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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}
}
  • Electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry coupled with capillary reverse phase LC (RPLC) was used to characterize intact proteins from the large subunit of the yeast ribosome. High mass measurement accuracy, achieved by''mass locking'' with an internal standard from a dual ESI source, allowed identification of ribosomal proteins. Analyses of the intact proteins revealed information on co-translational and post-translational modifications of the ribosomal proteins that included loss of the initiating methionine, acetylation, methylation, and proteolytic maturation. High resolution separations permitted differentiation of protein isoforms having high structural similarity as well as proteins from their modifiedmore » forms, facilitating unequivocal assignments. The study identified 42 of the 43 core large ribosomal subunit proteins and 58 (of 64 possible) core large subunit protein isoforms having unique masses in a single analysis. These results demonstrate the basis for the high-throughput analyses of complex mixtures of intact proteins, which we believe will be an important complement to other approaches for defining protein modifications and their changes resulting from physiological processes or environmental perturbations.« less
  • No abstract prepared.
  • Currently, most proteomic studies rely on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect and identify constituent peptides of enzymatically digested proteins obtained from various organisms and cell types. However, sample preparation methods for isolating membrane proteins typically involve the use of detergents, chaotropes, or reducing reagents that often interfere with electrospray ionization (ESI). To increase the identification of integral membrane proteins by LC-ESI-MS/MS, a sample preparation method combining carbonate extraction and surfactant-free organics solvent-assisted solubilization and proteolysis was developed and used to target the membrane subproteome of Deinococcus radiodurans. Out of 503 proteins identified, 135 were recognized as hydrophobic basedmore » on their positive grand average of hydropathicity values that covers 15% of the theoretical hydrophobic proteome. Using the PSORT algorithm, 268 identified proteins were recognized as integral membrane proteins covering 21% and 43% of the predicted integral cytoplasmic and outer membrane proteins, respectively. Of the integral cytoplasmic membrane proteins containing four or more predicted transmembrane domains (TMDs), 65% were identified by detecting at least one peptide spanning a TMD using LC-MS/MS. The extensive identification of highly hydrophobic proteins containing multiple TMDs confirms the efficacy of the described sample preparation protocol to isolate and solubilize integral membrane proteins and validates the method for large-scale analysis of bacterial membrane subproteomes using LC-ESI-MS/MS.« less
  • The throughput for proteomics measurements that provide broad protein coverage is limited by the quality and speed of both the separations and the subsequent mass analysis; present analysis times can range anywhere from hours to days (or longer). We have explored the basis for ultrahigh-throughput proteomics measurements using high-speed reversed-phase liquid chromatography (RPLC) combined with high accuracy mass spectrometric measurements. Time-of-flight (TOF) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometers were evaluated in conjunction with 0.8-µm porous C18 particle-packed RPLC using 50 µm i.d. capillary columns for identifying peptides using the Accurate Mass and Time (AMT) tag approach. Peptidemore » RPLC relative retention (elution) times could be correlated to within 5% to elution times that differed by at least 25-fold in speed, which allowed peptides to be identified using AMT tags identified from much slower RPLC-MS/MS analyses. When coupled with RPLC, the mass spectrometers operated at fast spectrum acquisition speeds (e.g., 0.2 sec for TOF and either 0.3 or 0.6 sec for FTICR), and peptide mass measurement accuracies of better than ±15 ppm were obtained. Ion population control during fast separations limited the mass accuracies obtained with FTICR, but the use of fast regulation of ion populations using automated gain control improved the mass accuracies. The detection of low abundance species was somewhat suppressed for fast analyses. The proteome coverage obtained using AMT tags was limited by the separation peak capacity, the sensitivity of the MS, and the accuracy of both the mass measurements and the relative RPLC peptide elution times. Experimental results demonstrated that accuracies of 5% for the RPLC relative elution times and better than ±15 ppm for mass measurements were sufficient for confident identification of >2800 peptides and >760 proteins from >13,000 different detected species from a Shewanella oneidensis tryptic digest.. The TOF instrumentation was found to be preferable for faster separations (of <120 sec), while FTICR MS was more effective for analysis times of >150 sec due to the improved mass accuracies achievable with longer spectrum acquisition times. The present work demonstrates the feasibility of very high throughput proteomics measurements and indicates additional significant improvements in throughput are achievable by further increasing the speed of high peak capacity separations, as well by increasing the measurement sensitivity and the accuracy of mass measurements.« less
  • No abstract prepared.