Amino acid identification and sequence analysis of peptides by reaction mass spectrometry

Fast atom bombardment mass spectrometry (FAB-MS) is applied to distinguish N- terminal series ions from C-terminal series ions of a peptide by on-probe acetylation, it provides valuable information about the sequence of an unknown peptide. The FAB mass spectra contain a number of characteristic ions at low-mass region in addition to the sequence ions at high-mass region. It was found that the ions below m/z 200 are characteristic of the amino acid composition of the peptide, from which the amino acid composition of the peptide could be estimated. Additionally, mixture analysis is also discussed.

Identification in Lupin Seed of a Serine- Endopeptidase Activity Cleaving between Twin Arginine Pairs and Causing Limited Proteolysis of Seed Storage Proteins

The occurrence of twin-arginine motifs （-R-R-） in the amino acid sequences of animal pro-proteins frequently defines the cleavage site（s） for their structural/functional maturation. No information is available on the presence and possible biological meaning of these motifs in the seed storage proteins. In this work, a novel endopeptidase activity with cleavage specificity to twin-arginine pairs has been detected in mature dry Lupinus albus seeds. The endopeptidase was tested with a number of endogenous and exogenous protein substrates, which were selected according to the pres- ence of one or more twin-arginine residue motifs in their amino acid sequences. The observed hydrolysis patterns were limited and highly specific. Partial proteolysis led to stable polypeptide fragments that were characterized by 1- and 2-D electrophoresis. Selected polypeptides were submitted to N-terminal amino acid sequencing and mass spectrometry anal- yses, These approaches, supported by bioinformatic analysis of the available sequences, allowed the conclusion that the polypeptide cleavage events had occurred at the peptide bonds comprised between twin-arginine residue pairs with all tested protein substrates. The endopeptidase activity was inhibited by 4-（2-AminoEthyl）Benzene-Sulphonyl Fluoride hy- drochloride （AEBSF）, leupeptin, and serine proteinase protein inhibitors, while it was not affected by pepstatin, trans- EpoxysuccinyI-L-leucylamido（4-guanidino）butane （E64）, and ethylenediaminetetraacetic acid （EDTA）, thus qualifying the Arg-Arg cleaving enzyme as a serine endopeptidase. The structural features of storage proteins from lupin and other legume seeds strongly support the hypothesis that the occurrence of an endopeptidase activity cleaving -R-R- bonds may be functional to facilitate their degradation at germination and possibly generate polypeptide fragments with specific biological activity.

Selection and determination of specific and protective antigens of Mycobacterium tuberculosis

Objective To select and identify the specific and protective antigens of Mycobacterium tuberculosis (M. tuberculosis) for searching a new approach to diagnose and treat tuberculosis Methods Extract and culture filtrates of M tuberculosis were obtained by ultrasonic treatment and millipore membrane filtration, respectively The protein samples were tested with monoclonal antibody (MAb) and patient sera The proteins showing positive reaction were sequenced on Beckman /LF 3200/peptide amino acid sequencer Results Proteins of M tuberculosis with molecular weight of 31?ku and 30?ku showed positive results when reacted with anti M tuberculosis MAb and sera of tuberculosis patients, but not with normal mouse serum and healthy human sera N terminal sequences of the 31?ku and 30?ku antigen were Ala Glu Val Asp Trp Leu Val Phe Ala Val and Phe Ser Arg Pro Gly Leu Pro Val Glu Try respectively Conclusion 31?ku and 30?ku proteins of M tuberculosis are immune protective proteins They play important or dominant roles in determination of immunorecognization