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Midinfrared-matrix-assisted laser desorption/ionization mass spectrometry (mid-IR-MALDI MS) with a laser emission in the 6 [micro]m wavelength range, which utilizes energy absorption at the C=O double-bond stretch region, was applied to biomolecular analysis. The softness of IRMALDI MS was evident in the negative ion mode yielding clean mass spectra of [[M - H].sup.-] ions for acidic biomolecules with sulfate, phosphate, or carboxylate groups, resulting in better sensitivity than ultraviolet (UV)-MALDI MS. There was no substantial loss of sialic acid due to the prompt fragmentation occurring in IRMALDI of sialylated glycoconjugates such as gangliosides. Furthermore, the advantage of the low photon energy of IR is that, for the first time, intact protonated molecules of S-nitrosylated peptides can be detected by MALDI MS. In the analysis of redox-sensitive molecules including methylene blue and riboflavin, reductive hydrogenation was minimal, suggesting few hydrogen radicals to have formed in the plume, in contrast to UV-MALDI. In conjunction with a potent new matrix, oxamide, requiring smaller laser fluence, distinct features of the 6 [micro]m IR wavelength range are anticipated to remove one of the limitations of MALDI MS for biomolecular analysis.

Tumor necrosis factor-[alpha] (TNF-[alpha]) signaling through the I[kappa]B kinase (IKK) complex attenuates insulin action via the phosphorylation of insulin receptor substrate 1 (IRS-1) at [Ser.sup.307]. However, the precise molecular mechanism by which the IKK complex phosphorylates IRS-1 is unknown. In this study, we report nuclear factor [kappa]B essential modulator (NEMO)/IKK-[gamma] subunit accumulation in membrane ruffles followed by an interaction with IRS-1. This intracellular trafficking of NEMO requires insulin, an intact actin cytoskeletal network, and the motor protein Myo1c. Increased Myo1c expression enhanced the NEMO-IRS-1 interaction, which is essential for TNF-[alpha]-induced phosphorylation of [Ser.sup.307]-IRS-1. In contrast, dominant inhibitory Myo1c cargo domain expression diminished this interaction and inhibited IRS-1 phosphorylation. NEMO expression also enhanced TNF-[alpha]-induced [Ser.sup.307]-1RS-1 phosphorylation and inhibited glucose uptake. In contrast, a deletion mutant of NEMO lacking the IKK-[beta]-binding domain or silencing NEMO blocked the TNF-[alpha] signal. Thus, motor protein Myo1c and its receptor protein NEMO act cooperatively to form the IKK-IRS-1 complex and function in TNF-[alpha]-induced insulin resistance.

In glycoproteomics, key structural issues, protein identification, locations of glycosylation sites, and evaluation of the glycosylation site microheterogeneity should be easily evaluated in a large number of glycoproteins, while mass spectrometry (MS) provides substantial information about individual purified glycoproteins. Considering that structural issues are elucidated by studying glycopeptides and that the tandem MS of a tryptic peptide composed of several amino acid residues is enough for protein identification, construction of an MS-based method handling tryptic glycopeptides would be of considerable benefit in research. To this end, a simple and efficient method, utilizing hydrophilic binding of carbohydrate matrixes such as cellulose and Sepharose to oligosaccharides, was successfully applied to the isolation of tryptic glycopeptides. Both peptide and oligosaccharide structures were elucidated by multiple-stage tandem MS ([MS.sup.n]) of the ions generated by matrix-assisted laser desorption/ionization (MALDI), as follows. The MALDI ion trap mass spectrum of a tryptic glycopeptide mixture from N-linked glycoproteins was composed of the [M + [H].sup.+] ions of component glycopeptides. Collision-induced dissociation (CID) of the glycopeptide [M + [H].sup.+] ion generated saccharide-spaced peaks, with an interval of, for example, 146, 162, and 203 Da, and their fragment ions corresponding to the peptide and peptide + N-acetylglucosamine (G1cNAc) species in the [MS.sup.2] spectrum. The saccharide-spaced ladder served to outline oligosaccharide structures, which were then selected as precursors for subsequent [MS.sup.n] analyses. The peptide or peptide + G1cNAc ions in the [MS.sup.2] spectrum or the corresponding ions abundant in the [MS.sup.1] spectrum were subjected to CID for determination of peptide sequences, to identify proteins and their glycosylation sites. The strategy, isolation of glycopeptides followed by [MS.sup.n] analysis, efficiently characterized the structures of [[beta].sub.2]-glycoprotein I with four N-glycosylation sites and was applied to an analysis of total serum glycoproteins.

