Amino acid sequences analysis/identification

The Edman reaction enabled amino acid sequence analysis to be automated. Mass spectrometry provides a sensitive and versatile tool for determining primary strucmre and for the identification of post-translational modifications. 

Combination of two immobilized enzyme columns with HPLC/thermospray MS can be useful for amino acid sequencing and identification. The use of an endopeptidase bioreactor followed by HPLC separation then an exopeptidase column and MS detection can enable sequencing of 3-5 amino acids of each endopeptidase hydrolysis product. The trypsin, hydrolysis/HPLC/ carboxypeptidase A, B, and Y (1 1 1) hydrolysis/ thermospray MS analysis assist in the sequencing of Y-endorphin (Figure 2C,C ). 

A critical input in unraveling the catalytic mechanism of epoxide hydrolases has come from the identification of essential residues by a variety of techniques such as analysis of amino acid sequence relationships with other hydrolases, functional studies of site-directed mutated enzymes, and X-ray protein crystallography (e.g., [48][53][68 - 74]). As schematized in Fig. 10.6, the reaction mechanism of microsomal EH and cytosolic EH involves a catalytic triad consisting of a nucleophile, a general base, and a charge relay acid, in close analogy to many other hydrolases (see Chapt. 3). 

Different authors used RP-HPLC and UV detection to monitor peptide formation during cheese ripening [174-178], providing valuable information about proteolysis. When large hydrophobic peptide need to be separated an lEC represents the best choice [179]. Nevertheless, the identification of these peptides is essential for the complete understanding of the proteolytic process. The peptides eluted from the LC column can be subjected to ESl-MS for molecular weight determination and MS/MS for amino acid sequence determination, which allow rapid peptide identification [172]. HPLC-ESl-MS and MS/MS techniques have been successfully used for peptide mass fingerprint purposes for sequence analysis of purified albumin from Theobroma cacao seeds [180,181]. 

MALDI-TOF provides limited capabilities for mixture analysis, LC/MS methods are used to provide more detailed interrogation of protein expression and peptide sequence. The use of LC/MS approaches for protein identification in conjunction with 2-DGE offers distinct advantages such as the ability to handle low picomole (miniaturized) level samples, enhanced separation, detection, the amenability to N-terminally blocked proteins, and fast analysis. The LC/MS methods for protein characterization focus on four distinct goals (1) confirmation of putative sequence, (2) identification of amino acid modifications, (3) identification of known proteins, and (4) sequence determination of unknown proteins. 

AA Analysis EMBL P1R Protein identification based on amino acid sequence... 

---