation sites were identified using this approach. This integrative approach was performed using only 10 μL of human serum for both N-glycosylation site assignment and site microheterogeneity determination.

Comparison of MS/MS Methods for Characterization of DNA/cisplatin Adducts

Xu, Zhe; Shaw, Jared B.; Brodbelt, Jennifer S.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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The development of activation/dissociation techniques such as ultraviolet photodissociation (UVPD), infrared multiphoton dissociation (IRMPD) and electron transfer dissociation (ETD) as alternatives to collision induced dissociation (CID) has extended the range of strategies for characterizing biologically relevant molecules. Here, we describe a comprehensive comparison of CID, IRMPD, UVPD, ETD and hybrid processes termed ETcaD and ET-IRMPD (and analogous hybrid methods in the negative mode NETcaD and NET-IRMPD) for generating sequence specific-fragment ions and allowing adduction sites to be pinpointed for DNA/cisplatin adducts. Among the six MS/MS methods, the numerous products generated by the IRMPD and UVPD techniques resulted in the most specific and extensive backbone cleavages. We conclude that IRMPD and UVPD methods generally offer the best characteristics for pinpointing the cisplatin adduction sites in the fragment-rich spectra.

Probing the Complementarity of FAIMS and Strong Cation Exchange Chromatography in Shotgun Proteomics

Creese, Andrew J.; Shimwell, Neil J.; Larkins, Katherine P. B.; Heath, John K.; Cooper, Helen J.
Fonte: Springer-Verlag Publicador: Springer-Verlag
Tipo: Artigo de Revista Científica
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High field asymmetric waveform ion mobility spectrometry (FAIMS), also known as differential ion mobility spectrometry, coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) offers benefits for the analysis of complex proteomics samples. Advantages include increased dynamic range, increased signal-to-noise, and reduced interference from ions of similar m/z. FAIMS also separates isomers and positional variants. An alternative, and more established, method of reducing sample complexity is prefractionation by use of strong cation exchange chromatography. Here, we have compared SCX-LC-MS/MS with LC-FAIMS-MS/MS for the identification of peptides and proteins from whole cell lysates from the breast carcinoma SUM52 cell line. Two FAIMS approaches are considered: (1) multiple compensation voltages within a single LC-MS/MS analysis (internal stepping) and (2) repeat LC-MS/MS analyses at different and fixed compensation voltages (external stepping). We also consider the consequence of the fragmentation method (electron transfer dissociation or collision-induced dissociation) on the workflow performance. The external stepping approach resulted in a greater number of protein and peptide identifications than the internal stepping approach for both ETD and CID MS/MS...

Characterization of Host-Cell Line Specific Glycosylation Profiles of Early Transmitted/Founder HIV-1 gp120 Envelope Proteins

Go, Eden P.; Liao, Hua-Xin; Alam, S. Munir; Hua, David; Haynes, Barton F.; Desaire, Heather
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Glycosylation plays an essential role in regulating protein function by modulating biological, structural, and therapeutic properties. However, due to its inherent heterogeneity and diversity, the comprehensive analysis of protein glycosylation remains a challenge. As part of our continuing effort in the analysis of glycosylation profiles of recombinant HIV-1 envelope-based immunogens, we evaluated and compared the host-cell specific glycosylation pattern of recombinant HIV-1 surface glycoprotein, gp120, derived from clade C transmitted/founder virus 1086.C expressed in Chinese hamster ovary (CHO) and human embryonic kidney containing T antigen (293T) cell lines. We used an integrated glycopeptide-based mass mapping workflow that includes a partial deglycosylation step described in our previous study1 with the inclusion of the fragmentation technique, electron transfer dissociation (ETD), to complement collision induced dissociation (CID). The inclusion of ETD facilitated the analysis by providing additional validation for glycopeptide identification and expanding the identified glycopeptides to include coverage of O-linked glycosylation. The site-specific glycosylation analysis shows that the transmitted/founder 1086.C gp120 expressed in CHO and 293T displayed distinct similarities and differences. For N-linked glycosylation...

UniNovo: a universal tool for de novo peptide sequencing

Jeong, Kyowon; Kim, Sangtae; Pevzner, Pavel A.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
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Motivation: Mass spectrometry (MS) instruments and experimental protocols are rapidly advancing, but de novo peptide sequencing algorithms to analyze tandem mass (MS/MS) spectra are lagging behind. Although existing de novo sequencing tools perform well on certain types of spectra [e.g. Collision Induced Dissociation (CID) spectra of tryptic peptides], their performance often deteriorates on other types of spectra, such as Electron Transfer Dissociation (ETD), Higher-energy Collisional Dissociation (HCD) spectra or spectra of non-tryptic digests. Thus, rather than developing a new algorithm for each type of spectra, we develop a universal de novo sequencing algorithm called UniNovo that works well for all types of spectra or even for spectral pairs (e.g. CID/ETD spectral pairs). UniNovo uses an improved scoring function that captures the dependences between different ion types, where such dependencies are learned automatically using a modified offset frequency function.

