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Molecular & Cellular Proteomics 8:846-856, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

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

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

From the Department of Chemistry and Volen Center for Complex Systems and Department of Computer Science, National Center of Behavioral Genomics, Brandeis University, Waltham, Massachusetts 02454, ^¶ University of Massachusetts Medical School, Worcester, Massachusetts 01655, and ^** Bruker Daltonics Inc., Billerica, Massachusetts 01821

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.

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