Differential Label-free Quantitative Proteomic Analysis of Shewanella oneidensis Cultured under Aerobic and Suboxic Conditions by Accurate Mass and Time Tag Approach

Yufeng Shen,Dwayne A. Elias,Ruihua Fang,Jim K. Fredrickson,Carrie D. Goddard,Ronald J. Moore,Stephen J. Callister,Martin Mcintosh,Matthew E. Monroe,Richard D. Smith,Yuri A. Gorby,Mary S. Lipton,Pei Wang,Joshua N. Adkins

doi:10.1074/mcp.m500301-mcp200

Abstract

We describe the application of LC-MS without the use of stable isotope labeling for differential quantitative proteomic analysis of whole cell lysates of Shewanella oneidensis MR-1 cultured under aerobic and suboxic conditions. LC-MS/MS was used to initially identify peptide sequences, and LC-FTICR was used to confirm these identifications as well as measure relative peptide abundances. 2343 peptides covering 668 proteins were identified with high confidence and quantified. Among these proteins, a subset of 56 changed significantly using statistical approaches such as statistical analysis of microarrays, whereas another subset of 56 that were annotated as performing housekeeping functions remained essentially unchanged in relative abundance. Numerous proteins involved in anaerobic energy metabolism exhibited up to a 10-fold increase in relative abundance when S. oneidensis was transitioned from aerobic to suboxic conditions.

Highlights

We describe the application of LC-MS without the use of stable isotope labeling for differential quantitative proteomic analysis of whole cell lysates of Shewanella oneidensis MR-1 cultured under aerobic and suboxic conditions
A linear correlation between the amount of an analyte and its peak area can be obtained by using a sufficiently low LC flow rate and a small amount of sample because ESI approaches optimum (ϳ100%) efficiency under such conditions [7,8,9]. This correlation has been demonstrated with simple mixtures of a few analytes [7, 8] and with peptides from mixtures of several proteins spiked into serum [10, 11] in the case of complex biological samples in which thousands of peptides are measured in one LC-MS analysis, many peptides co-elute
Differential Quantitative Analysis Using the AMT Tag Approach—The three steps used in the present differential quantitative analysis of S. oneidensis MR-1 using the AMT tag approach are summarized in Fig. 1

Summary

Introduction

We describe the application of LC-MS without the use of stable isotope labeling for differential quantitative proteomic analysis of whole cell lysates of Shewanella oneidensis MR-1 cultured under aerobic and suboxic conditions. These peptide PMT tags were stored in a relational database with their calculated accurate masses and NETs. Second, tryptic digests of proteins from S. oneidensis cultured under aerobic and suboxic conditions were analyzed by LC-FTICR. The ARs of peptides and their corresponding proteins were calculated to compare the S. oneidensis proteomes under aerobic and suboxic conditions.

Results

Conclusion