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Mass spectrometry Quantitative proteomics Label-free quantification Secretome Secreted proteins Cytokines Interleukins Interferons Growth factors Sample preparation
Abstract:
Secreted proteins such as cytokines, interleukins, growth factors, and hormones have pleiotropic functions and facilitate intercellular communication in organisms. Quantification of these proteins conventionally relies on antibody-based methods, i.e., enzyme-linked immunosorbent assays (ELISA), whose large-scale use is limited by availability, specificity, and affordability.
Here, we describe an experimental and bioinformatics workflow to comprehensively quantify cellular protein secretion by mass spectrometry. Secreted proteins are collected in vitro or ex vivo, digested with proteases and the resulting peptide mixtures are analyzed in single liquid chromatography–mass spectrometry (LC-MS/MS) runs. Label-free quantification and bioinformatics analysis is conducted in the MaxQuant and Perseus computational environment. Our workflow allows the quantification of thousands of secreted proteins spanning a concentration range of four orders of magnitude and permits the systems-level characterization of secretory programs as well as the discovery of proteins with unexpected extracellular functions.
