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Title: Proteomic profiling of intact proteins using WAX-RPLC 2-D separations and FTICR mass spectrometry

Abstract

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. 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 ~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. This work aimed at optimizing intact protein detection for profiling proteins at a level that incorporates their modification complement. 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.

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org.:
USDOE
OSTI Identifier:
902954
Report Number(s):
PNNL-SA-50906
20496; 400412000
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Proteome Research, 6(2):602-610; Journal Volume: 6; Journal Issue: 2
Country of Publication:
United States
Language:
English
Subject:
comparative proteomics; FTICR MS; intact proteins; post-translational modifications; Environmental Molecular Sciences Laboratory

Citation Formats

Sharma, Seema, Simpson, David C., Tolic, Nikola, Jaitly, Navdeep, Mayampurath, Anoop M., Smith, Richard D., and Pasa-Tolic, Liljiana. Proteomic profiling of intact proteins using WAX-RPLC 2-D separations and FTICR mass spectrometry. United States: N. p., 2007. Web. doi:10.1021/pr060354a.
Sharma, Seema, Simpson, David C., Tolic, Nikola, Jaitly, Navdeep, Mayampurath, Anoop M., Smith, Richard D., & Pasa-Tolic, Liljiana. Proteomic profiling of intact proteins using WAX-RPLC 2-D separations and FTICR mass spectrometry. United States. doi:10.1021/pr060354a.
Sharma, Seema, Simpson, David C., Tolic, Nikola, Jaitly, Navdeep, Mayampurath, Anoop M., Smith, Richard D., and Pasa-Tolic, Liljiana. Thu . "Proteomic profiling of intact proteins using WAX-RPLC 2-D separations and FTICR mass spectrometry". United States. doi:10.1021/pr060354a.
@article{osti_902954,
title = {Proteomic profiling of intact proteins using WAX-RPLC 2-D separations and FTICR mass spectrometry},
author = {Sharma, Seema and Simpson, David C. and Tolic, Nikola and Jaitly, Navdeep and Mayampurath, Anoop M. and Smith, Richard D. and Pasa-Tolic, Liljiana},
abstractNote = {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. 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 ~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. This work aimed at optimizing intact protein detection for profiling proteins at a level that incorporates their modification complement. 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.},
doi = {10.1021/pr060354a},
journal = {Journal of Proteome Research, 6(2):602-610},
number = 2,
volume = 6,
place = {United States},
year = {Thu Feb 01 00:00:00 EST 2007},
month = {Thu Feb 01 00:00:00 EST 2007}
}