Tandem MS Analysis of Selenamide-Derivatized Peptide Ions

Zhang, Yun; Zhang, Hao; Cui, Weidong; Chen, Hao
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Our previous study showed that selenamide reagents such as ebselen and N-(phenylseleno) phthalimide (NPSP) can be used for selective and rapid derivatization of protein/peptide thiols in high conversion yield. This paper reports the systematic investigation of MS/MS dissociation behaviors of selenamide-derivatized peptide ions upon collision induced dissociation (CID) and electron transfer dissociation (ETD). In the positive ion mode, derivatized peptide ions exhibit tag-dependent CID dissociation pathways. For instance, ebselen-derivatized peptide ions preferentially undergo Se–S bond cleavage upon CID to produce a characteristic fragment ion, the protonated ebselen (m/z 276), which allows selective identification of thiol peptides from protein digest as well as selective detection of thiol proteins from protein mixture using precursor ion scan (PIS). In contrast, NPSP-derivatized peptide ions retain their phenylselenenyl tags during CID, which is useful in sequencing peptides and locating cysteine residues. In the negative ion CID mode, both types of tags are preferentially lost via the Se–S cleavage, analogous to the S–S bond cleavage during CID of disulfide-containing peptide anions. In consideration of the convenience in preparing selenamide-derivatized peptides and the similarity of Se–S of the tag to the S–S bond...

Staphylococcus aureus Clinical Isolates: Antibiotic Susceptibility, Molecular Characteristics, and Ability to Form Biofilm

Indrawattana, N.; Sungkhachat, O.; Sookrung, N.; Chongsa-nguan, M.; Tungtrongchitr, A.; Voravuthikunchai, S. P.; Kong-ngoen, T.; Kurazono, H.; Chaicumpa, W.
Fonte: Hindawi Publishing Corporation Publicador: Hindawi Publishing Corporation
Tipo: Artigo de Revista Científica
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Periodic monitoring of Staphylococcus aureus characteristics in a locality is imperative as their drug-resistant variants cause treatment problem. In this study, antibiograms, prevalence of toxin genes (sea-see, seg-ser, seu, tsst-1, eta, etb, and etd), PFGE types, accessory gene regulator (agr) groups, and ability to form biofilm of 92 S. aureus Thailand clinical isolates were investigated. They were classified into 10 drug groups: groups 1–7 (56 isolates) were methicillin resistant (MRSA) and 8–10 (36 isolates) were methicillin sensitive (MSSA). One isolate did not have any toxin gene, 4 isolates carried one toxin gene (seq), and 87 isolates had two or more toxin genes. No isolate had see, etb, or tsst-1; six isolates had eta or etd. Combined seg-sei-sem-sen-seo of the highly prevalent egc locus was 26.1%. The seb, sec, sel, seu, and eta associated significantly with MSSA; sek was more in MRSA. The sek-seq association was 52.17% while combined sed-sej was not found. Twenty-three PFGE types were revealed, no association of toxin genes with PFGE types. All four agr groups were present; agr group 1 was predominant (58.70%) but agr group 2 strains carried more toxin genes and were more frequent toxin producers. Biofilm formation was found in 72.83% of the isolates but there was no association with antibiograms. This study provides insight information on molecular and phenotypic markers of Thailand S. aureus clinical isolates which should be useful for future active surveillance that aimed to control a spread of existing antimicrobial resistant bacteria and early recognition of a newly emerged variant.

Front-End Electron Transfer Dissociation: A New Ionization Source

Earley, Lee; Anderson, Lissa C.; Bai, Dina L.; Mullen, Christopher; Syka, John E. P.; English, A. Michelle; Dunyach, Jean-Jacques; Stafford, George C.; Shabanowitz, Jeffrey; Hunt, Donald F.; Compton, Philip D.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Electron transfer dissociation (ETD), a technique that provides efficient fragmentation while depositing little energy into vibrational modes, has been widely integrated into proteomics workflows. Current implementations of this technique, as well as other ion–ion reactions like proton transfer, involve sophisticated hardware, lack robustness, and place severe design limitations on the instruments to which they are attached. Described herein is a novel, electrical discharge-based reagent ion source that is located in the first differentially pumped region of the mass spectrometer. The reagent source was found to produce intense reagent ion signals over extended periods of time while having no measurable impact on precursor ion signal. Further, the source is simple to construct and enables implementation of ETD on any instrument without modification to footprint. Finally, in the context of hybrid mass spectrometers, relocation of the reagent ion source to the front of the mass spectrometer enables new approaches to gas phase interrogation of intact proteins.

