Proteins micro sequencing

The tunicate gonads that were harvested for protein extraction never saw my colleague s laboratory and were worked up here in acid-cleaned glassware. The implication is that two laboratories on separate continents would have acquired the same contaminant, once as mRNA and once as a protein Micro-sequencing is a legitimate and well-established method and quite powerful in the hands of an expert. Contamination can be a problem, but it is not an insurmountable one, and fear beyond reason must not be allowed to paralyze progress. 

Subsequently, proteolytic fragments of the rabbit renal 25-kDa amiloride-binding protein were micro-sequenced and found to have high sequence homology with rat and human NAD(P)H quinone oxidoreductase. Indeed, enzymatic assays revealed that renal brush border membrane vesicles contain significant NADPH quinone oxidoreductase activity. Presumably NAD(P)H quinone oxidoreductase coincidentally binds amiloride analogs with the same rank order as the Na /H exchanger [39]. 

Two methods are used to capture proteomes 2D electrophoresis (Section 7.3) and the SELDI protein chip system (Section 7.5). These methods are complemented by the good old micro-sequencing (Section 7.6) and different mass spectroscopic methods (Sections 7.4 and 7.6.5). After all, via partial sequences and the exact MW the proteins of a spectrum can be unambiguously identified in databases. 

J. Y. Chang, D. Brauer and B. Wittman-Liebold, Micro-Sequence Analysis of Peptids and Proteins using 4-NN-dimethyl-aminoazobenzene 4 -isothiocyanate/phenylisothiocyanate Double Coupling Method, FEBS Letters, 93 205-214 (1983). 

Biocatalysis refers to catalysis by enzymes. The enzyme may be introduced into the reaction in a purified isolated form or as a whole-cell micro-organism. Enzymes are highly complex proteins, typically made up of 100 to 400 amino acid units. The catalytic properties of an enzyme depend on the actual sequence of amino acids, which also determines its three-dimensional structure. In this respect the location of cysteine groups is particularly important since these form stable disulfide linkages, which hold the structure in place. This three-dimensional structure, whilst not directly involved in the catalysis, plays an important role by holding the active site or sites on the enzyme in the correct orientation to act as a catalyst. Some important aspects of enzyme catalysis, relevant to green chemistry, are summarized in Table 4.3. 

Gel Electrophoresis. This is becoming a more commonly used procedure for purifying proteins, nucleic acids, nucleoproteins, polysaccharides and carbohydrates. The gels can be electroblotted onto membranes and the modem procedures of identifying, sequencing (proteins and nucleic acids) and amplifying (nucleic acids) on sub-micro scales have made this technique of separation a very important one. (See D.Patel Gel Electrophoresis, J.Wiley-Lis, Inc., 1994). 

M., Bay, S., Hennessy, K., Plastic microfluidic devices for DNA sequencing and protein separations. Micro Total Analysis Systems, Proceedings 5th pZAS1 Symposium, Monterey, CA, Oct. 21-25, 2001, 19-21. 

The sequencing strategy follows the order 1) preparation of pure protein, 2) selective cleavage of peptide bond 3) isolation of cleaved peptide fragments, 4) analysis of primary structure on amino acid sequencer, 5) determination of entire primary structure. Although this order has not changed since our primary structure determination work on AspAT carried out in early 1970, many improvements have been made, e.g. preparation of micro scale amount of sample and introduction of high sensitivity analytical instruments. These improvements have shortened analysis time and lowered the sample amount of required. 

The demand for these methods in peptide and protein separations in micro-quantity will increase. A new device which combines electrophoretic apparatus and a mass spectrometer or a sequencer may be developed in the near future. 

The protein sequencer used for this study was an Applied Biosystems Model 477A, used without the Model 120A PTH analyzer, liie sequencer was fitted with an Applied Biosystems Micro cartridge, but all other hardware was as supplied by the maniifacturer. Reaction and conversion cycles were modified to accommodate this configuration. Some modified solvents and reagents were used and will be discussed below. 

C.L. Gatlin, J.K. Eng, S.T. Cross, J.C. Detter, J.R. Yates, III, Automated identif cation of amino acid sequence variations in proteins by HPLC-micro-ESI-MS-MS, Anal. Chem., 72 (2000) 757. 

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