From coarse to fine: the absolute Escherichia coli proteome under diverse growth conditions.

Matteo Mori,Zhongge Zhang,Ben C Collins,Christina Ludwig,Terence Hwa,Ruedi Aebersold,Deok‐Sun Lee,Jean‐Benoît Lalanne,Olga T Schubert,Alexander Schmidt,Hiroyuki Okano,Amir Banaei‐Esfahani,Gene‐Wei Li

doi:10.15252/msb.20209536

Abstract

Accurate measurements of cellular protein concentrations are invaluable to quantitative studies of gene expression and physiology in living cells. Here, we developed a versatile mass spectrometric workflow based on data‐independent acquisition proteomics (DIA/SWATH) together with a novel protein inference algorithm (xTop). We used this workflow to accurately quantify absolute protein abundances in Escherichia coli for > 2,000 proteins over > 60 growth conditions, including nutrient limitations, non‐metabolic stresses, and non‐planktonic states. The resulting high‐quality dataset of protein mass fractions allowed us to characterize proteome responses from a coarse (groups of related proteins) to a fine (individual) protein level. Hereby, a plethora of novel biological findings could be elucidated, including the generic upregulation of low‐abundant proteins under various metabolic limitations, the non‐specificity of catabolic enzymes upregulated under carbon limitation, the lack of large‐scale proteome reallocation under stress compared to nutrient limitations, as well as surprising strain‐dependent effects important for biofilm formation. These results present valuable resources for the systems biology community and can be used for future multi‐omics studies of gene regulation and metabolic control in E. coli.

Highlights

Proteins are one of the key molecular players in living cells, directly affecting cell behavior through myriads of activities
We developed a versatile workflow for relative and absolute quantification of E. coli proteomes across many samples using DIA/ SWATH mass spectrometry
All 34 samples were measured by dependent acquisition (DDA)-based mass spectrometry on a quadrupole-time-of-flight mass spectrometer (TripleTOF 5600, Sciex)

Summary

Introduction

Proteins are one of the key molecular players in living cells, directly affecting cell behavior through myriads of activities They are controlled, regulated, and fine-tuned in time and space through various mechanisms, including protein synthesis, turnover, posttranslational modifications, and protein–protein interactions. A specific challenge for systemslevel studies is the reliable quantification of thousands of proteins, including proteins at low concentrations, across large sample cohorts from a variety of different growth conditions, phenotypes, or strains (Rost et al, 2015) Both relative protein quantification (allowing cross-sample comparisons for the same protein) and absolute protein quantification (allowing cross-protein comparisons in the same sample) provide crucial information on the activity of a 2021 The Authors.

Methods

Results

Conclusion