DIGESTIF: A Universal Quality Standard for the Control of Bottom-Up Proteomics Experiments

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.