Multiplexed Protease Specificity Profiling Using Isobaric Labeling.

The determination of a protease's cleavage site specificity is one of the major goals in degradomics. In the last years, the use of proteome-derived peptide libraries and liquid chromatography-mass spectrometry (LC-MS) in a proteomic identification of protease cleavage sites (PICS) experiment became popular for that purpose.In this chapter, we offer a step-by-step protocol for the execution of a quantitative proteomic identification of protease cleavage sites (Q-PICS) experiment, which enables the relative quantification of proteolytic events by isobaric labeling, e.g., with tandem mass tags (TMT). In this way, the cleavage site specificity and activity of a protease can be compared under different reaction conditions (e.g., buffer, pH, temperature, inhibitor). Multiplexing can further be used to analyze replicate experiments in parallel, decreasing instrument times and work effort significantly, or to perform internal controls.

From top-down to bottom-up: Time-dependent monitoring of proteolytic protein degradation by LC-MS.

The understanding of proteolytic processes includes manifold aspects, ranging from the characterization of proteases and their corresponding substrates to the localization of cleavage sites. The analysis of protease-catalyzed reactions at a single time-point in many cases excludes the identification of intermediate cleavage products of potential biological function. To overcome this problem, proteolysis has to be monitored over time. For that purpose, we established a straight-forward two-step approach. First, Tricine-SDS-PAGE separation of the proteolytic products is applied to survey the proteolytic reaction. In the second step, the reaction mixture is analyzed by an LC-MS set-up. An optimized chromatographic separation coupled to electrospray Orbitrap mass spectrometry allowed the simultaneous monitoring of intact substrates, intermediates and cleavage products of lower molecular weight. The applicability of the strategy was shown on the example of the gastric protease pepsin and its physiologically relevant substrate hen egg white lysozyme, one of the major egg allergens. While lysozyme-derived cysteine-free peptides were cleaved comparatively fast, disulfide bonds protected connected peptides from rapid peptic proteolysis. Two previously identified potential IgE-binding motifs were observed as disulfide-linked cleavage products. In summary, the presented approach is not only ideally suited for the simulation of gastro-intestinal digestion, which is of high interest in food research, but can be transferred to any protease-substrate pair of interest. Furthermore the strategy can be exploited to deduce the effect of post-translational modifications on proteolysis.

Targeted mass spectrometry for the analysis of nutritive modulation of catalase and heme oxygenase-1 expression.

Comprehensive physiological food assessment requires recording of activity profiles. To elucidate the nutritive regulation of antioxidant enzymes, a generally applicable targeted MS method was established for the expression analysis of catalase and then adapted to heme oxygenase-1. Before tryptic digestion, target proteins were prefractionated by off-gel IEF of stimulated and control cell lysate. Targeted proteome analysis was achieved by LC coupled with scheduled selected reaction monitoring MS using 2 proteotypic peptides per protein and 3-4 transitions per peptide. Relative quantification was performed by stable isotope labeling by amino acids in cell culture (SILAC). The assay showed good correlation with results by Western blot. Linearity, precision, and sensitivity were even improved (LC/SRM vs. Western blot: 3 vs. 1 orders of magnitude, RSD 3.7-13.7% vs. 18.4%, LOD 0.2 vs. 1.6µg/mL). The developed method indicated that coffee does not modulate catalase expression in macrophages (T7cat 103±22%, T17cat 103±16%, p>0.05 vs. control), but leads to a highly significant increase of heme oxygenase-1 expression (T15Ho-1 420±24%, T22Ho-1 364±37%, p<0.001 vs. control, p>0.05 T15Ho-1 vs. T22Ho-1). In regard to multiplex options of the method, targeted proteome analysis can be a valuable tool for the comprehensive analysis of cellular effects of food components. BIOLOGICAL SIGNIFICANCE: In the present study, targeted mass spectrometry was applied to determine the influence of food components on the expression of antioxidative enzymes. The results implicate that targeted proteomics may develop into a valuable tool in food science and nutrition to determine the physiological effects of nutrients. In contrast to conventional methods for expression analysis, targeted proteome analysis can be applied to monitor the effects of a food component on a broad range of cellular targets in parallel. Additionally, proteins or protein modifications can be addressed which elude immunochemical methods.

LC-MS based cleavage site profiling of the proteases ADAM10 and ADAM17 using proteome-derived peptide libraries.

A Disintegrin and Metalloproteinase 10 (ADAM10) and ADAM17 catalyze ectodomain shedding of a number of cell surface proteins important for embryonic development and tissue homeostasis. Changes in the expression levels or dysregulated proteolytic activity of ADAM10 and ADAM17 have been shown to play important roles in multiple diseases such as inflammation, cancer, and neurodegenerative disorders. Despite the well documented substrate repertoire of ADAM10 and ADAM17, little is known about their cleavage site specificity. We optimized Q-PICS (Quantitative Proteomics for the Identification of Cleavage Sites) to elucidate the cleavage site specificity of recombinant murine ADAM10 and ADAM17. Two different yeast proteome-derived peptide libraries were used and samples were analyzed by LC-MALDI and LC-ESI MS in parallel. We show that the largest difference in the cleavage site specificities of ADAM10 and ADAM17 is at the P1' site: while both enzymes cleave N-terminal of leucine, only ADAM10 shows additional preference toward aromatic amino acids, whereas ADAM17 exhibits the highest preference for valine. Together with further amino acid preferences more adjacent to the scissile bond, our data is in good agreement with ADAM10/17 cleavage sites previously identified in native substrates. Overall, the precise identification of ADAM10 and ADAM17 cleavage site specificity provides the basis for better substrate identification in vivo and the generation of specific inhibitors or activity based probes.