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Selected/multiple reaction monitoring-mass spectrometry (SRM/MRM) is an analytical method that is frequently combined to the use of stable isotope-labeled standard (SIS) peptides for absolute protein quantification. The application of SRM/MRM is a relatively recent development in the proteomics field for analysis of biological samples (plasma, urine, cell/tissue lysates) targeting to a large extent biomarker validation. Although MRM generally by-passes the use of antibodies (being linked to sub-optimal assay specificity in many cases), bioanalytical validation of MRM protocols has not been robustly applied because of sensitivity issues, in contrary to antibody-based methods. In this chapter, we will discuss the points that should be addressed for MRM method development in clinical proteomics applications.

Application of various proteomics methodologies have been implemented for the global and targeted proteome analysis of many different types of biological samples such as tissue, urine, plasma, serum, blood, and cell lines. Among the aforementioned biological samples, tissue has an exceptional role into clinical research and practice. Disease initiation and progression is usually located at the tissue level of different organs, making the analysis of this material very important for the understanding of the disease pathophysiology. Despite the significant advances in the mass spectrometry instrumentation, tissue proteomics still faces several challenges mainly due to increased sample complexity and heterogeneity. However, the most prominent challenge is attributed to the invasive procedure of tissue sampling which restricts the availability of fresh frozen tissue to minimal amounts and limited number of samples. Application of GeLC-MS sample preparation protocol for tissue proteomics analysis can greatly facilitate making up for these difficulties. In this chapter, a step by step guide for the proteomics analysis of minute amounts of tissue samples using the GeLC-MS sample preparation protocol, as applied by our group in the analysis of multiple different types of tissues (vessels, kidney, bladder, prostate, heart) is provided.

Proteomic study of clinical samples aims at the better understanding of physiological and pathological conditions, as well as the discovery of diagnostic and prognostic markers for the latter. Quantitative and/or qualitative variations of body fluid proteome may reflect health- or disease-associated events connected to the adjacent or distant body regions of the fluid production/secretion/excretion and/or systemic reactions to the presence of disease. Sample collection and preparation is a critical step in order to obtain useful and valid information in clinical proteomics analysis. In this chapter, we present the current protocols and guidelines for human body fluid collection and storage, prior to proteomic analysis. A variety of body fluids that are currently being used in proteomic analysis and have potential interest in clinical practice are presented including blood plasma and serum, urine, cerebrospinal fluid, cerumen, nasal secretions, saliva, tears, breast milk, bronchoalveolar fluid, nipple aspirate fluid, amniotic fluid, bile, cervico-vaginal fluid, and seminal plasma. With no doubt these body fluids differ in the extent of their application in clinical proteomics investigations, hence in some cases the presented SOPs are established following more extensive testing (e.g., plasma, serum, urine, CSF) than others (nasal secretions, saliva, tears, breast milk, bronchoalveolar fluid, nipple aspirate fluid, amniotic fluid, bile, cervico-vaginal fluid, and seminal plasma). However, even in these latter cases, the presented protocols were reported by at least two independent groups according to the literature. We hope they can thus serve as a reliable guide for sample collection based on our current knowledge in the field and excellent starting points for proteomics investigators. It should also be pointed that variations to these protocols exist and their further refinement in the future is foreseen following the evolution of the proteomics technologies.

The high complexity of the total cellular proteome underscores the need for a more targeted investigation of particular subcellular fractions as a means to detect the changes at the level of low abundance proteins. However, this approach requires the application of an enrichment strategy. In this chapter, we present the protocols, which have been used for the analysis of secretome from cell lines, targeting the investigation of protein expression changes.