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Proteins are the carriers of biological structure and function, which are involved in almost all the physiological processes in organisms. The qualitative and quantitative analysis of proteins can not only help us understand the mechanisms of life activities, but also screen for the proteins with abnormal expression compared to healthy controls as biomarkers of diseases as well as environmental exposure to harmful substances, thus facilitating to unravel the mechanisms of disease generation and development. Tandem mass spectrometry (MS/MS) is currently the main tool for high-throughput proteomics analysis. It can characterize the proteins by determining the molecular mass of digestion products, locating modification sites, and obtaining sequence information. However, acidic amino acid residues often have a significant impact on the cleavage process of peptides, limiting the performance of MS/MS. For example, for the phosphopeptides containing abundance of negatively charged carboxyl and phosphoryl groups, their ionization is not efficient in the positive mode, and neutral loss is easy to occur during the cleavage process, resulting in a low coverage of product ions in the MS/MS spectra. In this paper, the carboxyl groups from the R group of aspartic acid and glutamic acid as well as the C-terminus of peptide were neutralized using dimethylamine derivatization under mild condition. Accordingly, the fragmentation features of peptides with altered charge properties were studied in collision-induced dissociation (CID) mode. Further, the peptides were labeled using light and heavy isotopic dimethylamine, respectively, thus enabling to verify the features based on the same ion with different labels. The results showed that the precursor ion signal from the derivatized peptide was significantly enhanced compared to that of the native peptide. And, the derived peptides could yield a variety of a, b, y, and z ions, and some reported ions in MS/MS analysis, such as the ion at m/z 129.10/135.14 produced by the peptides with glutamic acid or lysine residue at C-terminus, which facilitating to determine the C-terminal amino acid of protein. While, the peptides proned to lose the derivative groups located at the C-terminus, yielding a series of ions at m/z ［yn-45.06］ or ［yn-73.05] In addition, the neutralization of carboxyl groups could suppress the loss of phosphoryl groups in phosphopeptides to a certain extent, thus improving the resolution efficiency of the MS/MS spectra. Overall, the derivatization method based on carboxyamidation can provide abundant information for the sequence determination and quantitative analysis of peptides due to the altered fragmentation features in CID mode. [ABSTRACT FROM AUTHOR]

Obtaining compounds with specificity for certain isoforms of ion channels is a significant problem of curent physiology and pharmacology. In a series of papers, we have shown that the α-hairpinin fold can serve as a template for the rational design of peptide ligands of potassium channels. Here, we used molecular modeling to optimize the structure of the previously obtained Tk-hefu-10 peptide, a selective KV1.3 channel blocker, with a half-maximal inhibitory concentration (IC50) of ≈150 nM. Molecular dynamics simulation of the Tk-hefu-10–KV1.3 complex provided information on the interaction of individual amino acid residues of the peptide and channel, and the analysis of these interactions made it possible to propose amino acid substitutions in the structure of Tk-hefu-10 to increase its affinity. Novel Tk-hefu-12 peptide is a truncated analog of Tk-hefu-10 by one residue with five substitutions; it is characterized by an IC50 value of ≈70 nM against KV1.3. In addition, there are no methionine residues in the structure of Tk-hefu-12, which makes it possible to obtain Tk-hefu peptides using cyanogen bromide. [ABSTRACT FROM AUTHOR]