Figure 1. Overview of the LC-MS/MS shotgun proteomics pipeline.

Primary tumor tissue is sectioned and used for macrodissection to enrich for tumor cell content. This preparation is used for protein isolation and subsequent digestion with trypsin to create peptide mixtures. Peptides are then separated into 15 fractions based on isoelectric point and each of these fractions is further separated using high-performance liquid chromatography (HPLC), in-line with the mass spectrometer. Ions obtained by electrospray are captured and individually collided with an inert gas to create fragmentation patterns (MS scans) that are unique for each peptide. The MS scans are compared with predicted fragmentation patterns from the known human proteome and positively identified peptides are then assembled into groups of proteins. The number of times each peptide was observed (spectral counts) serves as a measure for the abundance of the protein in the original mixture and these counts are used for quasi-likelihood modeling to compare protein levels between tissue types. Protein expression levels can be used for hierarchical cluster analysis and represented in heat maps. The link with biological data is made by enrichment analyses against biological databases (Gene Ontology, GO; Kyoto Encyclopedia of Genes and Genomes, KEGG, etc.)