Cleavage specificity analysis of six type II transmembrane serine proteases (TTSPs) using PICS with proteome-derived peptide libraries.

Background: Type II transmembrane serine proteases (TTSPs) are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors.

Methodology/principal Finding: To profile the active site specificities of the TTSPs, we applied Proteomic Identification of protease Cleavage Sites (PICS).

Identification and relative quantification of native and proteolytically generated protein C-termini from complex proteomes: C-terminome analysis.

Proteome-wide analysis of protein C-termini has long been inaccessible, but is now enabled by a newly developed negative selection strategy we term C-terminomics. In this procedure, amine- and carboxyl groups of full-length proteins are chemically protected. After trypsin digestion, N-terminal and internal tryptic peptides - but not C-terminal peptides - posses newly formed, unprotected C-termini that are removed by coupling to the high-molecular-weight polymer poly-allylamine.

Proteomic analyses reveal an acidic prime side specificity for the astacin metalloprotease family reflected by physiological substrates.

Astacins are secreted and membrane-bound metalloproteases with clear associations to many important pathological and physiological processes. Yet with only a few substrates described their biological roles are enigmatic. Moreover, the lack of knowledge of astacin cleavage site specificities hampers assay and drug development.

Characterization of the prime and non-prime active site specificities of proteases by proteome-derived peptide libraries and tandem mass spectrometry.

To link cleaved substrates in complex systems with a specific protease, the protease active site specificity is required. Proteomic identification of cleavage sites (PICS) simultaneously determines both the prime- and non-prime-side specificities of individual proteases through identification of hundreds of individual cleavage sequences from biologically relevant, proteome-derived peptide libraries. PICS also identifies subsite cooperativity.

Proteome-wide analysis of protein carboxy termini: C terminomics.

As proteome-wide C-terminal sequence analysis has been largely intractable, we developed a polymer-based enrichment approach to profile protein C-terminal peptides by mass spectrometry and identified hundreds of C-terminal peptides in the Escherichia coli proteome. We isotopically labeled GluC protease-digested and undigested samples and identified GluC substrates and their cleavage sites by quantification of neo-C-terminal peptides. Our method thus enables global annotation of protein C-terminal posttranslational modifications, including proteolytic truncations.

Characterization of the CopR regulon of Lactococcus lactis IL1403.

To identify components of the copper homeostatic mechanism of Lactococcus lactis, we employed two-dimensional gel electrophoresis to detect changes in the proteome in response to copper. Three proteins upregulated by copper were identified: glyoxylase I (YaiA), a nitroreductase (YtjD), and lactate oxidase (LctO). The promoter regions of these genes feature cop boxes of consensus TACAnnTGTA, which are the binding site of CopY-type copper-responsive repressors.

Copper induction of lactate oxidase of Lactococcus lactis: a novel metal stress response.

Lactococcus lactis IL1403, a lactic acid bacterium widely used for food fermentation, is often exposed to stress conditions. One such condition is exposure to copper, such as in cheese making in copper vats. Copper is an essential micronutrient in prokaryotes and eukaryotes but can be toxic if in excess.

Improved protocol for chromatofocusing on the ProteomeLab PF2D.

Beckman-Coulter has recently introduced the ProteomeLab PF2D for 2-D liquid separation of protein samples. The system features separation in the first dimension by chromatofocusing, followed by RP chromatography in the second dimension, allowing the analysis of complex proteomics samples. When used by the standard protocol, reproducibility and column life times are limited, making the use of the instrument very costly.

Characterization of the DNA-binding site in the ferric uptake regulator protein from Escherichia coli by UV crosslinking and mass spectrometry.

Ferric uptake regulator protein (Fur) is activated by its cofactor iron to a state that binds to a specific DNA sequence called 'Fur box'. Using mass spectrometry-based methods, we showed that Tyr 55 of Escherichia coli Fur, as well as the two thymines in positions 18 and 19 of the consensus Fur Box, are involved with binding. A conformational model of the Fur-DNA complex is proposed, in which DNA is in contact with each H4 [A52-A64] Fur helix.