The relationship between peptide structure and electron transfer dissociation (ETD) is important for structural analysis by mass spectrometry. In the present study, the formation, structure and reactivity of the reaction intermediate in the ETD process were examined using a quadrupole ion trap mass spectrometer equipped with an electrospray ionization source. ETD product ions of zwitterionic tryptophan (Trp) and Trp-containing dipeptides (Trp-Gly and Gly-Trp) were detected without reionization using non-covalent analyte complexes with Ca2+ and 18-crown-6 (18C6). In the collision-induced dissociation, NH3 loss was the main dissociation pathway, and loss related to the dissociation of the carboxyl group was not observed. This indicated that Trp and its dipeptides on Ca2+(18C6) adopted a zwitterionic structure with an NH3+ group and bonded to Ca2+(18C6) through the COO− group. Hydrogen atom loss observed in the ETD spectra indicated that intermolecular electron transfer from a molecular anion to the NH3+ group formed a hypervalent ammonium radical, R-NH3, as a reaction intermediate, which was unstable and dissociated rapidly through N-H bond cleavage. In addition, N-C α bond cleavage forming the z1 ion was observed in the ETD spectra of Trp-GlyCa2+(18C6) and Gly-TrpCa2+(18C6). This dissociation was induced by transfer of a hydrogen atom in the cluster formed via an N-H bond cleavage of the hypervalent ammonium radical and was in competition with the hydrogen atom loss. The results showed that a hypervalent radical intermediate, forming a delocalized hydrogen atom, contributes to the backbone cleavages of peptides in ETD. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

To develop a new experimental teaching material for improving the sanitary consciousness, the number of bacteria colonies cultured on shirts and blouses were measured by using the food stamp method. A comparison has been made between the number of colonies in collars, sleeves, abdomens, and back of the shirts and that of the blouses respectively. The significant differences were observed between the number of colonies on the collars and the sleeves, and between that on the collar and the back of male and female students. The number of colonies on the collar of the female students was higher than that of the male students, and that of the back of the male students was higher than that of the female students. For all of both male and female students, the number of colonies on the sleeve was positively correlated with that on the abdomen and the back. Many colonies have been found on the collar, the abdomen, and the back of the students who didn't put on underwear in the winter. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor-α (TNF-α) signaling through the IκB kinase (IKK) complex attenuates insulin action via the phosphorylation of insulin receptor substrate 1 (IRS-1) at Ser[sup 307]. However, the precise molecular mechanism by which the IKK complex phosphorylates IRS-1 is unknown. In this study, we report nuclear factor κB essential modulator (NEMO)/IKK-γ subunit accumulation in membrane ruffles followed by an interaction with IRS-1. This intracellular trafficking of NEMO requires insulin, an intact actin cytoskeletal network, and the motor protein Myo1c. Increased Myo1c expression enhanced the NEMO-IRS-1 interaction, which is essential for TNF-α-induced phosphorylation of Ser[sup 307]-IRS-1. In contrast, dominant inhibitory Myo1c cargo domain expression diminished this interaction and inhibited IRS-1 phosphorylation. NEMO expression also enhanced TNF-α-induced Ser[sup 307]-IRS-1 phosphorylation and inhibited glucose uptake. In contrast, a deletion mutant of NEMO lacking the IKK-β-binding domain or silencing NEMO blocked the TNF-α signal. Thus, motor protein Myo1c and its receptor protein NEMO act cooperatively to form the IKK-IRS-1 complex and function in TNF-α-induced insulin resistance. [ABSTRACT FROM AUTHOR]