Peptide, amino acid sequence analysis

Since the Schiff base formation is reversible, it should be reduced by sodium borohydride for the fixation of the label. The rate of the reduction of the Schiff base becomes slow as the number of the phosphate groups of the label increases. However, except for adenylate kinase, the NP -PL bound to the proteins were easily fixed by borohydride reduction. After reductive fixation, labeled proteins are cleaved by appropriate methods. The labeled lysine is cleaved by neither trypsin nor lysyl endopeptidase. There are at least three ways to detect the labeled peptide during isolation 1) use of radioactive reagent, 2) use of radioactive sodium borohydride for reduction of the Schiff base, and 3) use of fluorescence derived from the pyridoxyl moiety of the reagent (excitation at 295 nm and emission at 390 nm at acidic pH). The labeled lysyl residue is not positively identified in the amino acid sequence analysis. However, the presence of the label in the peptide isolated can be confirmed by the presence of pyridoxyl lysine in the amino acid analysis. 

Purification of T from retinal rod outer segments and of G0 from brain, provided yields of these proteins that were sufficient for partial amino acid sequence analysis of their proteolytic fragments. This analysis revealed that a subunits of G proteins, while quite distinct from each other in general terms, are nevertheless similar. A partial sequence of 21 amino acids was determined to be common in bovine rod cell T and bovine brain G0 [168], On the basis of this sequence, and other amino acid sequence analysis, four laboratories cloned cDNAs coding for transducin. The deduced amino acid structure of three of the cDNAs is the same [169-171] the fourth differed [172]. Peptide-directed antibodies designed to distinguish between the two cloned forms localized one to rod cells (T-r) and the other to cone cells (T-c) [173],... 

The amino-terminal amino acid sequence analysis of each fraction revealed that every fraction was comprised of three kinds of peptides with different proportions. One of them showed the same amino-terminal sequence, G-N-I-Q-V-E-N-Q-A-I-P-D-P-, as observed in the previous experiment, and the other two peptides had shortened sequences, N-Q-A-I-P-D-P- and Q-A-I-P-D-P-, in which six or seven amino acid residues were deleted from the amino-terminal of the former sequence. However, all active fractions possessed similar amino acid compositions and, in addition, after V8 protease digestion, showed almost identical peptide mapping patterns on HPLC. 

Proteins and Amino Acids Total protein in food and feed samples is commonly determined by Kjeldahl (acid digestion/titration) or Dumas (pyrolysis) or elemental analysis.14 FIPLC can separate major proteins and furnish protein profiles and speciation information. HPLC can be used to further characterize specific proteins via peptide mapping and amino acid sequence analysis. HPLC modes used for protein include IEC, SEC, RPC, and affinity chromatography with typical UV detection at 215 nm or MS analysis. Details on protein separations are discussed in the life sciences section. 

It is important to determine the total amino acid composition of a peptide before the amino acid sequence analysis is undertaken. This helps to establish the final order of amino acids in the peptide. The understanding of amino acid composition also helps to resolve the situation of whether the peptide contains two adjacent glycine (mol Wt = 57 daltons) residues or just one aspargine (mol. Wt = 114 daltons). This dilemma is based on the molecular weight data alone. 

In order to characterize and confirm the analytic structure in the case of r-DNA technology-derived proteins or peptides, amino acid sequencing is the method of choice. Both the overall amino acid composition as well as N- and or C-terminal amino acid sequencing are useful and well-established tools in protein chemistry. Amino-terminal analysis reveals information about the primary structure, homogeneity and occurrence of cleavages in the polypeptide. The... 

Peptide mapping and amino acid sequence analysis of radiolabeled peptides indicate that... 

Microcolumn reverse-phase HPLC electrospray ionization tandem mass spectrometry (ESI-MS/MS) is a rapid and sensitive technique for the analysis of complex mixtures of peptides. This technique is used to determine the amino acid sequence of unknown peptides, to verify the structure of proteins, and to determine posttrans-lational modifications (see also the article by Beth L. Gillece-Castro). In particular, the strength of this approach is the analysis of peptides in complicated mixtures, such as amino acid sequence analysis of peptides isolated from class I and II major histocompatibility T-cell receptor complexes (Hunt et al., 1992a). 

Modern methods of amino-acid and peptide analysis, have enabled the complete amino-acid sequence of a number of proteins to be worked out. The grosser structure can be determined by X-ray diffraction procedures. Proteins have molecular weights ranging from about 6 000 000 to 5 000 (although the dividing line between a protein and a peptide is ill defined). Edible proteins can be produced from petroleum and nutrients under fermentation. 

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