Inserting Pre-Analytical Chromatographic Priming Runs Significantly Improves Targeted Pathway Proteomics With Sample Multiplexing.

GoDig, a recent platform for targeted pathway proteomics without the need for manual assay scheduling or synthetic standard peptides, is a relatively flexible and easy-to-use method that uses tandem mass tags (TMT) to increase sample throughput up to 18-fold relative to label-free targeted proteomics. Though the protein quantification success rate of GoDig is generally high, the peptide-level success rate is more limited, hampering the extension of GoDig to assays of harder-to-quantify proteins and site-specific phenomena. In order to guide the optimization of GoDig assays as well as improvements to the GoDig platform, we created GoDigViewer, a new stand-alone software that provides detailed visualizations of GoDig runs. GoDigViewer guided the implementation of "priming runs," an acquisition mode with significantly higher success rates due to improved elution order calibration. In this mode, one or more chromatographic priming runs are automatically performed to determine accurate and precise target elution orders, followed by analytical runs which quantify targets. Using priming runs, peptide-level quantification success rates exceeded 97% for a list of 400 peptide targets and 95% for a list of 200 targets that are usually not quantified using untargeted mass spectrometry. We used priming runs to establish a quantitative assay of 125 macroautophagy proteins that had a >95% success rate and revealed differences in macroautophagy protein expression profiles across four human cell lines.