Abstract
Data-Independent Acquisition (DIA) is a method to improve consistent identification and precise quantitation of peptides and proteins by mass spectrometry (MS). The targeted data analysis strategy in DIA relies on spectral assay libraries that are generally derived from a priori measurements of peptides for each species. Although Escherichia coli (E. coli) is among the best studied model organisms, so far there is no spectral assay library for the bacterium publicly available. Here, we generated a spectral assay library for 4,014 of the 4,389 annotated E. coli proteins using one- and two-dimensional fractionated samples, and ion mobility separation enabling deep proteome coverage. We demonstrate the utility of this high-quality library with robustness in quantitation of the E. coli proteome and with rapid-chromatography to enhance throughput by targeted DIA-MS. The spectral assay library supports the detection and quantification of 91.5% of all E. coli proteins at high-confidence with 56,182 proteotypic peptides, making it a valuable resource for the scientific community. Data and spectral libraries are available via ProteomeXchange (PXD020761, PXD020785) and SWATHAtlas (SAL00222-28).
Highlights
Background & SummaryAchieving systems-wide reliable identification and precise quantification of peptides and proteins remains a challenge for many organisms[1]
To overcome this bias and achieve broad proteome coverage, dependent acquisition (DDA) is often combined with different fractionation techniques such as off-gel electrophoresis (OGE), high pH chromatography and ion exchange chromatography[1,6] or paired with ion mobility gas-phase separation[7,8,9]
We demonstrate the utility of this spectral assay library to consistently quantify the E. coli proteome with minimal technical variability and rapid-chromatography workflows accelerating data acquisition up to 4-fold for increased sample throughput
Summary
Achieving systems-wide reliable identification and precise quantification of peptides and proteins remains a challenge for many organisms[1]. To reach deep proteome coverage, these studies generally measured many experimental conditions and fractionated samples These DDA based methods are expensive, time-intense, and technically complex, which limits their implementation for routine use across laboratories[6]. The library was generated from 209 measurements of unfractionated and fractionated E. coli cell lysates using OGE and differential ion mobility (DMS), overexpressed proteins, and synthetic peptides enabling deep proteome coverage on SCIEX TripleTOF 5600+ and 6600 instruments. This library has been statistically validated with MAYU31 and its quality assessed with the spectral library tool DIALib-QC32 (www.swathatlas.org) (Fig. 1). Sample Type ASKA(-) overexpressed library Whole cell lysate (WCL) Whole cell lysate (WCL) Whole cell lysate (WCL) Whole cell lysate (WCL) Synthetic peptides Total
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