Over the past few decades, we have welcomed a vast array of novel technologies that have enabled the comprehensive analyses of genomes, transcriptomes, and to a lesser extent, proteomes. Although the technical challenges associated with proteomics have offset the promise of rapid biomarker discovery, we forge ahead based on the notion that protein biomarkers have specifically altered oncology clinic practice more than any other type of molecule currently has. Considering that proteins are the functional units within cells, their expression being altered would more likely directly reflect changes in tumour biology. However, roadblocks associated with intra-patient biological variability and the large dynamic range of proteins in traditionally analysed biofluids (plasma and urine) are important challenges.

Traditional proteomics studies have employed antibody-based detection strategies, two-dimensional gel electrophoresis, surface enhanced laser desorption ionization, and other rudimentary technologies, but these have led to incremental advances in the field. Mass spectrometry (MS) is an ever evolving and increasingly powerful methodology that enables comprehensive proteomic analysis of complex biofluids. In this methods collection, we will present the most important and current improvements in MS-based clinical proteomics with a particular focus on cancer biomarkers. We will address the challenges and solutions associated with clinically relevant samples types, best practice sample preparation and quantitation strategies, and MS setup, operation, and data analysis. Beyond finding novel, clinically-relevant biomarkers, rigorous validation is essential for improving early diagnosis, risk stratification, and monitoring patient treatment response.