Pathway-Informed Discovery and Targeted Proteomic Workflows Using Mass Spectrometry

Recent advancements in mass spectrometry (MS) and data analysis software have enabled new strategies for biological discovery using proteomics. Proteomics has evolved from routine discovery and identification of proteins to integrated multi-omics projects relating specific proteins to their genes and metabolites. Using additional information, such as that contained in biological pathways, has enabled the use of targeted protein quantitation for monitoring fold changes in expression as well as biomarker discovery. Here we discuss a full proteomic workflow from discovery proteomics on a quadrupole Time-of-Flight (Q-TOF) MS to targeted proteomics using a triple quadrupole (QQQ) MS. A discovery proteomics workflow encompassing acquisition of data-dependent proteomics data on a Q-TOF and protein database searching will be described which uses the protein abundances from identified proteins for subsequent statistical analysis and pathway visualization. From the active pathways, a protein target list is created for use in a peptide-based QQQ assay. These peptides are used as surrogates for target protein quantitation. Peptide-based QQQ assays provide sensitivity and selectivity allowing rapid and robust analysis of large batches of samples. These quantitative results are then statistically compared and visualized on the original biological pathways with a more complete coverage of proteins in the studied pathways.