Dataset Information

Opening a SWATH Window on Posttranslational Modifications: Automated Pursuit of Modified Peptides.

ABSTRACT: Posttranslational modifications of proteins play an important role in biology. For example, phosphorylation is a key component in signal transduction in all three domains of life, and histones can be modified in such a variety of ways that a histone code for gene regulation has been proposed. Shotgun proteomics is commonly used to identify posttranslational modifications as well as chemical modifications from sample processing. However, it favors the detection of abundant peptides over the repertoire presented, and the data analysis usually requires advance specification of modification masses and target amino acids, their number constrained by available computational resources. Recent advances in data independent acquisition mass spectrometry technologies such as SWATH-MS enable a deeper recording of the peptide contents of samples, including peptides with modifications. Here, we present a novel approach that applies the power of SWATH-MS analysis to the automated pursuit of modified peptides. With the new SWATHProphet(PTM) functionality added to the open source SWATHProphet software, precursor ions consistent with a modification are identified along with the mass and localization of the modification in the peptide sequence in a sensitive and unrestricted manner without the need to anticipate the modifications in advance. Using this method, we demonstrate the detection of a wide assortment of modified peptides, many unanticipated, in samples containing unpurified synthetic peptides and human urine, as well as in phospho-enriched human tissue culture cell samples.

SUBMITTER: Keller A

PROVIDER: S-EPMC4813695 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Opening a SWATH Window on Posttranslational Modifications: Automated Pursuit of Modified Peptides.

Keller Andrew A Bader Samuel L SL Kusebauch Ulrike U Shteynberg David D Hood Leroy L Moritz Robert L RL

Molecular & cellular proteomics : MCP 20151224 3

Posttranslational modifications of proteins play an important role in biology. For example, phosphorylation is a key component in signal transduction in all three domains of life, and histones can be modified in such a variety of ways that a histone code for gene regulation has been proposed. Shotgun proteomics is commonly used to identify posttranslational modifications as well as chemical modifications from sample processing. However, it favors the detection of abundant peptides over the reper ...[more]

PMID: 26704149

Similar Datasets

Project description:Histone post-translational modifications (PTMs) have a fundamental function in chromatin biology, as they model chromatin structure and recruit enzymes involved in gene regulation, DNA repair, and chromosome condensation. High throughput characterization of histone PTMs is mostly performed by using nano-liquid chromatography coupled to mass spectrometry. However, limitations in speed and stochastic sampling of data dependent acquisition methods in MS lead to incomplete discrimination of isobaric peptides and loss of low abundant species. In this work, we analyzed histone PTMs with a data-independent acquisition method, namely SWATH™ analysis. This approach allows for MS/MS-based quantification of all analytes without upfront assay development and no issues of biased and incomplete sampling. We purified histone proteins from human embryonic stem cells and mouse trophoblast stem cells before and after differentiation, and prepared them for MS analysis using the propionic anhydride protocol. Results on histone H3 peptides verified that sequential window acquisition of all theoretical mass spectra could accurately quantify peptides (<9% average coefficient of variation, CV) over four orders of magnitude, and we could discriminate isobaric and co-eluting peptides (e.g. H3K18ac and H3K23ac) using MS/MS-based quantification. This method provided high sensitivity and precision, supported by the fact that we could find significant differences for remarkably low abundance PTMs such as H3K9me2S10ph (relative abundance <0.02%). We performed relative quantification for few sample peptides using different fragment ions and observed high consistency (CV <15%) between the fragments. This indicated that different fragment ions can be used independently to achieve the same peptide relative quantification. Taken together, sequential window acquisition of all theoretical mass spectra proved to be an easy-to-use MS acquisition method to perform high quality MS/MS-based quantification of histone-modified peptides.

Dataset Information

Opening a SWATH Window on Posttranslational Modifications: Automated Pursuit of Modified Peptides.

Publications

Opening a SWATH Window on Posttranslational Modifications: Automated Pursuit of Modified Peptides.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets