Amino acid residue identification/sequence

ScoFFONE, E., and A. Fontana Identification of Specific Amino Acid Residues. Protein Sequence Determination. Methods and Techniques (S. B. Needleman, Ed.), p. 185-210. New York Springer. 1970. 

An alternative approach to peptide sequencing uses a dry method in which the whole sequence is obtained from a mass spectrum, thereby obviating the need for multiple reactions. Mass spec-trometrically, a chain of amino acids breaks down predominantly through cleavage of the amide bonds, similar to the result of chemical hydrolysis. From the mass spectrum, identification of the molecular ion, which gives the total molecular mass, followed by examination of the spectrum for characteristic fragment ions representing successive amino acid residues allows the sequence to be read off in the most favorable cases. 

Determining the order of amino acids involves the sequential removal and identification of successive amino acid residues from one or the other free terminal of the polypeptide chain. However, in practice it is extremely difficult to get the required specific cleavage reaction of the desired products to proceed with 100% yield. This obstacle becomes significant when sequencing long polypeptides, because the fraction of the total material of minimum polypeptide chain length becomes constantly smaller as the successive removal of terminal residues continues. Conversely, the amino acid released from the polypeptide chain becomes increasingly contaminated with amino acids released from previously unreacted chains. 

Stabilization is maintained by several hydrogen bonds. Interesting details include a single m-amide linkage between MeLeu-9 and MeLeu-10. Furthermore, the L-configuration of MeBmt-1 was confirmed and the still unknown partial sequence of residues 7 and 8 was determined as L-alanine-D-alanine. The absolute configuration of iodocyclosporin A and hence that of cyclosporin A were deduced from the reliable identification of the L-amino-acid residues. 

ApoSAA, normally a trace component of plasma, is an acute-phase plasma protein, that is, one that is elevated in a variety of disease states (R18). Its identification is interesting. A small protein of 76 residues, now called protein AA, was identified during the study of the proteins present in extracellular amyloid deposits in the type of amyloidosis particularly associated with inflammation (B24, H36, Lll, S38), Antibodies to protein AA reacted with two AA-related proteins in plasma, one of approximate Mr 180,000 (SAA) and the other found in HDL of Mr 14,000-15,000 or 12,000 (apoSAA) (A19, B25, B26, L12, L15). The N-terminal 76-amino-acid portion of apoSAA is identical to that of amyloid protein AA (E8). Human apoSAA has now been sequenced and has been shown to consist of 104 amino acid residues (B27). Further studies in man have demonstrated microheterogeneity in apoSAA (B18, B19, M30) and Benditt et al. describe specific amino acid substitutions (B27, P6). Shore et al. have described a second similar threonine-poor apolipoprotein, apparently a dimer of Mr 40,000... 

The structure and properties of peptides and proteins depend critically upon the sequence of amino acids in the peptide chain. The first complete amino acid sequence of a protein, that of insulin (51 amino acid residues), was determined by F. Sanger in 1953. The process is now performed using automated protein sequencers, and involves step-by-step identification of amino acids at the N terminus of the protein using a chemical process known as Edman degradation. 

Fig. 6.10 Identification of Cdk4-specific amino acid residues [20]. (A) Amino acid residues around the ATP binding pocket. (a)Sequence identity confined to around the ATP binding pocket of Cdk4 (b) sequence...

Automated carboxy-terminal (C-terminal) protein sequence analysis enables the direct and unambiguous confirmation of the C-terminal sequence of native and expressed proteins, the detection and characterization of protein processing at the C-terminus, the identification of post-translational proteolytic cleavages, and partial sequence information on amino-terminally blocked protein samples. In order for C-terminal sequence analysis to be of immediate benefit, each of the 20 common amino acid residues must be detectable. Additionally, the scope of typically analyzable protein samples must span a usefully broad molecular weight range and degree of structural complexity. 

Sequence information can also be obtained using Edman degradation to remove amino-terminal residues from a peptide, to produce a collection of peptides. MALDI-TOF can then be used to obtain the peptides masses, and the sequence determined by mass difference between consecutive peptides. This methodology is called protein ladder sequencing, and allows information to be obtained for up to 30 residues. This method is useful for the identification of posttranslational modifications, such as phosphorylated amino acid residues.24... 

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