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Analysis of phosphotyrosine signaling in glioblastoma identifies STAT5 as a novel downstream target of ΔEGFR

Chumbalkar, Vaibhav; Latha, Khatri; Hwang, YeoHyeon; Maywald, Rebecca; Hawley, Lauren; Sawaya, Raymond; Diao, Lixia; Baggerly, Keith; Cavenee, Webster K.; Furnari, Frank B.; Bogler, Oliver
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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An in-frame deletion mutation in Epidermal Growth Receptor (EGFR), ΔEGFR is a common and potent oncogene in glioblastoma (GBM), promoting growth and survival of cancer cells. This mutated receptor is ligand independent and constitutively active. Its activity is low in intensity and thought to be qualitatively different from acutely ligand stimulated wild type receptor implying that the preferred downstream targets of ΔEGFR play a significant role in malignancy. To understand the ΔEGFR signal we compared it to that of a kinase-inactivated mutant of ΔEGFR and wild-type EGFR with shotgun phosphoproteomics using an electron-transfer dissociation (ETD) enabled ion trap mass spectrometer. We identified and quantified 354 phosphopeptides corresponding to 249 proteins. Among the ΔEGFR-associated phosphorylations were the previously described Gab1, c-Met and Mig-6, and also novel phosphorylations including that of STAT5 on Y694/9. We have confirmed the most prominent phosphorylation events in cultured cells and in murine xenograft models of glioblastoma. Pathway analysis of these proteins suggests a preference for an alternative signal transduction pathway by ΔEGFR compared to wild type EGFR. This understanding will potentially benefit the search for new therapeutic targets for ΔEGFR expressing tumors.

Autophosphorylation in the Leucine-Rich Repeat Kinase 2 (LRRK2) GTPase Domain Modifies Kinase and GTP-Binding Activities

Webber, Philip J.; Smith, Archer D.; Sen, Saurabh; Renfrow, Matthew B.; Mobley, James A.; West, Andrew B.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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The LRRK2 protein has both GTPase and kinase activities and mutation in either enzymatic domain can cause late-onset Parkinson’s disease (PD). Nucleotide binding in the GTPase domain may be required for kinase activity and residues in the GTPase domain are potential sites for autophosphorylation, suggesting a complex mechanism of intrinsic regulation. To further define the effects of LRRK2 autophosphorylation, we applied a technique optimal for detection of protein phosphorylation, electron transfer dissociation (ETD), and identified autophosphorylation events exclusively nearby the nucleotide binding pocket in the GTPase domain. PD-linked mutations alter kinase activity but did not alter autophosphorylation site specificity or sites of phosphorylation in a robust in vitro substrate myelin basic protein. Amino-acid substitutions in the GTPase domain have large effects on kinase activity, as insertion of the GTPase-associated R1441C pathogenic mutation together with the G2019S kinase-domain mutation resulted in a multiplicative increase (~7-fold) in activity. Removal of a conserved autophosphorylation site (T1503) by mutation to an alanine residue resulted in greatly decreased GTP-binding and kinase activity. While autophosphorylation likely serves to potentiate kinase activity...

Determination of staphylococcal exotoxins, SCCmec types, and genetic relatedness of Staphylococcus intermedius group isolates from veterinary staff, companion animals, and hospital environments in Korea

Youn, Jung-Ho; Koo, Hye Cheong; Ahn, Kuk Ju; Lim, Suk-Kyung; Park, Yong Ho
Fonte: The Korean Society of Veterinary Science Publicador: The Korean Society of Veterinary Science
Tipo: Artigo de Revista Científica
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The Staphylococcus (S.) intermedius group (SIG) has been a main research subject in recent years. S. pseudintermedius causes pyoderma and otitis in companion animals as well as foodborne diseases. To prevent SIG-associated infection and disease outbreaks, identification of both staphylococcal exotoxins and staphylococcal cassette chromosome mec (SCCmec) types among SIG isolates may be helpful. In this study, it was found that a single isolate (one out of 178 SIG isolates examined) harbored the canine enterotoxin SEC gene. However, the S. intermedius exfoliative toxin gene was found in 166 SIG isolates although the S. aureus-derived exfoliative toxin genes, such as eta, etb and etd, were not detected. SCCmec typing resulted in classifying one isolate as SCCmec type IV, 41 isolates as type V (including three S. intermedius isolates), and 10 isolates as non-classifiable. Genetic relatedness of all S. pseudintermedius isolates recovered from veterinary staff, companion animals, and hospital environments was determined by pulsed-field gel electrophoresis. Strains having the same band patterns were detected in S. pseudintermedius isolates collected at 13 and 18 months, suggesting possible colonization and/or expansion of a specific S. pseudintermedius strain in a veterinary hospital.

