Abstract：
:A "one-pot" alternative method for processing proteins and isolating peptide mixtures from bacterial samples is presented for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and data reduction. The conventional in-solution digestion of the protein contents of bacteria is compared to a small disposable filter unit placed inside a centrifuge vial for processing and digestion of bacterial proteins. Each processing stage allows filtration of excess reactants and unwanted byproduct while retaining the proteins. Upon addition of trypsin, the peptide mixture solution is passed through the filter while retaining the trypsin enzyme. The peptide mixture is then analyzed by LC-MS/MS with an in-house BACid algorithm for a comparison of the experimental unique peptides to a constructed proteome database of bacterial genus, specie, and strain entries. The concentration of bacteria was varied from 10 × 10(7) to 3.3 × 10(3) cfu/mL for analysis of the effect of concentration on the ability of the sample processing, LC-MS/MS, and data analysis methods to identify bacteria. The protein processing method and dilution procedure result in reliable identification of pure suspensions and mixtures at high and low bacterial concentrations.
journal_name
J Proteome Resjournal_title
Journal of proteome researchauthors
Jabbour RE,Deshpande SV,Stanford MF,Wick CH,Zulich AW,Snyder APdoi
10.1021/pr101086asubject
Has Abstractpub_date
2011-02-04 00:00:00pages
907-12issue
2eissn
1535-3893issn
1535-3907journal_volume
10pub_type
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