Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is an intracellular enzyme that has been proposed to metabolize peptides within cells, thereby affecting antigen presentation and G protein-coupled receptor signal transduction. However, only a small number of intracellular substrates of EP24.15 have been reported previously. Here we have identified over 100 peptides in human embryonic kidney 293 (HEK293) cells that are derived from intracellular proteins; many but not all of these peptides are substrates or products of EP24.15. First, cellular peptides were extracted from HEK293 cells and incubated in vitro with purified EP24.15. Then the peptides were labeled with isotopic tags and analyzed by mass spectrometry to obtain quantitative data on the extent of cleavage. A related series of experiments tested the effect of overexpression of EP24.15 on the cellular levels of peptides in HEK293 cells. Finally, synthetic peptides that corresponded to 10 of the cellular peptides were incubated with purified EP24.15 in vitro, and the cleavage was monitored by high pressure liquid chromatography and mass spectrometry. Many of the EP24.15 substrates identified by these approaches are 9 -11 amino acids in length, supporting the proposal that EP24.15 can function in the degradation of peptides that could be used for antigen presentation. However, EP24.15 also converts some peptides into products that are 8 -10 amino acids, thus contributing to the formation of peptides for antigen presentation. In addition, the intracellular peptides described here are potential candidates to regulate protein interactions within cells.Intracellular protein turnover is a crucial step for cell functioning, and if this process is impaired, the elevated levels of aged proteins usually lead to the formation of intracellular insoluble aggregates that can cause severe pathologies (1). In mammalian cells, most proteins destined for degradation are initially tagged with a polyubiquitin chain in an energy-dependent process and then digested to small peptides by the 26 S proteasome, a large proteolytic complex involved in the regulation of cell division, gene expression, and other key processes (2, 3). In eukaryotes, 30 -90% of newly synthesized proteins may be degraded by proteasomes within minutes of synthesis (3, 4). In addition to proteasomes, other extralysosomal proteolytic systems have been reported (5, 6). The proteasome cleaves proteins into peptides that are typically 2-20 amino acids in length (7). In most cases, these peptides are thought to be rapidly hydrolyzed into amino acids by aminopeptidases (8 -10). However, some intracellular peptides escape complete degradation and are imported into the endoplasmic reticulum where they associate with major histocompatibility complex class I (MHC-I) 3 molecules and traffic to the cell surface for presentation to the immune system (10 -12). Additionally, based on the fact that free peptides added to the intracellular milieu can regulate cellular functions mediated by protein interactions such as gene reg...

Quantitative mass spectrometry is often performed using isotopically-labeled samples. While the 4-trimethylammoniumbutyryl (TMAB) labels have many advantages over other isotopic tags, only two forms have previously been synthesized (i.e. a heavy form containing 9 deuteriums and a light form without deuterium). In the present report, two additional forms containing 3 and 6 deuteriums have been synthesized and tested. These additional isotopic tags perform identically to the previously reported tags; peptides labeled with the new TMAB reagents co-elute from reverse phase HPLC columns with peptides labeled with the lighter and heavier TMAB reagents. Altogether, these 4 tags allow for multivariate analysis in a single liquid chromatography/mass spectrometry analysis, with each isotopically tagged peptide differing in mass by 3 Da per tag incorporated. The synthetic scheme is described in simple terms so that a biochemist without specific training in organic chemistry can perform the synthesis. The interpretation of tandem mass spectrometry data for the TMAB-labeled peptides is also described in more detail. The additional TMAB isotopic reagents described here, together with the additional description of the synthesis and analysis should allow these labels to be more widely used for proteomics and peptidomics analyses.

CPA4 (carboxypeptidase A4) is a member of the metallocarboxypeptidase family. CPA4 was originally found in a screen of mRNAs up-regulated by sodium butyrate-induced differentiation of cancer cells. Further studies suggested a relation between CPA4 and prostate cancer aggressiveness. In the present study, we determined that CPA4 is secreted from cells as a soluble proenzyme (pro-CPA4) that can be activated by endoproteases, such as trypsin. Three complementary approaches were used to study the substrate specificity of CPA4; kinetic analysis was performed using a new series of chromogenic substrates and some biologically relevant peptides, the cleavage of synthetic peptides was tested individually, and the cleavage of a mixture of >100 mouse brain peptides was examined using a quantitative peptidomics mass spectrometry-based approach. CPA4 was able to cleave hydrophobic C-terminal residues with a preference for Phe, Leu, Ile, Met, Tyr, and Val. However, not all peptides with C-terminal hydrophobic residues were cleaved, indicating the importance of additional residues within the peptide. Aliphatic, aromatic, and basic residues in the P1 position have a positive influence on the cleavage specificity. In contrast, acidic residues, Pro, and Gly have a negative influence in the P1 position. Some of the peptides identified as CPA4 substrates (such as neurotensin, granins, and opioid peptides) have been previously shown to function in cell proliferation and differentiation, potentially explaining the link between CPA4 and cancer aggressiveness. Taken together, these studies suggest that CPA4 functions in neuropeptide processing and regulation in the extracellular environment. Carboxypeptidases (CPs)3 hydrolyze a single amino acid from the C terminus of peptides and proteins. Metallo-CPs use a catalytic mechanism in which nucleophilic attack on a peptide bond is mediated by a Zn 2ϩ -activated water molecule (1). Most metallo-CPs are classified based on amino acid sequence, structure, and function into subfamilies within clan MC, family M14 (2). These subfamilies include M14A, also known as the CPA/ CPB subfamily; M14B, also known as the CPN/CPE subfamily; M14C, which includes ␥-D-glutamyl-(L)-meso-diaminopimelate peptidase I from Bacillus sphaericus; and a proposed fourth subfamily, including cytosolic CPs (3, 4). Metallo-CPs have also been divided into subgroups based on substrate specificity: A-like enzymes with preference for hydrophobic C-terminal amino acids; B-like enzymes, which cleave C-terminal basic residues; CPs with preference for glutamate in the C-terminal position; and CPs with a broad substrate specificity.CPA4 (carboxypeptidase A4) belongs to the M14A subfamily of carboxypeptidases. The pancreatic members of this subfamily (CPA1, CPA2, and CPB) act in the degradation of dietary proteins in the digestive tract. Other members of this subfamily display a wide spectrum of physiological roles. Mast cell carboxypeptidase (recently renamed CPA3) is found in the secretory granules of mast cells and plays a role ...

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