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DIGESTIF: A universal quality standard for the control of bottom-up proteomics experiments


Lebert, Dorothée; Louwagie, Mathilde; Goetze, Sandra; Picard, Guillaume; Ossola, Reto; Duquesne, Caroline; Basler, Konrad; Ferro, Myriam; Rinner, Oliver; Aebersold, Ruedi; Garin, Jérôme; Mouz, Nicolas; Brunner, Erich; Brun, Virginie (2015). DIGESTIF: A universal quality standard for the control of bottom-up proteomics experiments. Journal of Proteome Research, 14(2):787-803.

Abstract

In bottom-up mass spectrometry-based proteomics analyses, variability at any step of the process, particularly during sample proteolysis, directly affects the sensitivity, accuracy, and precision of peptide detection and quantification. Currently, no generic internal standards are available to control the quality of sample processing steps. This makes it difficult to assess the comparability of MS proteomic data obtained under different experimental conditions. Here, we describe the design, synthesis, and validation of a universal protein standard, called DIGESTIF, that can be added to any biological sample. The DIGESTIF standard consists of a soluble recombinant protein scaffold to which a set of 11 artificial peptides (iRT peptides) with good ionization properties has been incorporated. In the protein scaffold, the amino acids flanking iRT peptide cleavage sites were selected either to favor or hinder protease cleavage. After sample processing, the retention time and relative intensity pattern of the released iRT peptides can be used to assess the quality of sample workup, the extent of digestion, and the performance of the LC-MS system. Thus, DIGESTIF can be used to standardize a broad spectrum of applications, ranging from simple replicate measurements to large-scale biomarker screening in biomedical applications.

Abstract

In bottom-up mass spectrometry-based proteomics analyses, variability at any step of the process, particularly during sample proteolysis, directly affects the sensitivity, accuracy, and precision of peptide detection and quantification. Currently, no generic internal standards are available to control the quality of sample processing steps. This makes it difficult to assess the comparability of MS proteomic data obtained under different experimental conditions. Here, we describe the design, synthesis, and validation of a universal protein standard, called DIGESTIF, that can be added to any biological sample. The DIGESTIF standard consists of a soluble recombinant protein scaffold to which a set of 11 artificial peptides (iRT peptides) with good ionization properties has been incorporated. In the protein scaffold, the amino acids flanking iRT peptide cleavage sites were selected either to favor or hinder protease cleavage. After sample processing, the retention time and relative intensity pattern of the released iRT peptides can be used to assess the quality of sample workup, the extent of digestion, and the performance of the LC-MS system. Thus, DIGESTIF can be used to standardize a broad spectrum of applications, ranging from simple replicate measurements to large-scale biomarker screening in biomedical applications.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:07 Faculty of Science > Institute of Molecular Life Sciences
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2015
Deposited On:16 Jan 2015 09:44
Last Modified:05 Apr 2016 18:51
Publisher:American Chemical Society
ISSN:1535-3893
Additional Information:This document is the Accepted Manuscript version of a Published Work that appeared in final form in [Journal of proteome research], copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see [http://pubs.acs.org/doi/abs/10.1021/pr500834z].
Publisher DOI:https://doi.org/10.1021/pr500834z
PubMed ID:25495225

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