Mass Analysis Of Proteins Research Articles

Cytotoxic activity of coagulase-negative staphylococci in bovine mastitis.

Secreted toxins play important roles in the pathogenesis of bacterial infections. In this study, we examined the presence of secreted cytotoxic factors of coagulase-negative staphylococci (CoNS) from bovine clinical and subclinical mastitis. A 34- to 36-kDa protein with cell-rounding cytotoxic activity was found in many CoNS strains, especially in Staphylococcus chromogenes strains. The protein caused cell detachment and cell rounding in several cell lines, including HEp-2, Int 407, CHO-K1, and Y-1 cells. Native protein recovered from nondenatured polyacrylamide gel electrophoresis showed both cytotoxic activity and casein hydrolysis activity. The purified protein had a pH optimal at 7.2 to 7.5 and a pI of 5.1 and was heat labile. The proteolytic activity could be inhibited by zinc and metal specific inhibitors such as 1, 10-phenanthroline and EDTA, indicating that it is a metalloprotease. Protein mass analysis and peptide sequencing indicated that the protein is a novel metalloprotease. Different bacterial strains expressed variable levels of 34- to 36-kDa protease, which may provide an indication of strain virulence.

Sample preparation techniques for peptides and proteins analyzed by MALDI-MS.

Protein mass analysis and peptide mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has emerged as a powerful tool for identification and characterization of proteins. The method involves (1) the mass spectrometric analysis of the intact protein, and, in particular, (2) the proteolytic cleavage of the latter, followed by mass determination of the generated peptides. The subsequent identification of the protein is performed by matching the set of measured peptide masses against the calculated masses of theoretical digest, based on protein or cDNA sequence databases (, , ). The detailed characterization of primary stmcture and covalent modifications of the protein is performed by differential peptide mapping, i.e., by comparative analysis of different proteolytic digests prior to and after application of, for example, phosphatases () or glycosidases ().

Formation of multiply charged ions from large molecules using massive‐cluster impact

Massive-cluster impact is demonstrated to be an effective ionization technique for the mass analysis of proteins as large as 17 kDa. The design of the cluster source permits coupling to both magnetic-sector and quadrupole mass spectrometers. Mass spectra are characterized by the almost total absence of chemical background and a predominance of multiply charged ions formed from 100% glycerol matrix. The number of charge states produced by the technique is observed to range from +3 to +9 for chicken egg lysozyme (14,310 Da). The lower m/z values provided by higher charge states increase the effective mass range of analyses performed with conventional ionization by fast-atom bombardment or liquid secondary ion mass spectrometry.