Proteomic Identification of Surface Proteins

After the cell-surface associated protein fraction was separated by SDS-PAGE, protein bands were stained with Coomassie Brilliant Blue and excised. Peptides were prepared for LC-MS/MS analysis by in-gel digestion with trypsin. LC-MS/MS analysis was performed using a LTQ-orbitrap XL system (Thermo Scientific), as described by Tanaka et al. [68 (link)]. The MS/MS spectra of the identified peptides were searched against our own database, which contains SpaA, flagellin, and CmoA sequences, and against non-redundant protein sequences in the National Center for Biotechnology Information (NCBI) database, using a MASCOT server (Matrix Science).

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Kobayashi K., Kanesaki Y, & Yoshikawa H. (2016). Genetic Analysis of Collective Motility of Paenibacillus sp. NAIST15-1. PLoS Genetics, 12(10), e1006387.

Publication 2016

LTQ-orbitrap XL system MASCOT server

Cell Coomassie brilliant blue Digestion Flagellin Ms ms Peptides Protein Protein sequences Sds page Spaa Trypsin

Corresponding Organization : Nara Institute of Science and Technology

Other organizations : Tokyo University of Agriculture

Top 5 similar protocols

Variable analysis

independent variables

Separation of cell-surface associated protein fraction by SDS-PAGE
In-gel digestion of excised protein bands with trypsin

dependent variables

Identification of peptides by LC-MS/MS analysis

control variables

Use of LTQ-orbitrap XL system for LC-MS/MS analysis, as described by Tanaka et al. [68]
Searching the MS/MS spectra against a database containing SpaA, flagellin, and CmoA sequences, and against the NCBI non-redundant protein database using a MASCOT server

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