Discovery and Characterization of the Crustacean Hyperglycemic Hormone Precursor Related Peptides (CPRP) and Orcokinin Neuropeptides in the Sinus Glands of the Blue Crab Callinectes sapidus Using Multiple Tandem Mass Spectrometry Techniques

Hui, Limei; Cunningham, Robert; Zhang, Zichuan; Cao, Weifeng; Jia, Chenxi; Li, Lingjun
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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The crustacean sinus gland (SG) is a well-defined neuroendocrine site that produces numerous hemolymph-borne agents including the most complex class of endocrine signaling molecules—neuropeptides. Via a multifaceted mass spectrometry (MS) approach, 70 neuropeptides were identified including orcokinins, orcomyotropin, crustacean hyperglycemic hormone (CHH) precursor-related peptides (CPRPs), red pigment concentrating hormone (RPCH), pigment dispersing hormone (PDH), proctolin, RFamides, RYamides, and HL/IGSL/IYRamide. Among them, 15 novel orcokinins, 9 novel CPRPs, one novel orcomyotropin, one novel Ork/Orcomyotropin-related and one novel PDH were de novo sequenced via collision induced dissociation (CID) from the SG of a model organism Callinectes sapidus. Electron transfer dissociation (ETD) was used for sequencing of intact CPRPs due to their large size and charge state. Capillary isoelectric focusing (CIEF) was employed for separation of members of the orcokinin family which is one of the most abundant neuropeptide families observed in the SG. Collectively, our study represents the most complete characterization of neuropeptides of the SG and provides a foundation for future investigation of the physiological function of neuropeptides in the SG of C. sapidus.

University of Nebraska Medical Center Mass Spectrometry and Proteomics Core Facility

Ciborowski, P.; Wojtkiewicz, M.
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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The UNMC Mass Spectrometry and Proteomics Core Facility offers a broad range of services, such as ESI and MALDI protein identification using Mascot and Sequest Algorithms, iTRAQ-based quantitative proteomics, MRM protein quantitation, phosphoproteomics profiling and molecular weight determination for proteins, peptides and small molecules. The facility is equipped with LTQ Orbitrap ETD, LTQ Velos, 4800 MALDI TOF-TOF, 4000 Q TRAP, all with supporting nano-LC systems. Although the majority of users are investigators from UNMC, we also provide services for other outside academics and corporations. For further information, visit our website: www.unmc.edu/mspcf.

Hydrogen/Deuterium Exchange and Electron-Transfer Dissociation Mass Spectrometry Determine the Interface and Dynamics of Apolipoprotein E Oligomerization

Huang, Richard Y-C.; Garai, Kanchan; Frieden, Carl; Gross, Michael L.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Apolipoprotein E, a 34 kDa protein, plays a key role in triglyceride and cholesterol metabolism. Of the three common isoforms (ApoE2, 3 and 4), only ApoE4 is a risk factor for Alzheimer’s Disease. All three isoforms of wild-type ApoE self-associate to form oligomers, a process that may have functional consequences. Although the C-terminal domain, residues 216–299, of ApoE is believed to mediate self-association, the specific residues involved in this process are not known. Here we report the use of hydrogen/deuterium exchange (H/DX) coupled with enzymatic digestion to identify those regions in the sequence of full-length apoE involved in oligomerization. For this determination, we compared the results of H/DX of the wild-type proteins and those of monomeric forms obtained by modifying four residues in the C-terminal domain. The three wild type and mutant isoforms show similar structures based on their similar H/DX kinetics and extents of exchange. Regions of the C-terminus (residues 230–270) of the ApoE isoforms show significant differences of deuterium uptake between oligomeric and monomeric forms, confirming that oligomerization occurs at these regions. To achieve single amino acid resolution, we examined the extents of H/DX by using electron transfer dissociation (ETD) fragmentation of peptides representing selected regions of both the monomeric and the oligomeric forms of ApoE4. From these experiments...