N- and O-Glycosylation in the Murine Synaptosome*

Trinidad, Jonathan C.; Schoepfer, Ralf; Burlingame, Alma L.; Medzihradszky, Katalin F.
Fonte: The American Society for Biochemistry and Molecular Biology Publicador: The American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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We present the first large scale study characterizing both N- and O-linked glycosylation in a site-specific manner on hundreds of proteins. We demonstrate that a lectin-affinity fractionation step using wheat germ agglutinin enriches not only peptides carrying intracellular O-GlcNAc, but also those bearing ER/Golgi-derived N- and O-linked carbohydrate structures. Liquid chromatography-MS (LC/MS) analysis with high accuracy precursor mass measurements and high sensitivity ion trap electron-transfer dissociation (ETD) were utilized for structural characterization of glycopeptides. Our results reveal both the identity of the precise sites of glycosylation and information on the oligosaccharide structures possible on these proteins. We report a novel iterative approach that allowed us to interpret the ETD data set directly without making prior assumptions about the nature and distribution of oligosaccharides present in our glycopeptide mixture. Over 2500 unique N- and O-linked glycopeptides were identified on 453 proteins. The extent of microheterogeneity varied extensively, and up to 19 different oligosaccharides were attached at a given site. We describe the presence of the well-known mucin-type structures for O-glycosylation, an EGF-domain-specific fucosylation and a rare O-mannosylation on the transmembrane phosphatase Ptprz1. Finally...

Does Electron Capture Dissociation Cleave Protein Disulfide Bonds?

Ganisl, Barbara; Breuker, Kathrin
Fonte: WILEY-VCH Verlag Publicador: WILEY-VCH Verlag
Tipo: Artigo de Revista Científica
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Peptide and protein characterization by mass spectrometry (MS) relies on their dissociation in the gas phase into specific fragments whose mass values can be aligned as ‘mass ladders’ to provide sequence information and to localize possible posttranslational modifications. The most common dissociation method involves slow heating of even-electron (M+n H)n+ ions from electrospray ionization by energetic collisions with inert gas, and cleavage of amide backbone bonds. More recently, dissociation methods based on electron capture or transfer were found to provide far more extensive sequence coverage through unselective cleavage of backbone N–Cα bonds. As another important feature of electron capture dissociation (ECD) and electron transfer dissociation (ETD), their unique unimolecular radical ion chemistry generally preserves labile posttranslational modifications such as glycosylation and phosphorylation. Moreover, it was postulated that disulfide bond cleavage is preferred over backbone cleavage, and that capture of a single electron can break both a backbone and a disulfide bond, or even two disulfide bonds between two peptide chains. However, the proposal of preferential disulfide bond cleavage in ECD or ETD has recently been debated. The experimental data presented here reveal that the mechanism of protein disulfide bond cleavage is much more intricate than previously anticipated.

Proteomic Analysis and Molecular Modeling Characterize the Iron-Regulatory Protein, Hemojuvelin/Repulsive Guidance Molecule c

Nili, Mahta; David, Larry; Elferich, Johannes; Shinde, Ujwal; Rotwein, Peter
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/05/2013 Português
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Hemojuvelin (HJV) plays a key role in iron metabolism in mammals by regulating expression of the liver-derived hormone, hepcidin, which controls systemic iron uptake and release. Mutations in HJV cause juvenile hemochromatosis, a rapidly progressing iron overload disorder in humans. HJV, also known as repulsive guidance molecule c (RGMc), is a member of the three-protein RGM family. RGMs are GPI-linked glycoproteins that share ~50% amino acid identity and several structural motifs, including the presence of 14 cysteines in analogous locations. Unlike RGMa and RGMb, HJV/RGMc is composed of both single-chain and two-chain isoforms. To date there is no structural information for any member of the RGM family. Here we have mapped the disulfide bonds in mouse HJV/RGMc using a proteomics strategy combining sequential mass spectrometry (MS) steps composed of electron transfer dissociation (ETD) and collision-induced dissociation (CID), in which ETD induces cleavage of disulfide linkages, and CID establishes disulfide bond assignments between liberated peptides. Our results identify an HJV/RGMc molecular species containing 4 disulfide linkages. We predict using ab initio modeling that this molecule is a single-chain HJV/RGMc isoform. Our observations outline a general approach using tandem MS and ab initio molecular modeling to define unknown structural features in proteins.

Gas-phase Ion Isomer Analysis Reveals the Mechanism of Peptide Sequence Scrambling

Jia, Chenxi; Wu, Zhe; Lietz, Christopher B.; Liang, Zhidan; Cui, Qiang; Li, Lingjun
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Peptide sequence scrambling during mass spectrometry-based gas-phase fragmentation analysis causes misidentification of peptides and proteins. Thus, there is a need to develop an efficient approach to probing the gas-phase fragment ion isomers related to sequence scrambling and the underlying fragmentation mechanism, which will facilitate the development of bioinformatics algorithm for proteomics research. Herein, we report on the first use of electron transfer dissociation (ETD)-produced diagnostic fragment ions to probe the components of gas-phase peptide fragment ion isomers. In combination with ion mobility spectrometry (IMS) and formaldehyde labeling, this novel strategy enables qualitative and quantitative analysis of b-type fragment ion isomers. ETD fragmentation produced diagnostic fragment ions indicative of the precursor ion isomer components, and subsequent IMS analysis of b ion isomers provided their quantitative and structural information. The isomer components of three representative b ions (b9, b10, and b33 from three different peptides) were accurately profiled by this method. IMS analysis of the b9 ion isomers exhibited dynamic conversion among these structures. Furthermore, molecular dynamics simulation predicted theoretical drift time values which were in good agreement with experimentally measured values. Our results strongly support the mechanism of peptide sequence scrambling via b ion cyclization...