Quantitative Peptidomics Using Reductive Methylation of Amines.

A number of different approaches have been used for quantitative peptidomics. In this protocol, we describe the method in which peptides are reacted with formaldehyde and sodium cyanoborohydride, which converts primary and secondary amines into tertiary amines. By using different combinations of regular reagents, deuterated reagents (2H), and reagents containing deuterium and 13C, it is possible to produce five isotopically distinct forms of the methylated peptides, which can be quantified by mass spectrometry. Peptides with free N-termini that are primary amines incorporate two methyl groups using this procedure, which differ by 2 Da for each of the five isotopic combinations. Peptides that contain unmodified lysine residues incorporate additional pairs of methyl groups, leading to larger mass differences between isotopic forms. The reagents are commercially available, relatively inexpensive, and chemically stable.

Peptidomics of Zebrafish Brain in a 6-OHDA-Induced Neurodegeneration Model.

Bioactive peptides such as neuropeptides and peptide hormones are largely understood in their involvement in a variety of physiologic systems. In addition to the neuropeptides produced and processed by the classic secretory pathway, intracellular peptides (InPeps) have shown biological activity in studies involving different organisms. A model that has become attractive in many research fields is the zebrafish (Danio rerio), which has allowed correlating behavioral responses or physiological processes with underlying molecular pathways or signaling cascades, improving the understanding of homeostasis mechanisms of the central nervous system, as well as pathological processes such as neurodegenerative diseases. Here, we provide a detailed description of the protocol of treatment with 6-OHDA, which mimics some features of Parkinson's Disease, as well as the validation of the treatment by evaluation of the locomotor activity and the protocol of peptide extraction followed by isotopic labeling to peptide relative quantitation by mass spectrometry.

Methods for Intracellular Peptidomic Analysis.

Peptides have broad biological significance among different species. Intracellular peptides are considered a particular class of bioactive peptides, whose generation is initiated by proteasomal degradation of cytosolic, nuclear, or mitochondrial proteins. To extract and purify intracellular peptides, which may apply for biological peptides in general, it is important to consider the initial source: tissue, cell, or fluid. First, it is important to proceed fast with inactivation of proteases and/or peptidases commonly present in the biological source of peptides, which might rapidly degrade peptides during the initial process of extraction. The incubation of biological tissues, cells, and fluids at 80 °C for up to 20 min have been sufficient to fully inactivate proteases or peptidases activities. It is particularly important not to acidify the samples at high temperature, because it can lead to nonspecific hydrolysis reactions; particularly, the Asp-Pro peptide bond can be cleaved at acidic environments and elevated temperatures. Unfortunately, not every sample can have proteinases and peptidases denatured by heating the biological source of intracellular peptides. Plasma, for example, when heated at temperatures higher than 55 °C can clot and trap peptides within the fibrin net. Therefore, alternative conditions for inactivating proteinases and peptidases must apply for plasma samples. In this chapter, the most successful methods used in our laboratory to extract intracellular peptides are described.

Interferon-gamma activity is potentiated by an intracellular peptide derived from the human 19S ATPase regulatory subunit 4 of the proteasome.

Hundreds of intracellular peptides that are neither antigens nor neuropeptides are present in mammalian cells and tissues. These peptides correspond to fragments of cytosolic, nuclear or mitochondrial proteins. Proteasome inhibition affects the levels of the intracellular peptides in human cell lines. Here, the effect of immuneproteasome expression on the intracellular peptide profile was evaluated, and its functional significance was investigated. The expression of the immuneproteasome in HeLa cells was induced by interferon gamma treatment, and the relative concentrations of the intracellular peptides were compared to those of the control cells using isotope labeling and electron spray mass spectrometry. One of the peptides identified, VGSELIQKY (EL28), corresponds to amino acids 251-259 of the human 19S ATPase regulatory subunit 4. This peptide was increased in the extracts of HeLa cells that had been treated with interferon gamma compared to those of control cells. In vitro, EL28 increased the chymotrypsin, trypsin and caspase-like proteasome activities. In vivo, when covalently linked to a cell-penetrating peptide, EL28 potentiated the ability of interferon gamma to stimulate the expression of the immuneproteasome ß5i subunit and to increase the proliferation of CD8+ T-cells. The EL28/cell-penetrating peptide construct also improved and positively modulated the secondary IgG anti-bovine serum albumin immune responsiveness elicited in high antibody-responder mice. Together, these results suggest that EL28 is a functional intracellular peptide that can potentiate interferon gamma activity. BIOLOGICAL SIGNIFICANCE: The functional identification of EL28 advances our understanding regarding the bioactive peptides generated by limited proteolysis within cells.