Genotypes and Toxin Gene Profiles of Staphylococcus aureus Clinical Isolates from China

Xie, Yanping; He, Yiping; Gehring, Andrew; Hu, Yu; Li, Qiongqiong; Tu, Shu-I; Shi, Xianming
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 15/12/2011 Português
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A total of 108 S. aureus isolates from 16 major hospitals located in 14 different provinces in China were characterized for the profiles of 18 staphylococcal enterotoxin (SE) genes, 3 exfoliatin genes (eta, etb and etd), and the toxic shock syndrome toxin gene (tsst) by PCR. The genomic diversity of each isolate was also evaluated by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and accessory gene regulator (agr) typing. Of these strains, 90.7% (98/108) harbored toxin genes, in which tsst was the most prevalent toxin gene (48.1%), followed by sea (44.4%), sek (42.6%) and seq (40.7%). The see and etb genes were not found in any of the isolates tested. Because of high-frequency transfer of toxin gene-containing mobile genetic elements between S. aureus strains, a total of 47 different toxin gene combinations were detected, including a complete egc cluster in 19 isolates, co-occurrence of sea, sek and seq in 38 strains, and sec and sel together in 11 strains. Genetic typing by PFGE grouped all the strains into 25 clusters based on 80% similarity. MLST revealed 25 sequence types (ST) which were assigned into 16 clonal complexes (CCs) including 2 new singletons. Among these, 11 new and 6 known STs were first reported in the S. aureus strains from China. Overall...

Coordination Sphere Tuning of the Electron Transfer Dissociation Behavior of Cu(II)-Peptide Complexes

Dong, Jia; Vachet, Richard W.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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In contrast to previous electron capture dissociation (ECD) studies, we find that electron transfer dissociation (ETD) of Cu(II)-peptide complexes can generate c- and z- type product ions when the peptide has a sufficient number of strongly coordinating residues. Double-resonance experiments, ion-molecule reactions, and collision-induced dissociation (CID) prove that the c and z product ions are formed via typical radical pathways without the associated reduction of Cu(II), despite the high second ionization energy of Cu. A positive correlation between the number of Cu(II) binding groups in the peptide sequence and the extent of c and z ion formation was also observed. This trend is rationalized by considering that the recombination energy of Cu(II) can be lowered by strong binding ligands to an extent that enables electron transfer to non-Cu sites (e.g. protonation sites) to compete with Cu(II) reduction, thereby generating c/z ions in a manner similar to that observed for protonated (i.e. non-metalated) peptides.

Increasing peptide identifications and decreasing search times for ETD spectra by pre-processing and calculation of parent precursor charge

Sridhara, Viswanadham; Bai, Dina L; Chi, An; Shabanowitz, Jeffrey; Hunt, Donald F; Bryant, Stephen H; Geer, Lewis Y
Fonte: BioMed Central Publicador: BioMed Central
Tipo: Artigo de Revista Científica
Publicado em 09/02/2012 Português
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Genotypes, Exotoxin Gene Content, and Antimicrobial Resistance of Staphylococcus aureus Strains Recovered from Foods and Food Handlers

Argudín, M. A.; Mendoza, M. C.; González-Hevia, M. A.; Bances, M.; Guerra, B.; Rodicio, M. R.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /04/2012 Português
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Staphylococcal food poisoning, one of the most common food-borne diseases, results from ingestion of one or more staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus in foods. In the present study, 64 S. aureus isolates recovered from foods and food handlers, associated or not associated with food-poisoning outbreaks in Spain, were investigated. They were assigned to 31 strains by spa typing, multilocus sequence typing (MLST), exotoxin gene content, and antimicrobial resistance. The strains belonged to 10 clonal complexes (CCs): CC5 (29.0%), CC30 (25.8%), CC45 (16.1%), CC8, CC15 (two strains each), CC1, CC22, CC25, CC59, and CC121 (one strain each). They contained hemolysin genes (90.3%); lukED (77.4%); exfoliatin genes eta, etd (6.5% each), and etb (3.2%); tst (25.8%); and the following enterotoxin or enterotoxin-like genes or clusters: sea (38.7%), seb (12.9%), sec (16.1%), sed-selj with or without ser (22.9%), selk-selq (6.5%), seh, sell, selp (9.7% each), egc1 (32.3%), and egc2 (48.4%). The number of se and sel genes ranged from zero to 12. All isolates carrying tst, and most isolates with genes encoding classical enterotoxins (SEA, SEB, SEC, and SED), expressed the corresponding toxin(s). Two CC5 isolates from hamburgers (spa type t002...

