title: Body Fluid Degradomics and Characterization of Basic N-Terminome.
abstract: Rapid improvements in instrumentation and data analysis make mass spectrometry-based proteomics the method of choice for global characterization of proteomes and discovery of protein-based biomarkers. On the contrary to tissue biopsies, body fluids-e.g., blood, wound fluid, urine, and saliva-are noninvasive and easy to collect and process. However, they are very complex and present high dynamic ranges of protein concentrations, rendering direct shotgun proteomics analysis as inefficient for identification of low-abundance proteins in these specimens. Sample prefractionation, immunoaffinity depletion of highly abundant proteins, and enrichment of posttranslational modifications (PTM) are common strategies for proteome simplification of body fluids. Combinatorial peptide ligand libraries (CPLL) relatively deplete high-abundance proteins by binding equimolar amounts of protein species in the sample and provide an elegant species-independent alternative to immunoaffinity-based approaches. By cleaving target proteins, proteases catalyze an irreversible PTM, whereby uncontrolled proteolysis is associated with many diseases. Thus, proteolytic events represent powerful indicators for disease progression and their specific identification in body fluids holds great promises for establishment of novel biomarkers. Quantitative N-terminal enrichment strategies, such as terminal amine isotopic labeling of substrates (TAILS) detect protease-generated neo-N-termini with high specificity and increase coverage of low-abundance proteins by inherent proteome simplification. In this chapter, we describe a protocol that combines the CPLL technology with iTRAQ-based TAILS to systematically characterize the basic N-terminome of body fluid proteomes and its alterations in disease conditions that we have successfully applied to explore the wound fluid degradome at multiple time points after skin injury.
authors.affiliation: ETH Zurich, Institute of Molecular Health Sciences, Zurich, Switzerland.
authors.forename: F
authors.initials: F
authors.lastname: Sabino
authors.affiliation: ETH Zurich, Institute of Molecular Health Sciences, Zurich, Switzerland.
authors.forename: O
authors.initials: O
authors.lastname: Hermes
authors.affiliation: ETH Zurich, Institute of Molecular Health Sciences, Zurich, Switzerland. Electronic address: ulrich.aufdemkeller@biol.ethz.ch.
authors.forename: U
authors.initials: U
authors.lastname: Auf dem Keller
mesh_terms: D001826:Body Fluids / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*
chemical_list: D020543:Proteome
keywords: Body fluid; Combinatorial peptide ligand libraries; Degradome; N-Terminome; TAILS; iTRAQ