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

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. ABSTRACTIn 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 in 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 to either 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 up to large-scale biomarker screenings in biomedical applications.

Quality control in mass spectrometry‐based proteomics

“…DIGESTIF is a commercially available compound QC sample that can be used to evaluate the tryptic digestion efficiency . The DIGESTIF standard is assembled from a protein scaffold and artificial peptides, with the amino acids flanking the cleavage sites of these peptides selected to either favor or hinder proteolytic cleavage.…”

“…Instead, the reference RTs of the iRT peptides can be used to correct for variations in the RT of the other peptides detected in a single experiment or to align RTs across multiple experiments. Several QC standards containing iRT peptides have been proposed . These standards mostly vary slightly in the range of the LC gradient they can cover, but some standards have further advanced properties.…”

“…These standards mostly vary slightly in the range of the LC gradient they can cover, but some standards have further advanced properties. For example, the previously mentioned DIGESTIF standard can additionally be used to evaluate the tryptic digestion performance, while the RePLiCal standard consists of a synthetic protein that exclusively contains lysine‐terminating peptides, which ensures that proteolytic digestion by both trypsin and Lys‐C can be evaluated analogously …”

Clinical translation of MS-based, quantitative plasma proteomics: status, challenges, requirements, and potential

“…To date, a number of QCs have been commercialized for human plasma proteomics on an array of instrument platforms [96,97]. The ProteusQC ™ standard (Cambridge Isotope Laboratories), which contains an isotopically labeled mixture of human apolipoprotein A-I and a subset of its peptides, is a recent example.…”

A quality control of proteomic experiments based on multiple isotopologous internal standards

“…Furthermore, the preparation of samples using well-established procedures is necessary. These points have been widely recognized and several efforts have been undertaken in the past years towards the standardization of bottom-up proteomics LC-MS/MS analyses [1][2][3][4][5][6][7][8][9][10][11]. More specifically for quantitative analyses, proteomics can actually rely on the guidelines previously established in analytical and clinical chemistry [12,13].…”