Use of Synthetic Standard Peptides in Standardized Digests to Evaluate Both Sample and Instrument Suitability in Proteomics

Whole proteome digests are routinely used to diagnose chromatograph and mass spectrometer outputs to ensure suitability for the analysis of complex matrices, but their usage inherently fails to prove system reliability under varying "load" conditions. There is a need for a reliable, predictive tool that can explain variation in both instrument response and downstream identification results from a whole proteome analysis. We designed an experiment using a hybrid sample of standardized materials to create such an approach, which could then lead to a new system suitability test for bottom-up proteomics. The standard HeLa protein digest was combined with Promega 6 × 5 LC-MS/MS Peptide Reference Mix and diluted to create a range of sample mass loadings and reference peptide concentrations. Data were collected using data-dependent (DDA) and data-independent acquisition methods, and reference peptide peak abundances were correlated to the number of protein identifications (IDs), peptide groups (PGs), and peptide spectrum matches (PSMs) found by Proteome Discoverer. An asymptotic relationship explained decreasing IDs, PGs, and PSMs identified from the HeLa digest with decreasing 6 × 5 Peptide abundances. By linking the mass spectrometer measurement of ion abundance with downstream results obtained from a complex matrix, we successfully used the hybrid standardized sample to mathematically define new system suitability thresholds.