A Platform for Characterizing Therapeutic Monoclonal Antibody Breakdown Products by 2D Chromatography and Top-Down Mass Spectrometry

Mazur, Matthew T.; Seipert, Richard S.; Mahon, David; Zhou, Qinwei; Liu, Tun
Fonte: Springer US Publicador: Springer US
Tipo: Artigo de Revista Científica
Publicado em 12/05/2012 Português
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With the growing commercialization of therapeutic monoclonal antibodies developed for the treatment of various diseases comes the need for increased analytical scrutiny of the impurity components contained within such drug products. Traditionally, relatively low performance and throughput analytical techniques were employed for elucidating the product-related breakdown components derived from the original molecule, including N-terminal Edman sequencing and matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry. Although N-terminal sequencing provides a definitive starting point of an unknown breakdown product, the resolution and mass accuracy of MALDI-TOF instruments are often insufficient for unambiguous sequence characterization. Described here is the implementation of existing advanced analytical technologies, including high-performance mass spectrometry (LTQ-Orbitrap XL-ETD) and a chip-based nanoelectrospray autosampling robot (TriVersa NanoMate), for the thorough identification and characterization of breakdown products derived from a force-degraded monoclonal antibody. Many anticipated breakdown products were identified, including Fab fragment (48,325 Da) and heavy chain polypeptide hydrolysis product (15...

Identification of protein SUMOylation sites by mass spectrometry using combined microwave-assisted aspartic acid cleavage and tryptic digestion

Osula, Omoruyi; Swatkoski, Stephen; Cotter, Robert J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/2012 Português
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SUMO (Small-Ubiquitin-like MOdifier) is a post-translational modifier of protein substrates at lysine residues that conjugates to proteins in response to various changes in the cell. As a result of SUMO modification, marked changes in transcription regulation, DNA repair, subcellular localization, and mitosis, among other cellular processes, are known to occur. However, while the identification of ubiquitylation sites by mass spectrometry is aided in part by the presence of a small di-amino acid GlyGly “tag” that remains on lysine residues following tryptic digestion, SUMOylation poses a particular challenge as the absence of a basic residue near to the SUMO C-terminus results in a significant 27 or 32 amino acid sequence branch conjugated to the substrate peptide. MS/MS analyses of these branch peptides generally reveal abundant fragment ions resulting from cleavage of the SUMO tail, but which obscure those needed for characterizing the target peptide sequence. Other approaches for identifying SUMO substrates exist and include overexpression of the SUMO isoforms using an N-terminal histidine tag, as well as site-directed mutagenesis of the C-terminal end of the SUMO sequence. Here, we employ combined enzymatic/chemical approaches which serve to shorten the SUMO tag...

Quantitative Phosphoproteomic Analysis of Soybean Root Hairs Inoculated with Bradyrhizobium japonicum*

Nguyen, Tran Hong Nha; Brechenmacher, Laurent; Aldrich, Joshua T.; Clauss, Therese R.; Gritsenko, Marina A.; Hixson, Kim K.; Libault, Marc; Tanaka, Kiwamu; Yang, Feng; Yao, Qiuming; Paša-Tolić, Ljiljana; Xu, Dong; Nguyen, Henry T.; Stacey, Gary
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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Root hairs are single hair-forming cells on roots that function to increase root surface area, enhancing water and nutrient uptake. In leguminous plants, root hairs also play a critical role as the site of infection by symbiotic nitrogen fixing rhizobia, leading to the formation of a novel organ, the nodule. The initial steps in the rhizobia-root hair infection process are known to involve specific receptor kinases and subsequent kinase cascades. Here, we characterize the phosphoproteome of the root hairs and the corresponding stripped roots (i.e. roots from which root hairs were removed) during rhizobial colonization and infection to gain insight into the molecular mechanism of root hair cell biology. We chose soybean (Glycine max L.), one of the most important crop plants in the legume family, for this study because of its larger root size, which permits isolation of sufficient root hair material for phosphoproteomic analysis. Phosphopeptides derived from root hairs and stripped roots, mock inoculated or inoculated with the soybean-specific rhizobium Bradyrhizobium japonicum, were labeled with the isobaric tag eight-plex iTRAQ, enriched using Ni-NTA magnetic beads and subjected to nanoRPLC-MS/MS1 analysis using HCD and decision tree guided CID/ETD strategy. A total of 1625 unique phosphopeptides...

