Analyzing Various Biological Fluids with a Single Automated Proteomic Workflow for Biomarker Discovery

Protein biomarker discovery in human biological fluids has greatly developed over the past two decades thanks to technological advances allowing deeper proteome coverage and higher sample throughput, among others. While blood samples are most commonly investigated due to their moderate ease of collection and high information content, other biological fluids such as cerebrospinal fluid (CSF) and urine are highly relevant for specific pathologies, such as brain and urologic diseases, respectively. Independently of the biofluid of interest, platforms that can robustly handle a large number of samples are essential in the discovery phase of a clinical study.We have previously described a scalable automated proteomic pipeline (ASAP²) for the sample preparation of hundreds to thousands of blood plasma, serum, and CSF samples before liquid chromatography (LC)-mass spectrometry (MS) analysis. Here, we describe how the workflow was further adapted to milk and urine samples, with small modifications at the beginning of the workflow. For blood and CSF samples, an optional immuno-affinity depletion step for abundant-protein removal constitutes the first step of the workflow. In the analysis of milk, a defatting step is incorporated before the samples are further processed with ASAP², while acetone precipitation is used for the analysis of urine samples. The main sample preparation steps then remain identical for all sample types, are automated on a liquid handling workstation, and include reduction of disulfide bridges, alkylation of free thiols, protein digestion, isobaric labeling of peptides, sample pooling, and purifications. The workflow is completed by LC-MS analysis of the samples and subsequent data processing.