Identifying peptide substrates that are efficiently cleaved by proteases gives insights into substrate recognition and specificity, guides development of inhibitors, and improves assay sensitivity. Peptide arrays and SAMDI mass spectrometry were used to identify a tetrapeptide substrate exhibiting high activity for the bacterial outer-membrane protease (OmpT). Analysis of protease activity for the preferred residues at the cleavage site (P1, P1') and nearest-neighbor positions (P2, P2') and their positional interdependence revealed FRRV as the optimal peptide with the highest OmpT activity. Substituting FRRV into a fragment of LL37, a natural substrate of OmpT, led to a greater than 400-fold improvement in OmpT catalytic efficiency, with a k /K value of 6.1×10 L mol s . Wild-type and mutant OmpT displayed significant differences in their substrate specificities, demonstrating that even modest mutants may not be suitable substitutes for the native enzyme.

Identifying peptide substrates that are efficiently cleaved by proteases gives insights into substrate recognition and specificity,guides development of inhibitors,and improves assays ensitivity.P eptide arrays and SAMDI mass spectrometry were used to identify at etrapeptide substrate exhibiting high activity for the bacterial outer-membrane protease (OmpT). Analysis of protease activity for the preferred residues at the cleavage site (P1, P1')a nd nearest-neighbor positions (P2, P2')a nd their positional interdependence revealed FRRV as the optimal peptide with the highest OmpT activity.S ubstituting FRRV into af ragment of LL37, an atural substrate of OmpT,l ed to ag reater than 400-fold improvement in OmpT catalytic efficiency,with ak cat /K m value of 6.1 10 6 Lmol À1 s À1 .Wild-type and mutant OmpT displayed significant differences in their substrate specificities,d emonstrating that even modest mutants mayn ot be suitable substitutes for the native enzyme.

Phosphatases, the enzymes responsible for dephosphorylating proteins, play critical roles in many cellular processes. While their importance is widely recognized, phosphatase activity and regulation remain poorly understood. Currently, there are few assays available that are capable of directly measuring phosphatase activity and specificity. We have previously introduced SAMDI (self-assembled monolayers on gold for matrix-assisted laser desorption/ionization) mass spectrometry as a technique to profile the substrate specificities of enzymes. SAMDI mass spectrometry assays are well suited to examine phosphatase activities and offer many advantages over current methods. This technique uses monolayers that terminate with a peptide or molecular enzyme substrate and allows for enzyme reactions to be performed on a surface that can easily be rinsed and analyzed by mass spectrometry without the need for analyte labeling. In this chapter, we describe the process of combining SAMDI mass spectrometry with peptide arrays to study the substrate specificities of two protein tyrosine phosphatases.

The opposing activities of phosphatases and kinases determine the phosphorylation status of proteins, yet kinases have received disproportionate attention in studies of cellular processes, with the roles of phosphatases remaining less understood. This paper describes the use of phosphotyrosine-containing peptide arrays together with MALDI mass spectrometry to directly profile phosphatase substrate selectivities. Twenty-two phosphotyrosine phosphatases were characterized with the arrays to give a profile of their specificities. An analysis of the data revealed that certain residues in the substrates had a conserved effect on activity for all enzymes tested, including the general rule that inclusion of a basic lysine or arginine residue on either side of the phosphotyrosine decreased activity. This insight also provides a new perspective on the role of a R1152Q mutant in the insulin receptor, which is known to exhibit a lower phosphorylation level, and which this work suggests may be due to an increased activity towards phosphatase enzymes. The use of SAMDI-MS to provide a rapid and quantitative assay of phosphatase enzymes will be important to gaining a more complete understanding of the biochemistry and biology of this important enzyme class.

Protein oxidation by reactive oxygen species has been associated with aging and neurodegenerative disorders, and histidine is one of the major oxidation targets due to its metal-chelating property and susceptibility to metal-catalyzed oxidation. 2-Oxohistidine, the major product of histidine oxidation, has been recently identified as a stable marker of oxidative damage in biological systems, but its biophysical and biochemical properties are understudied, partly because of difficulties in its chemical synthesis. We developed an efficient method to generate a 2-oxohistidine side chain using metal-catalyzed oxidation, applicable to both monomers and peptides. By optimizing reagent ratios and pH buffering in Cu(2+) /ascorbate/O2 reaction system, we improved the yield more than tenfold compared to reported conditions, which allowed us to obtain homogeneously modified 2-oxohisidine peptides for further studies. Analysis of 2-oxohistidine-containing model peptides by liquid chromatography-tandem mass spectrometry demonstrated increased retention time in reverse-phase chromatography and general stability of 2-oxohistidine under electrospray ionization and collision-induced dissociation. Thus, large-scale analysis of 2-oxohistidine-modified proteome should be feasible using shotgun protein mass spectrometry, and we were able to observe such peptides in proteomics datasets. The feasibility of acquiring purified peptide probes and peptide antigens containing 2-oxohistidine will help advance the study of this non-enzymatic posttranslational modification.