PeptideShaker enables reanalysis of MS-derived proteomics data sets

Vaudel, Marc; Burkhart, Julia M.; Zahedi, René P.; Oveland, Eystein; Berven, Frode S.; Sickmann, Albert; Martens, Lennart; Barsnes, Harald

doi:10.1038/nbt.3109

Cited by 461 publications

(409 citation statements)

References 19 publications

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“…Carbamidomethylation of Cys was used as fixed modification and oxidation of Met and deamidation of Asn and Gln as variable modifications. PeptideShaker v.1.16.15 was used to import the results for peptide and protein inference [26]. Peptide-Spectrum Matches (PSMs), peptides and proteins were validated at a 1.0% False Discovery Rate estimated using the decoy hit distribution.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Trichuris muris secreted proteins and extracellular vesicles provides new insights into host–parasite communication

Eichenberger

Talukder

Field

et al. 2018

J of Extracellular Vesicle

107

142

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Whipworms are parasitic nematodes that live in the gut of more than 500 million people worldwide. Owing to the difficulty in obtaining parasite material, the mouse whipworm Trichuris muris has been extensively used as a model to study human whipworm infections. These nematodes secrete a multitude of compounds that interact with host tissues where they orchestrate a parasitic existence. Herein we provide the first comprehensive characterization of the excretory/secretory products of T. muris. We identify 148 proteins secreted by T. muris and show for the first time that the mouse whipworm secretes exosome-like extracellular vesicles (EVs) that can interact with host cells. We use an Optiprep® gradient to purify the EVs, highlighting the suitability of this method for purifying EVs secreted by a parasitic nematode. We also characterize the proteomic and genomic content of the EVs, identifying >350 proteins, 56 miRNAs (22 novel) and 475 full-length mRNA transcripts mapping to T. muris gene models. Many of the miRNAs putatively mapped to mouse genes are involved in regulation of inflammation, implying a role in parasite-driven immunomodulation. In addition, for the first time to our knowledge, colonic organoids have been used to demonstrate the internalization of parasite EVs by host cells. Understanding how parasites interact with their host is crucial to develop new control measures. This first characterization of the proteins and EVs secreted by T. muris provides important information on whipworm–host communication and forms the basis for future studies.

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Section: Methodsmentioning

confidence: 99%

Characterization of Trichuris muris secreted proteins and extracellular vesicles provides new insights into host–parasite communication

Eichenberger

Talukder

Field

et al. 2018

J of Extracellular Vesicle

107

142

View full text Add to dashboard Cite

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“…There are already several tools, including PRIDE Inspector 27 and PeptideShaker 72 that support this concept, but more are certainly needed. This is especially true for tools that link and display information from multiple resources in a meaningful way.…”

Section: Discussionmentioning

confidence: 99%

“…For validation at the dataset level, tools such as PRIDE Inspector 27 and PeptideShaker 72, can be used to inspect and reprocess the data, respectively. Note that PeptideShaker provides a direct connection to PRIDE datasets to enable their streamlined reprocessing.…”

Section: Introductionmentioning

confidence: 99%

Exploring the potential of public proteomics data

et al. 2015

Self Cite

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In a global effort for scientific transparency, it has become feasible and good practice to share experimental data supporting novel findings. Consequently, the amount of publicly available MS‐based proteomics data has grown substantially in recent years. With some notable exceptions, this extensive material has however largely been left untouched. The time has now come for the proteomics community to utilize this potential gold mine for new discoveries, and uncover its untapped potential. In this review, we provide a brief history of the sharing of proteomics data, showing ways in which publicly available proteomics data are already being (re‐)used, and outline potential future opportunities based on four different usage types: use, reuse, reprocess, and repurpose. We thus aim to assist the proteomics community in stepping up to the challenge, and to make the most of the rapidly increasing amount of public proteomics data.

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“…Raw mass spectrometric data were converted to MGF file format (35). Converted data were processed with SearchGUI and PeptideShaker by searching with X!Tandem against the Drosophila melanogaster or human proteome downloaded from UniProt (36,37). Methionine oxidation was a variable modification, with precursor mass tolerance of 10-20 ppm, fragment ion tolerance of 0.5 Da, and two maximum missed trypsin cleavages.…”

Section: Methodsmentioning

confidence: 99%

Streamlined discovery of cross-linked chromatin complexes and associated histone modifications by mass spectrometry

Zee

Alekseyenko

McElroy

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Posttranslational modifications (PTMs) are key contributors to chromatin function. The ability to comprehensively link specific histone PTMs with specific chromatin factors would be an important advance in understanding the functions and genomic targeting mechanisms of those factors. We recently introduced a cross-linked affinity technique, BioTAP-XL, to identify chromatin-bound protein interactions that can be difficult to capture with native affinity techniques. However, BioTAP-XL was not strictly compatible with similarly comprehensive analyses of associated histone PTMs. Here we advance BioTAP-XL by demonstrating the ability to quantify histone PTMs linked to specific chromatin factors in parallel with the ability to identify nonhistone binding partners. Furthermore we demonstrate that the initially published quantity of starting material can be scaled down orders of magnitude without loss in proteomic sensitivity. We also integrate hydrophilic interaction chromatography to mitigate detergent carryover and improve liquid chromatography-mass spectrometric performance. In summary, we greatly extend the practicality of BioTAP-XL to enable comprehensive identification of protein complexes and their local chromatin environment.chromatin complexes | affinity pulldown | liquid chromatography-mass spectrometry | BioTAP-XL | histone modifications C hromatin encompasses the subset of proteins and RNAs in complex with DNA to form the chromosomes within eukaryotic nuclei. As the primary protein constituents of chromatin, histones organize genomic DNA into nucleosomes and display an extensive array of posttranslational modifications (PTMs) (1). It is increasingly evident that specific histone PTMs are selectively recognized by specific nonhistone proteins that are themselves regulators of many nuclear events such as epigenetic silencing (2, 3). Consequently, defects in these chromatin components often manifest in a number of human diseases (4).As specific interactions between modified histones and nonhistone proteins help distinguish complexes participating in distinct processes, the identification of protein and modification-dependent interactions provides key insights into chromatin biology. Various chromatography and pulldown methods have been developed to identify binding partners. In a typical native pulldown experiment, nuclei from hypotonically lysed cells are digested with micrococcal nuclease (MNase). Extracted chromatin is subjected to immunoprecipitation to isolate the desired complexes. If using a tag such as FLAG, complexes are then competitively eluted and identified. Several drawbacks to native pulldowns as described here are that MNase leaves behind a substantial insoluble nuclear pellet and degrades associating RNAs, that salt extraction of the digested nucleosomes risks dissociation of the tagged bait from its interacting partners, and that most tags do not have sufficient affinity for their respective antibody to permit highly stringent washes. The result is often an incomplete picture of the chromatin...

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PeptideShaker enables reanalysis of MS-derived proteomics data sets

Cited by 461 publications

References 19 publications

Characterization of Trichuris muris secreted proteins and extracellular vesicles provides new insights into host–parasite communication

Characterization of Trichuris muris secreted proteins and extracellular vesicles provides new insights into host–parasite communication

Exploring the potential of public proteomics data

Streamlined discovery of cross-linked chromatin complexes and associated histone modifications by mass spectrometry

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