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Phosphorylation is an important post‐translational modification on proteins involved in many cellular processes; however, understanding of the regulation and mechanisms of global phosphorylation remains limited. Herein, we utilize self‐assembled monolayers on gold for matrix‐assisted laser desorption/ionization mass spectrometry (SAMDI‐MS) with three phosphorylated peptide arrays to profile global phosphatase activity in cell lysates derived from five mammalian cell lines. Our results reveal significant differences in the activities of protein phosphatases on phospho‐ serine, threonine, and tyrosine substrates and suggest that phosphatases play a much larger role in the regulation of global phosphorylation on proteins than previously understood. [ABSTRACT FROM AUTHOR]

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 kcat/ Km value of 6.1×106 L mol−1 s−1. 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. [ABSTRACT FROM AUTHOR]

Peptide arrays are used to identify highly active substrates for the OmpT protease, as described by M. Mrksich, B. Liedberg, et al. in their Communication on page 16531 ff. By preparing the peptide arrays on self‐assembled monolayers, mass spectrometry (the SAMDI method) was used to characterize the extent of cleavage of each peptide. A commonly used mutant form of the protease, although regarded as having identical activity, instead had an altered specificity for its substrates. [ABSTRACT FROM AUTHOR]

Peptidanordnungen, identifizieren hoch aktive Substrate für die OmpT‐Protease. M. Mrksich, B. Liedberg et al. beschreiben in ihrer Zuschrift auf S. 16758 die Herstellung der Peptidanordnungen auf selbstorganisierten Monoschichten und die massenspektrometrische Charakterisierung des Ausmaßes an Spaltung für jedes Peptid mithilfe der SAMDI‐Methode. Eine häufig eingesetzte Protease‐Mutante, der man eine identische Aktivität zuschreibt, zeigte eine abweichende Substratspezifität. [ABSTRACT FROM AUTHOR]