Wheat Proteomics: Multistep MS/MS Identification

Each distinct protein spot was excised from at least three 2-DE gels in order to digest them separately with trypsin, thermolysin and chymotrypsin. MS/MS analysis of enzymatic digests of protein spots was carried out with a QSTAR Pulsar i quadrupole time-of-flight mass spectrometer (Applied Biosystems/MDS Sciex, Toronto, Canada) that was equipped with a nano-electrospray source and nano-flow liquid chromatograph [27 (link)]. Automatic determination of the appropriate collision energy (relative to m/z) was carried out by the Analyst QS 1.1 software. When analyzing samples digested either with chymotrypsin or thermolysin the intercept of the collision energy values was decreased by eight units relative to that used with trypsin. The spectra from each digest were used to interrogate a "SuperWheat" database (Version # 100211) as described in [59 (link)]. For this study, the "SuperWheat" database was constructed by concatenating the following publicly available databases; NCBI non-redundant green plant protein sequences (download date: 2/11/2010) [84 ], nucleotide sequences translated in all six reading frames of contigs from TaGI Releases 10.0 and 11.0 [63 ], US Wheat Genome Project [85 ], HarvEST 1.14 (WI all NSF "stringent" assembly from 05/08/04) [86 ], NCBI Unigene Build #55 [84 ], and all ESTs from Butte 86 developing grain, as well as translated sequences (reading frame only) of 94 Butte 86 contigs (Additional file 2), including those for alpha-gliadins and gamma-gliadins [12 (link),13 (link)]. Additionally the database contained a list of proteins known to be common laboratory contaminants [87 ] and sequences for thermolysin. The "SuperWheat" database contained 2,094,746 protein sequences. Two search engines, X!Tandem [57 (link),87 ] and Mascot version 2.1 (Matrix Science, London, UK) [88 ,89 (link)] were used to match the peptide mass spectra to spectra generated in silico from database peptides. It has been demonstrated that Mascot and X!tandem yield somewhat different results [59 (link),70 (link)]. Mascot identified significantly more tryptic peptides than did X!tandem, while X!tandem identified more peptides from proteins digested with thermolysin or chymotrypsin. Scaffold Version 2_02_04 [62 ] was used to assemble and visualize MS/MS derived peptide and protein identifications. A "subset" database was generated from the initial search of the SuperWheat database by exporting from Scaffold all protein sequences that had a 20% or greater probability of being a match. Appended to these 2,134 sequences was an equal number of decoy protein sequences from the archaeobacter Jannaschia sp, translated sequences from the set of Butte 86 contigs not already included in the subset database, and the set of common protein contaminants. A "second pass" search [90 (link),91 (link)] was conducted with both search engines and the results assembled and validated with Scaffold. Identifications of proteins were required to meet the following criteria: at least two peptides having a parent mass tolerance threshold of less than or equal to 100 ppm and a greater than 90% peptide probability as specified by the Peptide Prophet algorithm [92 ]. Scaffold Version 3.00.03 was used to compile the final set of MS/MS based peptide and protein identifications, using the MUDPIT algorithm to independently analyze the data for each spot. The false discovery rate was generally found to be 0.0% under the filter settings used. The data associated with this manuscript may be downloaded from ProteomeCommons.org Tranche using the following hash:
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