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Webb I.K.,Purdue University | Mentinova M.,Purdue University | Mentinova M.,Caprion Proteomics Inc. | McGee W.M.,Purdue University | McLuckey S.A.,Purdue University
Journal of the American Society for Mass Spectrometry | Year: 2013

Gas-phase intra-molecular crosslinking of protein ubiquitin cations has been demonstrated via ion/ion reactions with anions of a homobifunctional N-hydroxysulfosuccinimide (sulfo-NHS) ester reagent. The ion/ion reaction between multiply-protonated ubiquitin and crosslinker monoanions produces a stable, charge-reduced complex. Covalent crosslinking is indicated by the consecutive loss of 2 molecules of sulfo-NHS under ion trap collisional activation conditions. Covalent modification is verified by the presence of covalently crosslinked sequence ions produced by ion-trap collision-induced dissociation of the ion generated from the losses of sulfo-NHS. Analysis of the crosslinked sequence fragments allows for the localization of crosslinked primary amines, enabling proximity mapping of the gas-phase 3-D structures. The presence of two unprotonated reactive sites within the distance constraint of the crosslinker is required for successful crosslinking. The ability to covalently crosslink is, therefore, sensitive to protein charge state. As the charge state increases, fewer reactive sites are available and protein structure is more likely to become extended because of intramolecular electrostatic repulsion. At high charge states, the reagent shows little evidence for covalent crosslinking but does show evidence for 'electrostatic crosslinking' in that the binding of the sulfonate groups to the protein is sufficiently strong that backbone cleavages are favored over reagent detachment under ion trap collisional activation conditions. [Figure not available: see fulltext.] © 2013 American Society for Mass Spectrometry.

Canuel M.,University of Montreal | Sun X.,University of Montreal | Asselin M.-C.,University of Montreal | Paramithiotis E.,Caprion Proteomics Inc. | And 2 more authors.
PLoS ONE | Year: 2013

Elevated LDL-cholesterol (LDLc) levels are a major risk factor for cardiovascular disease and atherosclerosis. LDLc is cleared from circulation by the LDL receptor (LDLR). Proprotein convertase subtilisin/kexin 9 (PCSK9) enhances the degradation of the LDLR in endosomes/lysosomes, resulting in increased circulating LDLc. PCSK9 can also mediate the degradation of LDLR lacking its cytosolic tail, suggesting the presence of as yet undefined lysosomal-targeting factor(s). Herein, we confirm this, and also eliminate a role for the transmembrane-domain of the LDLR in mediating its PCSK9-induced internalization and degradation. Recent findings from our laboratory also suggest a role for PCSK9 in enhancing tumor metastasis. We show herein that while the LDLR is insensitive to PCSK9 in murine B16F1 melanoma cells, PCSK9 is able to induce degradation of the low density lipoprotein receptor-related protein 1 (LRP-1), suggesting distinct targeting mechanisms for these receptors. Furthermore, PCSK9 is still capable of acting upon the LDLR in CHO 13-5-1 cells lacking LRP-1. Conversely, PCSK9 also acts on LRP-1 in the absence of the LDLR in CHO-A7 cells, where re-introduction of the LDLR leads to reduced PCSK9-mediated degradation of LRP-1. Thus, while PCSK9 is capable of inducing degradation of LRP-1, the latter is not an essential factor for LDLR regulation, but the LDLR effectively competes with LRP-1 for PCSK9 activity. Identification of PCSK9 targets should allow a better understanding of the consequences of PCSK9 inhibition for lowering LDLc and tumor metastasis. © 2013 Canuel et al.

Becker C.H.,Caprion Proteomics Inc. | Bern M.,Palo Alto Research Center PARC
Mutation Research - Genetic Toxicology and Environmental Mutagenesis | Year: 2011

Proteomics is the study of proteins on a large scale, encompassing the many interests scientists and physicians have in their expression and physical properties. Proteomics continues to be a rapidly expanding field, with a wealth of reports regularly appearing on technology enhancements and scientific studies using these new tools. This review focuses primarily on the quantitative aspect of protein expression and the associated computational machinery for making large-scale identifications of proteins and their post-translational modifications. The primary emphasis is on the combination of liquid chromatography-mass spectrometry (LC-MS) methods and associated tandem mass spectrometry (LC-MS/MS). Tandem mass spectrometry, or MS/MS, involves a second analysis within the instrument after a molecular dissociative event in order to obtain structural information including but not limited to sequence information. This review further focuses primarily on the study of in vitro digested proteins known as bottom-up or shotgun proteomics. A brief discussion of recent instrumental improvements precedes a discussion on affinity enrichment and depletion of proteins, followed by a review of the major approaches (label-free and isotope-labeling) to making protein expression measurements quantitative, especially in the context of profiling large numbers of proteins. Then a discussion follows on the various computational techniques used to identify peptides and proteins from LC-MS/MS data. This review article then includes a short discussion of LC-MS approaches to three-dimensional structure determination and concludes with a section on statistics and data mining for proteomics, including comments on properly powering clinical studies and avoiding over-fitting with large data sets. © 2010 Elsevier B.V.

Borowsky B.,CHDI Management | Warner J.,CHDI Management | Leavitt B.R.,University of British Columbia | Tabrizi S.J.,University College London | And 7 more authors.
Neurology | Year: 2013

Objective: To evaluate plasma 8-hydroxy-deoxy-guanosine (8OHdG) levels as a potential biomarker of premanifest and early Huntington disease (HD). Methods: Personnel from 2 independent laboratories quantified 8OHdG in blinded longitudinal plasma samples taken 24 months apart from 160 TRACK-HD participants, as well as samples containing control plasma with added ("spiked") 8OHdG. One laboratory used a liquid chromatography- electrochemical array (LCECA) assay, and the other used liquid chromatography-mass spectrometry (LCMS). Results: The LCMS assay was more accurate than the LCECA assay for measurements of "spiked" 8OHdG levels in plasma. Neither assay demonstrated cross-sectional differences in plasma 8OHdG among controls, premanifest HD, and early symptomatic HD. Similarly, neither assay showed longitudinal changes in any disease group over 24 months. Conclusions: Plasma concentration of 8OHdG is not a biomarker of disease state or progression in HD. We recommend that future putative biomarker studies use blinded sample analysis, standard curves, independent analytical methods, and strict quality control of sample collection and storage. © 2013 American Academy of Neurology.

Dhaunchak A.S.,Montreal Neurological Institute | Dhaunchak A.S.,McGill University | Becker C.,Caprion Proteomics Inc. | Schulman H.,Caprion Proteomics Inc. | And 7 more authors.
Annals of Neurology | Year: 2012

Cerebrospinal fluid samples collected from children during initial presentation of central nervous system inflammation, who may or may not subsequently be diagnosed as having multiple sclerosis (MS), were subjected to large-scale proteomics screening. Unexpectedly, major compact myelin membrane proteins typically implicated in MS were not detected. However, multiple molecules that localize to the node of Ranvier and the surrounding axoglial apparatus membrane were implicated, indicating perturbed axon-glial interactions in those children destined for diagnosis of MS. Copyright © 2012 American Neurological Association.

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