Advancing the High Throughput Identification of Liver Fibrosis Protein Signatures Using Multiplexed Ion Mobility Spectrometry*
ABSTRACT Rapid diagnosis of disease states using less invasive, safer, and more clinically acceptable approaches than presently employed is a crucial direction for the field of medicine. While mass spectrometry (MS)-based proteomics approaches have attempted to meet these objectives, challenges such as the enormous dynamic range of protein concentrations in clinically relevant biofluid samples coupled with the need to address human biodiversity have slowed their employment. Herein, we report on the use of a new instrumental platform that addresses these challenges by coupling technical advances in rapid gas phase multiplexed ion mobility spectrometry (IMS) separations with liquid chromatography (LC) and MS to dramatically increase measurement sensitivity and throughput, further enabling future high throughput MS-based clinical applications. An initial application of the LC-IMS-MS platform analyzing blood serum samples from 60 post-liver transplant patients with recurrent fibrosis progression and 60 non-transplant patients illustrates its potential utility for disease characterization.
SourceAvailable from: Alexey L Chernobrovkin[Show abstract] [Hide abstract]
ABSTRACT: Based on conventional data-dependent acquisition strategy of shotgun proteomics, we present a new workflow DeMix, which significantly increases the efficiency of peptide identification for in-depth shotgun analysis of complex proteomes. Capitalizing on the high resolution and mass accuracy of Orbitrap-based tandem mass spectrometry, we developed a simple deconvolution method of "cloning" chimeric tandem spectra for co-fragmented peptides. Additional to a database search, a simple rescoring scheme utilizes mass accuracy and converts the unwanted co-fragmenting events into a surprising advantage of multiplexing. With the combination of cloning and rescoring, we obtained on average 9 peptide-spectrum matches (PSMs) per second on a Q-Exactive workbench, while the actual MS/MS acquisition rate was close to 7 spectra per second. This efficiency boost to 1.24 identified peptides per MS/MS spectrum enabled analysis of over 5,000 human proteins in single-dimensional LC-MS/MS shotgun experiments with an only two-hour gradient. These findings suggest a change in the dominant "one MS/MS spectrum - one peptide" paradigm for data acquisition and analysis in shotgun data-dependent proteomics. DeMix also demonstrated higher robustness than conventional approaches in terms of lower variation among the results of consecutive LC-MS/MS runs.Molecular & Cellular Proteomics 08/2014; 13(11). DOI:10.1074/mcp.O114.038877 · 7.25 Impact Factor
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ABSTRACT: Contemporary strategies that concentrate on only one or a handful of molecular targets limits the utility of the information gained for diagnostic and predictive purposes. Recent advances in the sensitivity, speed, and precision of measurements obtained from ion mobility coupled to mass spectrometry (IM-MS) have accelerated the utility of IM-MS in untargeted, discovery-driven studies in biology. Perhaps most evident is the impact that such wide-scale discovery capabilities have yielded in the areas of systems, synthetic, and chemical biology, where the need for comprehensive, hypothesis-driving studies from multidimensional and unbiased data is required. Copyright © 2014 Elsevier Ltd. All rights reserved.Current Opinion in Biotechnology 11/2014; 31. DOI:10.1016/j.copbio.2014.10.012 · 8.04 Impact Factor