Characterisation of the Virulence Factors and Genetic Types of Methicillin Susceptible Staphylococcus aureus from Patients and Healthy Individuals

Lim, King-Ting; Hanifah, Yasmin Abu; Yusof, Mohd Yasim Mohd; Thong, Kwai-Lin
Fonte: Springer-Verlag Publicador: Springer-Verlag
Tipo: Artigo de Revista Científica
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Methicillin sensitive Staphylococcus aureus is an important bacterial pathogen associated with hospital- and community-acquired infections leading to endocarditis, skin tissue infection and pneumonia. The objective of this study was to determine both the genetic characteristics of methicillin-sensitive S. aureus (MSSA) strains, and the occurrence of virulence factors produced by S. aureus strains isolated from UMMC and healthy students in the University from year 2009. Out of 429 nasal swab samples, 67 were MSSA. The prevalence of 21 different virulence genes among 67 Malaysian clinical and community MSSA strains was determined by PCR, and their genetic features were assessed by PCR-RFLP of coa gene, agr types, spa typing and PFGE. The five predominant virulence genes were ica (79 %), efb and fnbA (61 % each), sdrE (57 %) and hlg (45 %). Toxin genes (enterotoxin, etd and pvl) were significantly more common (P < 0.05) in clinical strains compared to community strains. Three agr genotypes were observed: agr type I (45 %), agr type III (25 %) and agr type II (19 %). All 67 MSSA strains were distinguished into 26 profiles by PCR-RFLP of coa, 55 pulsotypes and 21 spa types. Four novel spa types (t7312, t7581, t7582 and t7583) were observed. In conclusion...

Automated gas-phase purification for accurate, multiplexed quantification on a stand-alone ion trap mass spectrometer

Vincent, Catherine E.; Rensvold, Jarred W.; Westphall, Michael S.; Pagliarini, David J.; Coon, Joshua J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Isobaric tagging enables the acquisition of highly-multiplexed proteome quantification but is hindered by the pervasive problem of precursor interference. The elimination of co-isolated contaminants prior to reporter tag generation can be achieved through the use of gas-phase purification via proton transfer ion/ion reactions (QuantMode); however, the original QuantMode technique was implemented on the high resolution linear ion trap-Orbitrap hybrid mass spectrometer enabled with electron transfer dissociation (ETD). Here we extend this technology to stand-alone linear ion trap systems (trapQuantMode). Facilitated by the use of inlet beam-type activation (i.e., trapHCD) for production and observation of the low mass-to-charge reporter region, this scan sequence comprises three separate events to maximize peptide identifications, minimize duty cycle requirements, and increase quantitative accuracy, precision, and dynamic range. Significant improvements in quantitative accuracy were attained over standard methods when using trapQuantMode (trapQM) to analyze an interference model system comprising tryptic peptides of yeast that we contaminated with human peptides. Finally, we demonstrate practical benefits of this method by analysis of the proteomic changes that occur during mouse skeletal muscle myoblast differentiation. While trapQM’s reduced duty cycle led to the identification of fewer proteins than conventional operation (4...

Clinical evaluation of balloon dilation Eustachian tuboplasty in the Eustachian tube dysfunction

Jurkiewicz, Dariusz; Bień, Dominik; Szczygielski, Kornel; Kantor, Ireneusz
Fonte: Springer-Verlag Publicador: Springer-Verlag
Tipo: Artigo de Revista Científica
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The development of minimally invasive procedures such as the balloon dilation Eustachian tuboplasty (BET) is an alternative to the grommet tympanum membrane. BET is applied in the cases where, after elimination of all factors influencing the ET and middle ear functioning, no sufficient improvement is observed. The aim of this study was to present the therapeutic benefits of the BET method in the treatment of ETD caused by disorders in the middle ear ventilation. The BET procedure was offered to four patients (3 men and 1 woman) after subjective, physical, otorhinolaryngological and audiometric examinations including pure tone audiometry, tympanometry and pressure-swallow test. As the method was novel, preinterventional CT angiography of the carotid arteries was performed in all patients. Any complications were noticed during and after the procedure (bleeding or damage of regional mucosa) in any patients. Our clinical studies assessed the feasibility and safety of the BET during a short-term period—only a 6-week observation. Although patients revealed a significant improvement of ET score, longer long-term studies are necessary to determine whether this method will demonstrate lasting benefits and safety in the treatment of chronic Eustachian tube dysfunction. In other investigations...

