Proteomic profiling of intact proteins using WAX-RPLC 2-D separations and FTICR mass spectrometry.
We investigated the combination of weak anion exchange (WAX) fractionation and on-line reversed-phase liquid chromatography (RPLC) separation using a 12 T FTICR mass spectrometer for the detection of intact proteins from a Shewanella oneidensis MR-1 cell lysate. This work aimed at optimizing intact protein detection for profiling proteins at a level that incorporates their modification state. A total of 715 intact proteins were detected, and the combined results from the WAX fractions and the unfractionated cell lysate were aligned using LC-MS features to facilitate protein abundance measurements. Protein identifications and post-translational modifications were assigned for approximately 10% of the detected proteins by comparing intact protein mass measurements to proteins identified in peptide MS/MS analysis of an aliquot of the same fraction. Intact proteins were also detected for S. oneidensis lysates obtained from cells grown on 13C-, 15N-depleted media under aerobic and sub-oxic conditions. The strategy can be readily applied for measuring differential protein abundances and provides a platform for high-throughput selection of biologically relevant targets for further characterization.
Sharma
Seema
S
Pacific Northwest National Laboratory, Richland, WA 99352, USA.
Simpson
David C
DC
Tolić
Nikola
N
Jaitly
Navdeep
N
Mayampurath
Anoop M
AM
Smith
Richard D
RD
Pasa-Tolić
Ljiljana
L
D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002853:Chromatography, Liquid; D013058:Mass Spectrometry / Q000379:methods; D011499:Protein Processing, Post-Translational; D020592:Shewanella / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared
D001426:Bacterial Proteins