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A Mixed Integer Linear Optimization Framework for the Identification and Quantification of Targeted Post-translational Modifications of Highly Modified Proteins Using Multiplexed Electron Transfer Dissociation Tandem Mass Spectrometry*

DiMaggio, Peter A.; Young, Nicolas L.; Baliban, Richard C.; Garcia, Benjamin A.; Floudas, Christodoulos A.
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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Here we present a novel methodology for the identification of the targeted post-translational modifications present in highly modified proteins using mixed integer linear optimization and electron transfer dissociation (ETD) tandem mass spectrometry. For a given ETD tandem mass spectrum, the rigorous set of modified forms that satisfy the mass of the precursor ion, within some tolerance error, are enumerated by solving a feasibility problem via mixed integer linear optimization. The enumeration of the entire superset of modified forms enables the method to normalize the relative contributions of the individual modification sites. Given the entire set of modified forms, a superposition problem is then formulated using mixed integer linear optimization to determine the relative fractions of the modified forms that are present in the multiplexed ETD tandem mass spectrum. Chromatographic information in the mass and time dimension is utilized to assess the likelihood of the assigned modification states, to average several tandem mass spectra for confident identification of lower level forms, and to infer modification states of partially assigned spectra. The utility of the proposed computational framework is demonstrated on an entire LC-MS/MS ETD experiment corresponding to a mixture of highly modified histone peptides. This new computational method will facilitate the unprecedented LC-MS/MS ETD analysis of many hypermodified proteins and offer novel biological insight into these previously understudied systems.

Enhanced Peptide Identification by Electron Transfer Dissociation Using an Improved Mascot Percolator*

Wright, James C.; Collins, Mark O.; Yu, Lu; Käll, Lukas; Brosch, Markus; Choudhary, Jyoti S.
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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Peptide identification using tandem mass spectrometry is a core technology in proteomics. Latest generations of mass spectrometry instruments enable the use of electron transfer dissociation (ETD) to complement collision induced dissociation (CID) for peptide fragmentation. However, a critical limitation to the use of ETD has been optimal database search software. Percolator is a post-search algorithm, which uses semi-supervised machine learning to improve the rate of peptide spectrum identifications (PSMs) together with providing reliable significance measures. We have previously interfaced the Mascot search engine with Percolator and demonstrated sensitivity and specificity benefits with CID data. Here, we report recent developments in the Mascot Percolator V2.0 software including an improved feature calculator and support for a wider range of ion series. The updated software is applied to the analysis of several CID and ETD fragmented peptide data sets. This version of Mascot Percolator increases the number of CID PSMs by up to 80% and ETD PSMs by up to 60% at a 0.01 q-value (1% false discovery rate) threshold over a standard Mascot search, notably recovering PSMs from high charge state precursor ions. The greatly increased number of PSMs and peptide coverage afforded by Mascot Percolator has enabled a fuller assessment of CID/ETD complementarity to be performed. Using a data set of CID and ETcaD spectral pairs...

Glycoproteomic Analysis of the Secretome of Human Endothelial Cells*

Yin, Xiaoke; Bern, Marshall; Xing, Qiuru; Ho, Jenny; Viner, Rosa; Mayr, Manuel
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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Previous proteomics studies have partially unraveled the complexity of endothelial protein secretion but have not investigated glycosylation, a key modification of secreted and membrane proteins for cell communication. In this study, human umbilical vein endothelial cells were kept in serum-free medium before activation by phorbol-12-myristate-13 acetate, a commonly used secretagogue that induces exocytosis of endothelial vesicles. In addition to 123 secreted proteins, the secretome was particularly rich in membrane proteins. Glycopeptides were enriched by zwitterionic hydrophilic interaction liquid chromatography resins and were either treated with PNGase F and H218O or directly analyzed using a recently developed workflow combining higher-energy C-trap dissociation (HCD) with electron-transfer dissociation (ETD) for a hybrid linear ion trap–orbitrap mass spectrometer. After deglycosylation with PNGase F in the presence of H218O, 123 unique peptides displayed 18O-deamidation of asparagine, corresponding to 86 proteins with a total of 121 glycosylation sites. Direct glycopeptide analysis via HCD-ETD identified 131 glycopeptides from 59 proteins and 118 glycosylation sites, of which 41 were known, 51 were predicted, and 26 were novel. Two methods were compared: alternating HCD-ETD and HCD-product-dependent ETD. The former detected predominantly high-intensity...

High-throughput Database Search and Large-scale Negative Polarity Liquid Chromatography–Tandem Mass Spectrometry with Ultraviolet Photodissociation for Complex Proteomic Samples*

Madsen, James A.; Xu, Hua; Robinson, Michelle R.; Horton, Andrew P.; Shaw, Jared B.; Giles, David K.; Kaoud, Tamer S.; Dalby, Kevin N.; Trent, M. Stephen; Brodbelt, Jennifer S.
Fonte: The American Society for Biochemistry and Molecular Biology Publicador: The American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
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171.89906%
The use of ultraviolet photodissociation (UVPD) for the activation and dissociation of peptide anions is evaluated for broader coverage of the proteome. To facilitate interpretation and assignment of the resulting UVPD mass spectra of peptide anions, the MassMatrix database search algorithm was modified to allow automated analysis of negative polarity MS/MS spectra. The new UVPD algorithms were developed based on the MassMatrix database search engine by adding specific fragmentation pathways for UVPD. The new UVPD fragmentation pathways in MassMatrix were rigorously and statistically optimized using two large data sets with high mass accuracy and high mass resolution for both MS1 and MS2 data acquired on an Orbitrap mass spectrometer for complex Halobacterium and HeLa proteome samples. Negative mode UVPD led to the identification of 3663 and 2350 peptides for the Halo and HeLa tryptic digests, respectively, corresponding to 655 and 645 peptides that were unique when compared with electron transfer dissociation (ETD), higher energy collision-induced dissociation, and collision-induced dissociation results for the same digests analyzed in the positive mode. In sum, 805 and 619 proteins were identified via UVPD for the Halobacterium and HeLa samples...