Dataset Information

Parallel Reaction Monitoring on a Q Exactive Mass Spectrometer Increases Reproducibility of Phosphopeptide Detection in Bacterial Phosphoproteomics Measurements

ABSTRACT: Increasing number of studies report the relevance of protein Ser/Thr/Tyr phosphorylation in bacterial physiology, yet the analysis of this type of modification in bacteria still presents a considerable challenge. Unlike in eukaryotes, where tens of thousands of phosphorylation events likely occupy more than two thirds of the proteome, the abundance of protein phosphorylation is much lower in bacteria. Even the state-of-the-art phosphopeptide enrichment protocols fail to remove the high background of abundant unmodified peptides, leading to low signal intensity and undersampling of phosphopeptide precursor ions in consecutive data-dependent MS runs. Consequently, large-scale bacterial phosphoproteomic datasets often suffer from poor reproducibility and a high number of missing values. Here we explore the application of parallel reaction monitoring (PRM) on a Q Exactive mass spectrometer in bacterial phosphoproteome analysis, focusing especially on run-to-run sampling reproducibility. In multiple measurements of identical phosphopeptide-enriched samples, we show that PRM outperforms DDA in terms of detection frequency, reaching almost complete sampling efficiency, compared to 20% in DDA. We observe a similar trend over multiple rounds of (heterogeneous) phosphopeptide-enriched samples and conclude that PRM is a method of choice in bacterial phosphoproteomics projects where reproducible detection and quantification of a relatively small set of phosphopeptides is desired.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Escherichia Coli

SUBMITTER: Nicolas Nalpas

LAB HEAD: Boris Macek

PROVIDER: PXD008211 | Pride | 2018-06-05

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20161206_LG_Ecoli_Trypsin_Proteome_Shotgun_01.raw	Raw
	20161206_LG_Ecoli_Trypsin_Proteome_Shotgun_02.raw	Raw
	20161206_LG_Ecoli_Trypsin_Proteome_Shotgun_03.raw	Raw
	20161206_LG_Ecoli_Trypsin_Proteome_Shotgun_04.raw	Raw
	20161207_LG_Ecoli_Trypsin_Proteome_PRM_80peptides_lowintensity_01.raw	Raw

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Parallel reaction monitoring on a Q Exactive mass spectrometer increases reproducibility of phosphopeptide detection in bacterial phosphoproteomics measurements.

Taumer Christoph C Griesbaum Lena L Kovacevic Alen A Soufi Boumediene B Nalpas Nicolas C NC Macek Boris B

Journal of proteomics 20180329

Increasing number of studies report the relevance of protein Ser/Thr/Tyr phosphorylation in bacterial physiology, yet the analysis of this type of modification in bacteria still presents a considerable challenge. Unlike in eukaryotes, where tens of thousands of phosphorylation events likely occupy more than two thirds of the proteome, the abundance of protein phosphorylation is much lower in bacteria. Even the state-of-the-art phosphopeptide enrichment protocols fail to remove the high backgroun ...[more]

PMID: 29605292

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