McClintock C.S.,Oak Ridge National Laboratory |
McClintock C.S.,University of Tennessee at Knoxville |
Parks J.M.,Oak Ridge National Laboratory |
Bern M.,Palo Alto Research Center PARC |
And 3 more authors.
Journal of Proteome Research | Year: 2013
Redox proteomics has yielded molecular insight into diseases of protein dysfunction attributable to oxidative stress, underscoring the need for robust detection of protein oxidation products. Additionally, oxidative protein surface mapping techniques utilize hydroxyl radicals to gain structural insight about solvent exposure. Interpretation of tandem mass spectral data is a critical challenge for such investigations, because reactive oxygen species target a wide breadth of amino acids. Additionally, oxidized peptides may be generated in a wide range of abundances since the reactivity of hydroxyl radicals with different amino acids spans 3 orders of magnitude. Taken together, these attributes of oxidative footprinting pose both experimental and computational challenges to detecting oxidized peptides that are naturally less abundant than their unoxidized counterparts. In this study, model proteins were oxidized electrochemically and analyzed at both the intact protein and peptide levels. A multidimensional chromatographic strategy was utilized to expand the dynamic range of oxidized peptide measurements. Peptide mass spectral data were searched by the "hybrid" software packages Inspect and Byonic, which incorporate de novo elements of spectral interpretation into a database search. This dynamic search capacity accommodates the challenge of searching for more than 40 oxidative mass shifts that can occur in a staggering variety of possible combinatorial occurrences. A prevailing set of oxidized residues was identified with this comparative approach, and evaluation of these sites was informed by solvent accessible surface area gleaned through molecular dynamics simulations. Along with increased levels of oxidation around highly reactive "hotspot" sites as expected, the enhanced sensitivity of these measurements uncovered a surprising level of oxidation on less reactive residues. © 2013 American Chemical Society. Source
Yin X.,Kings College London |
Bern M.,Protein Metrics, Inc. |
Xing Q.,Kings College London |
Ho J.,Thermo Fisher Scientific |
And 2 more authors.
Molecular and Cellular Proteomics | Year: 2013
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-my-ristate-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 H 2 18O 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 H 2 18O, 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, multiply charged glycopeptides, whereas the latter preferentially selected precursors with complex/hybrid glycans for fragmentation. Validation was performed by means of glycoprotein enrichment and analysis of the input, the flow-through, and the bound fraction. This study represents the most comprehensive characterization of endothelial protein secretion to date and demonstrates the potential of new HCD-ETD workflows for determining the glycosylation status of complex biological samples. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Source
Protein Metrics, Inc. | Date: 2015-08-30
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 188.80K | Year: 2012
DESCRIPTION (provided by applicant): Due to the complexity of proteins and their biological production, characterization of protein pharmaceuticals ( biologics ) poses much more demanding analytical challenges than do small-molecule drugs. Consequently,many disparate biochemical analytical techniques are needed to characterize biologics. Because of the rapidly increasing power of mass spectrometry (MS), many of the measurements to characterize therapeutic proteins are now handled by MS, and that trend can be expected to continue owing to the accuracy and precision provided by state-of-the-art instrumentation. However, biotech companies currently make do with a patchwork of inefficient or ineffective software and manual data analysis for peptide identification, amino-acid-substitution evaluation, glycosylation characterization, disulfide-bond evaluation, and so forth. Current data analysis significantly lags behind the information content inherent in the complex data sets produced. We propose commercialdevelopment of software, named Byologic, which will make major improvements in the characterization of biologics via a range of mass spectrometric assays. Protein Metrics Inc. is a spin-off of Palo Alto Research Center where a variety of algorithms have been developed in recent years to address these analytical challenges. The proposed Phase I feasibility study will allow us to perform controlled studies to determine if MS- based assays and our algorithms are robust enough to satisfy the exacting standardsof the biotech industry. If so, we will then build robust and GMP-compliant Byologic in Phase II. Both generic ( biosimilar ) and innovator drug companies stand to gain from Byologic. Public health, and regulatory agencies like the FDA charged with protecting the public, stand to gain too because better characterization will improve quality control, safety, and increase the efficiency of drug development, leading to consumer and Government savings. PUBLIC HEALTH RELEVANCE: The proposed projectwill lead to commercial software for improved characterization of the detailed composition of therapeutic proteins. Better characterized protein drugs will lead to better quality assurance, enhanced public safety, more informed regulatory decisions, and lower overall costs.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 525.67K | Year: 2012
DESCRIPTION (provided by applicant): Molecular signatures collected from intact tissue sections by MALDI imaging mass spectrometry (MALDI IMS) have shown high potential for use as a prognostic or diagnostic pathology tool in the clinical setting. A majorobstacle to the widespread deployment of MALDI IMS, however, is the difficulty of identifying the proteins contributing to the signatures. Researchers have tried a number of approaches, including in situ digestion, MALDI TOF/TOF tandem mass spectrometry, and top-down proteomics on specific image regions. In preliminary work, we have obtained promising experimental results using top-down proteomics on intact proteins in the 2 - 20 kDa range. However, the lack of successful algorithms and software to identifythe proteins in IMS mass signatures poses a major bottleneck. In particular, available top-down proteomics software relies heavily on high-accuracy mass spectrometry. The requirement for high accuracy precludes the use of some of the most sensitive mass analyzers such as linear ion traps, especially useful for these very small and complex samples. Protein Metrics Inc. is a new software company building on six years of algorithms and software research at Palo Alto Research Center. We plan to extend Byonic,our next- generation proteomics search engine, to intact proteins up to about 20 kDa. For proteins larger than 20 kDa, we will also build software for middle-down proteomics, specifically for assembling large peptides (2 - 20 kDa) produced by limited digestion to recover the identity of the intact proteins observed in IMS. The proposed Phase I feasibility study will allow us to perform controlled studies to determine the best experimental and bioinformatics approaches. Phase II will then build commercial-grade software. The proposed project will advance the state of the art in imaging mass spectrometry. Translation of imaging mass spectrometry to routine clinical pathology use will advance the state-of-the-art in disease diagnosis and treatment, and advance medical imaging and public health. PUBLIC HEALTH RELEVANCE: The project will develop commercial software that will improve our ability to identify the proteins and modifications represented in imaging mass spectrometry molecular signatures. Project success will make imaging mass spectrometry much more useful as a clinical pathology tool.