Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells

A streamlined, robust sample-preparation method for mass spectrometry–based proteome analysis is reported. All sample preparation steps are carried out in a single enclosed reactor, reducing the potential for contamination and losses, and enabling comprehensive proteome coverage. Mass spectrometry (...

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Vydáno v:	Nature methods Ročník 11; číslo 3; s. 319 - 324
Hlavní autoři:	Kulak, Nils A, Pichler, Garwin, Paron, Igor, Nagaraj, Nagarjuna, Mann, Matthias
Médium:	Journal Article
Jazyk:	angličtina
Vydáno:	New York Nature Publishing Group US 01.03.2014 Nature Publishing Group
Témata:	38/56 631/1647/2067 631/337/475 631/45/475 82/16 82/58 82/81 Bioinformatics Biological Microscopy Biological Techniques Biomedical Engineering/Biotechnology Cells Contamination DNA Contamination Eukaryotes Eukaryotic Cells - metabolism Gene Dosage - genetics HeLa Cells Humans Life Sciences Mass spectrometry Numbers Peptides Physiological aspects Proteomics Proteomics - methods Reproducibility of Results Saccharomyces cerevisiae Proteins - genetics Yeasts Germany
ISSN:	1548-7091, 1548-7105, 1548-7105
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Popis
Shrnutí:	A streamlined, robust sample-preparation method for mass spectrometry–based proteome analysis is reported. All sample preparation steps are carried out in a single enclosed reactor, reducing the potential for contamination and losses, and enabling comprehensive proteome coverage. Mass spectrometry (MS)-based proteomics typically employs multistep sample-preparation workflows that are subject to sample contamination and loss. We report an in-StageTip method for performing sample processing, from cell lysis through elution of purified peptides, in a single, enclosed volume. This robust and scalable method largely eliminates contamination or loss. Peptides can be eluted in several fractions or in one step for single-run proteome analysis. In one day, we obtained the largest proteome coverage to date for budding and fission yeast, and found that protein copy numbers in these cells were highly correlated ( R 2 = 0.78). Applying the in-StageTip method to quadruplicate measurements of a human cell line, we obtained copy-number estimates for 9,667 human proteins and observed excellent quantitative reproducibility between replicates ( R 2 = 0.97). The in-StageTip method is straightforward and generally applicable in biological or clinical applications.
Bibliografie:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23 ObjectType-Undefined-3
ISSN:	1548-7091 1548-7105 1548-7105
DOI:	10.1038/nmeth.2834