An Integrated Proteomic Analysis of Major Isoaspartyl-Containing Proteins in the Urine of Wild Type and Protein L-Isoaspartate O-Methyltransferase-Deficient Mice

Dai, Shujia; Ni, Wenqin; Patananan, Alexander N.; Clarke, Steven G.; Karger, Barry L.; Zhou, Zhaohui Sunny
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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The formation of isoaspartyl residues (isoAsp or isoD) via either aspartyl isomerization or asparaginyl deamidation alters protein structure and potentially biological function. This is a spontaneous and non-enzymatic process, ubiquitous both in vivo and in non-biological systems, such as in protein pharmaceuticals. In almost all organisms, protein L-isoaspartate O-methyltransferase (PIMT, EC2.1.1.77) recognizes and initiates the conversion of isoAsp back to aspartic acid. Additionally, alternative proteolytic and excretion pathways to metabolize isoaspartyl-containing proteins have been proposed but not fully explored, largely due to the analytical challenges for detecting isoAsp. We report here the relative quantitation and site profiling of isoAsp in urinary proteins from wild type and PIMT-deficient mice, representing products from excretion pathways. First, using a biochemical approach, we found that the total isoaspartyl level of proteins in urine of PIMT-deficient male mice was elevated. Subsequently, the major isoaspartyl protein species in urine from these mice were identified as major urinary proteins (MUPs) by shotgun proteomics. To enhance the sensitivity of isoAsp detection, a targeted proteomic approach using electron transfer dissociation-selected reaction monitoring (ETD-SRM) was developed to investigate isoAsp sites in MUPs. Thirty-eight putative isoAsp modification sites in MUPs were investigated...

N-terminal Top-Down Protein Sequencing of the ABRF-PSRG2012 Study Samples by ETD-UHR-TOF Mass Spectrometry

Albers, Christian; Hartmer, Ralf; Jabs, Wolfgang; Baessmann, Carsten; Kaspar, Stephanie
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em //2012 Português
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MS based top-down protein sequencing (TDS) techniques are becoming increasingly important mainly because relevant sequence information such as N-terminal sequence assignment is obtained very quickly and accurately. This meets the demand of many growing areas like identity confirmation of biopharmaceuticals.

Limited Proteolysis Via Millisecond Digestions in Protease-Modified Membranes

Tan, Yu-Jing; Wang, Wei-Han; Zheng, Yi; Dong, Jinlan; Stefano, Giovanni; Brandizzi, Federica; Garavito, R. Michael; Reid, Gavin E.; Bruening, Merlin L.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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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/cm3) 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 ms 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 kD) 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 kD–10 kD) 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.

Increasing the multiplexing capacity of TMT using reporter ion isotopologues with isobaric masses

McAlister, Graeme C.; Huttlin, Edward L.; Haas, Wilhelm; Ting, Lily; Jedrychowski, Mark P.; Rogers, John C.; Kuhn, Karsten; Pike, Ian; Grothe, Robert A.; Blethrow, Justin D.; Gygi, Steven P.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Quantitative mass spectrometry methods offer near-comprehensive proteome coverage; however, these methods still suffer with regards to sample throughput. Multiplex quantitation via isobaric chemical tags (e.g., TMT and iTRAQ) provides an avenue for mass spectrometry based proteome quantitation experiments to move away from simple binary comparisons and towards greater parallelization. Herein, we demonstrate a straightforward method for immediately expanding the throughput of the TMT isobaric reagents from 6-plex to 8-plex. This method is based upon our ability to resolve the isotopic shift that results from substituting a 15N for a 13C. In an accommodation to the preferred fragmentation pathways of ETD, the TMT-127 and -129 reagents were recently modified such that a 13C was exchanged for a 15N. As a result of this substitution, the new TMT reporter ions are 6.32 mDa lighter. Even though the mass difference between these reporter ion isotopologues is incredibly small, modern high-resolution and mass accuracy analyzers can resolve these ions. Based on our ability to resolve and accurately measure the relative intensity of these isobaric reporter ions, we demonstrate that we are able to quantify across 8 samples simultaneously by combining the 13C and 15N containing reporter ions. Considering the structure of the TMT reporter ion...