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 MS(2) 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 MS(1), 2) using only MS(2), and 3) using the combination of MS(1) and MS(2). 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.
Highlights
Top-down and bottom-up mass spectrometry methods can generate gas phase fragments and use these to identify proteins
Evaluation of the Upper Mass Limit of Top-down MS—A frequently raised criticism of top-down MS is the mass limit imposed by the largest detectable protein mass, which in routine operation of FTMS instruments is 70 –100 kDa [53]
The second spectrum (10 mM ammonium acetate, pH 6.8) suggests that thyroglobulin fragmented at Gln1098 and fragmented further to give the sequence tag identified by BIG Mascot
Summary
Sample Preparation—Amyotrophic lateral sclerosis (ALS)-related [39, 40] human Cu/Zn-superoxide dismutase (SOD1) variants (A4V, L38V, G41S, G72S, D76Y, G85R, D90A, G93A, D124V, D125H, E133del, and S134N) were isolated and purified from baculovirustransfected Sf21 insect cells as described previously [41]. BIG Mascot requires a precursor ion mass input We bypassed this requirement by specifying a nominal 10,000-Da precursor mass and Ϯ1% (maximum) error tolerance and selecting “None” as the enzyme specificity. This setup allows an MS2-only search of all sequences and subsequences of proteins larger than 9,900 Da (10,000 Ϫ 100 Da difference is because of the tolerance window). Nterminal acetylation was specified as a fixed modification for all SOD1 variants Following these MS2 searches, an additional set of CAD “internal fragment” MS2 searches were performed. BIG Mascot displays the number of random sequences matched to data with a score above significance thresholds (identity and homology) and generates a separate report for decoy database search. In this study we report the score of the highest scoring random sequence to compare with the score of the BIG Mascot-identified protein sequence
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