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Liquid chromatography/mass spectrometry (LC/MS) has become a routine powerful technology in clinical proteomic studies for protein identification, protein characterization, and the discovery of biomarkers. In this chapter, we describe two protocol methods to analyze clinical patient samples using a resin-based depletion column followed by either protein In-Gel enzymatic digestion or protein In-Solution enzymatic digestion using a simple kit-based approach (i.e., using the PreOmics iST sample preparation kit), followed by analysis using one-dimensional reverse-phase chromatography (RPC) or high pH reversed-phase peptide fractionation., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

This chapter focuses on upstream immunodepletion of high-abundance proteins from plasma samples and subsequent analysis by fluorescence two-dimensional difference gel electrophoresis (2D-DIGE). The abundances of proteins in biofluid proteomes, such as serum, plasma, saliva, and bronchoalveolar lavage fluid (BALF), can exceed ten orders of magnitude. This substantial dynamic range is problematic for the detection of medium and low-abundance proteins by 2D-DIGE analysis. To increase the detection, quantification, and identification of medium-low-abundance proteins, the targeted depletion of known abundant proteins with antibody columns has been successfully employed. From the literature, it is clear that the performance of abundant protein depletion with immunodepletion columns has been successful in broadening the coverage of the biofluid proteome and facilitating the identification of disease-specific biomarkers. The task for a successful biomarker strategy involves the combination of a reproducible and robust fractionation method, coupled with a highly accurate quantitative method, a task that is exemplified by combining both immunodepletion and 2D-DIGE together to discover significant proteins associated with the disease phenotype., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

This chapter will focus on upstream immunodepletion of high abundant proteins from plasma samples and subsequent analysis by difference gel electrophoresis (DIGE). The abundances of proteins in biofluid proteomes, such as serum, plasma, saliva, and bronchoalveolar lavage fluid (BALF), can exceed 10 orders of magnitude. This substantial dynamic range is problematic for the detection of medium and low abundance proteins by DIGE analysis. To increase the detection, quantification, and identification of medium-low abundant proteins, the targeted depletion of known abundant proteins with antibody columns has been successfully employed. From the literature, it is clear that the performance of abundant protein depletion with immunodepletion columns has been successful in broadening the coverage of the biofluid proteome and facilitating the identification of disease-specific biomarkers. The task for a successful biomarker strategy involves the combination of a reproducible and robust fractionation method, coupled with a highly accurate quantitative method, a task that is exemplified by combining both immunodepletion and DIGE together to discover significant proteins associated with the disease phenotype.

Liquid chromatography-mass spectrometry (LC-MS) has become a routine powerful technology in clinical proteomic studies for protein identification, protein characterization and the discovery of biomarkers. In this chapter, we describe two protocol methods to analyze clinical patient samples using a resin based depletion column followed by either protein In-gel enzymatic digestion or protein in-solution enzymatic digestion and then analysis by one-dimensional reverse-phase chromatography or two-dimensional strong cation exchange (SCX)-reverse-phase chromatography (RPC).

Cell-free extracts made from Xenopus laevis eggs enable us to recapitulate many chromosomal events associated with cell cycle progression in a test tube. When sperm chromatin is incubated with these extracts, it is first duplicated within an assembled nucleus, and is then transformed into mitotic chromosomes, in each of which sister chromatids are juxtaposed with each other in a cohesin-dependent manner. Here we describe our protocols for assembling duplicated chromosomes using egg extracts, along with cytological and biochemical assays for addressing the molecular mechanisms of sister chromatid cohesion. A powerful approach involving immunodepletion of cohesin and its regulators is also included.

The COP9 signalosome (CSN) is an evolutionary conserved complex that is found in all eukaryotes, and implicated in regulating the activity of Cullin-RING ubiquitin Ligases (CRLs). Activity of CRLs is highly regulated; complexes are active when the cullin subunit is covalently attached to the ubiquitin like modifier, Nedd8. Neddylation/deneddylation cycles are required for proper CRLs activity, and deneddylation is performed by the CSN complex.We describe here a method utilizing resin-coupled antibodies to deplete the CSN from human cell extracts, and to obtain endogenous CSN complexes by immunopurification. In the first step, the cross-linked primary antibodies recognize endogenous CSN complexes, and deplete them from cell extract as the extract passes through the immunoaffinity column. The resulting "CSN-depleted extract" (CDP) is rich in neddylated cullins that can be used as a substrate for cullin-deneddylation assay for CSN complexes purified from various eukaryotes. Consequently, regeneration of the column results in dissociation of a highly purified CSN complex, together with its associated proteins. Immunopurification of the CSN from various human tissues or experimental conditions is advantageous for the generation of numerous CSN-interaction maps.

Mesenchymal stem cells (MSCs) were initially characterized as connective tissue progenitors resident in bone marrow, but have now been isolated from a variety of tissues and organs and shown to also exhibit potent tissue regenerative properties mediated largely via paracrine actions. These findings have spurred the development of MSC-based therapies for treating a diverse array of nonskeletal diseases. Although genetic and experimental rodent models of disease represent important tools for developing efficacious MSC-based therapies, development of reliable methods to isolate MSCs from mouse bone marrow has been hampered by the unique biological properties of these cells. Indeed, few isolation schemes afford high yields and purity while maintaining the genomic integrity of cells. We recently demonstrated that mouse MSCs are highly sensitive to oxidative stress, and long-term expansion of these cells in atmospheric oxygen selects for immortalized clones that lack a functional p53 protein. Herein, we describe a protocol for the isolation of primary MSCs from mouse bone marrow that couples immunodepletion with culture in a low-oxygen environment and affords high purity and yield while preserving p53 function.