A thorough evaluation of the quality, reproducibility, and variability of bottom-up proteomics data is necessary at every stage of a workflow, from planning to analysis. We share vignettes applying adaptable quality control (QC) measures to assess sample preparation, system function, and quantitative analysis. System suitability samples are repeatedly measured longitudinally with targeted methods, and we share examples where they are used on three instrument platforms to identify severe system failures and track function over months to years. Internal QCs incorporated at the protein and peptide levels allow our team to assess sample preparation issues and to differentiate system failures from sample-specific issues. External QC samples prepared alongside our experimental samples are used to verify the consistency and quantitative potential of our results during batch correction and normalization before assessing biological phenotypes. We combine these controls with rapid analysis (Skyline), longitudinal QC metrics (AutoQC), and server-based data deposition (PanoramaWeb). We propose that this integrated approach to QC is a useful starting point for groups to facilitate rapid quality control assessment to ensure that valuable instrument time is used to collect the best quality data possible. Data are available on Panorama Public and ProteomeXchange under the identifier PXD051318.

This paper includes 7 figures and 2 Supplemental Figures. The mass spectrometry data was collected between 2014 and 2023 in the MacCoss laboratory. The public GitHub Repository maccoss-sample-qc-system-suitability includes R scripts and R Markdown documents that were used to generate Figures 3-7 and Supplementary Figures 1 and 2. The figures include: (1) a schematic of quality controls used, (2) longitudinal tracking of system suitability, (3) the use of targeted system suitability methods to track system issues, (4) the use of internal controls to identify a sample preparation issue, (5) the use of internal controls to identify a system issue, (6) the use of internal controls and system suitability to a identify system issue, (7) a demonstration of external quality controls in large-scale human study, (S1) an expansion of the example in Figure 4 with an additional enolase peptide and two PRTC peptides, and (S2) an alternative way to view the vignette in Figure 6 with PRTC peptides split into two panels by run type.

Figure 1 is illustrative (no samples). The peptide internal QC in all figures is PRTC. The protein internal QC in all figures in yeast enolase (ENO). The system suitability sample is 600 fmol BSA and 150 fmol PRTC in all figures. A yeast digest was assessed for system suitability using DDA as part of Figure 3. In Figure 4 and Supplemental Figure 1, a pool of CSF was used for sample method development. Pooled human plasma was the sample matrix in Figure 5. Samples of mouse brain were run in Figure 6. Individual samples of human CSF were processed in Figure 7.