Web and Database Software for Identification of Intact Proteins Using “Top Down” Mass Spectrometry

Abstract

For the identification and characterization of proteins harboring posttranslational modifications (PTMs), a "top down" strategy using mass spectrometry has been forwarded recently but languishes without tailored software widely available. We describe a Web-based software and database suite called ProSight PTM constructed for large-scale proteome projects involving direct fragmentation of intact protein ions. Four main components of ProSight PTM are a database retrieval algorithm (Retriever), MySQL protein databases, a file/data manager, and a project tracker. Retriever performs probability-based identifications from absolute fragment ion masses, automatically compiled sequence tags, or a combination of the two, with graphical rendering and browsing of the results. The database structure allows known and putative protein forms to be searched, with prior or predicted PTM knowledge used during each search. Initial functionality is illustrated with a 36-kDa yeast protein identified from a processed cell extract after automated data acquisition using a quadrupole-FT hybrid mass spectrometer. A +142-Da delta(m) on glyceraldehyde-3-phosphate dehydrogenase was automatically localized between Asp90 and Asp192, consistent with its two cystine residues (149 and 153) alkylated by acrylamide (+71 Da each) during the gel-based sample preparation. ProSight PTM is the first search engine and Web environment for identification of intact proteins (https://prosightptm.scs.uiuc.edu/).

Modeling the resolution and sensitivity of FAIMS analyses

“…As most large ions, it belongs to C-type and passes FAIMS in P2 mode. Fast post-ionization separations of protein mixtures using FAIMS may find application in topdown proteomics [58,59], offering the same throughput advantage over condensed-phase methods as when separating proteolytic digests in bottom-up approaches. FAIMS separations of proteins are also largely orthogonal to both IMS and MS dimensions [40,42].…”

Construction of a hybrid quadrupole/fourier transform ion cyclotron resonance mass spectrometer for versatile MS/MS above 10 kDa

“…Experimental mass peaks lists, generated by a modified version of the THRASH algorithm [29], were analyzed for b, y, c, and z⅐ ions and sequence tag information [30,31] derived either by hand or with custom software for database retrieval and protein characterization available at http://prosightptm.scs.uiuc.edu/ [32]. The probability scores reported are with 50 ppm fragment ion tolerance, to accommodate externally calibrated ions, with a Ϯ1000 Da search window around the candidate protein mass to accommodate shifts associated with post-translational modifications.…”

“…The probability scores reported are with 50 ppm fragment ion tolerance, to accommodate externally calibrated ions, with a Ϯ1000 Da search window around the candidate protein mass to accommodate shifts associated with post-translational modifications. The theoretical masses for b/y and c/z⅐ fragment ions were calculated by the Ion Predictor program available in the Prosight PTM software and database suite [32]. Mass accuracies for all data are external calibration based on bovine ubiquitin except where system performance is evaluated, in which case internal calibration is performed.…”

“…SWIFT isolation of its 15ϩ ions followed by ECD gave an MS/MS spectrum (Figure 6b) with 375 resolved isotopic clusters resulting in 123 discrete fragment ion mass values. The "absolute mass" search function [32] in ProSight PTM was able to match 67 c and 56 z⅐ ions for N-acetylated P22943, a 12 kDa heat shock protein, with a resulting Pscore of 2.3 ϫ 10 Ϫ38 . Eighty-three of 107 backbone peptide linkages were cleaved to yield 11 sequence tags ranging from 3 to 14 amino acids in length, (Figure 6c).…”

What does the future hold for top down mass spectrometry?

“…For example, since 100% sequence coverage is obtained with Top Down MS, a larger number of PTMs (such as N-terminal modifications) and even single nucleotide polymorphisms need to be seriously considered in database searches. Up until the last year or so, ProSight PTM and earlier versions were the only software programs available for identifying proteins and their modified forms from the MS/MS spectra of intact proteins [67,68]. In ProSight, the user inputs the high-resolution neutral parent ion mass and all MS/MS fragment masses, and this data is then searched against a "shotgun annotated" proteome database or known protein sequences.…”