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Sensitive and Specific Identification of Wild Type and Variant Proteins from 8 to 669 kDa Using Top-down Mass Spectrometry*S⃞

By N. Murat Karabacak, Long Li, Ashutosh Tiwari, Lawrence J. Hayward, Pengyu Hong, Michael L. Easterling and Jeffrey N. Agar

Abstract

Top-down and bottom-up mass spectrometry methods can generate gas phase fragments and use these to identify proteins. Top-down methods, in addition, can provide the mass of the protein itself and therefore additional structural information. Despite the conceptual advantage of top-down methods, the market share advantage belongs to bottom-up methods as a result of their more robust sample preparation, fragmentation, and data processing methods. Here we report improved fragmentation and data processing methods for top-down mass spectrometry. Specifically we report the use of funnel-skimmer dissociation, a variation of nozzle-skimmer dissociation, and compare its performance with electron capture dissociation. We also debut BIG Mascot, an extended version of Mascot with incorporated top-down MS2 search ability and the first search engine that can perform both bottom-up and top-down searches. Using BIG Mascot, we demonstrated the ability to identify proteins 1) using only intact protein MS1, 2) using only MS2, and 3) using the combination of MS1 and MS2. We correctly identified proteins with a wide range of masses, including 13 amyotrophic lateral sclerosis-associated variants of the protein Cu/Zn-superoxide dismutase, and extended the upper mass limit of top-down protein identification to 669 kDa by identifying thyroglobulin

Topics: Research
Publisher: American Society for Biochemistry and Molecular Biology
OAI identifier: oai:pubmedcentral.nih.gov:2667363
Provided by: PubMed Central
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