Effective protein extraction combined with data independent acquisition analysis reveals a comprehensive and quantifiable insight into the proteomes of articular cartilage and subchondral bone.

OBJECTIVE: The objectives of this study was to establish a sensitive and reproducible method to map the cartilage and subchondral bone proteomes in quantitative terms, and mine the proteomes for proteins of particular interest in the pathogenesis of osteoarthritis (OA). The horse was used as a model animal. DESIGN: Protein was extracted from articular cartilage and subchondral bone samples from three horses in triplicate by pressure cycling technology or ultrasonication. Digested proteins were analysed by data independent acquisition based mass spectrometry. Data was processed using a pre-established spectral library as reference database (FDR 1%). RESULTS: We identified to our knowledge the hitherto most comprehensive quantitative cartilage (1758 proteins) and subchondral bone (1482 proteins) proteomes in all species presented to date. Both extraction methods were sensitive and reproducible and the high consistency of the identified proteomes (>97% overlap) indicated that both methods preserved the diversity among the extracted proteins. Proteome mining revealed a substantial number of quantifiable cartilage and bone matrix proteins and proteins involved in osteogenesis and bone remodeling, including ACAN, BGN, PRELP, FMOD, COMP, ACP5, BMP3, BMP6, BGLAP, TGFB1, IGF1, ALP, MMP3, and collagens. A number of proteins, including COMP and TNN, were identified in different protein isoforms with potential unique biological roles. CONCLUSION: We have successfully developed two sensitive and reproducible non-species specific workflows enabling a comprehensive quantitative insight into the proteomes of cartilage and subchondral bone. This facilitates the prospect of investigating the molecular events at the osteochondral unit in the pathogenesis of OA in future projects.

Exploring the role of extracellular matrix proteins to develop biomarkers of plaque vulnerability and outcome.

Cardiovascular disease (CVD) is the most common cause of death in industrialized countries. One underlying cause is atherosclerosis, which is a systemic disease characterized by plaques of retained lipids, inflammatory cells, apoptotic cells, calcium and extracellular matrix (ECM) proteins in the arterial wall. The biologic composition of an atherosclerotic plaque determines whether the plaque is more or less vulnerable, that is prone to rupture or erosion. Here, the ECM and tissue repair play an important role in plaque stability, vulnerability and progression. This review will focus on ECM remodelling in atherosclerotic plaques, with focus on how ECM biomarkers might predict plaque vulnerability and outcome.

Body Fluid Degradomics and Characterization of Basic N-Terminome.

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.