Polypeptides peptide sequencing using mass

In addition to molecvdar weight information. Fast Atom Bombardment Mass Spectrometry can also be used for sequencing polypeptides and oHgonucleotides. One such example is the study of the amino acid sequence in the ohgopeptide Antiamoebin I by Taylor et al. (30) by a combination of FAB and B/E-hnked scans. This peptide contains 16 amino acids. The peak at m/z 1670 is the quasi-molecular ion [M -t HF. A linked-scan experiment of the B/E type performed on this ion produced a peak at m/z 1422 which presented confirmatory evidence a loss of the terminal prohne.phenylalaninol.H group. A similar B/E scan from the latter ion produces ions at m/z 1337 and m/z 1244 for the loss of aminobutyric acid and hydroxyprohne.amino-isobutyric acid, respectively. This process can be repeated vmtil aU the amino acids are... 

It is assumed that the limited proteolysis phenomenon derives from the fact that a specific polypeptide chain segment of the compact, folded protein substrate is exposed and flexible so that it can fit the active site of the appropriate peptidase for an efficient and selective limited hydrolysis. There is no doubt that enhanced chain flexibility or segment mobility is the key feature of the site of peptide bond hydrolysis demonstrated by a clear-cut correlation between sites of proteolytic attack and sites of enhanced chain flexibility. The present availability of automatic, efficient and sensitive techniques of protein sequencing and, particularly, the recent dramatic advances of mass spectrometry 361 in the analysis of peptides and proteins, allows a more systematic use of the limited proteolysis approach as a simple first step in the elucidation of structure-dynamics-function relationships for novel proteins which are only available in minute amounts. 

A fragment of the lettuce VDE cDNA was amplified from lettuce cDNA by PCR and used to isolate a near full-length cDNA for lettuce VDE (Bugos and Yamamoto, 1996). The cDNA encoded a polypeptide of 473 amino acids with a calculated molecular mass of 54.4 kDa. This was confirmed from in vitro transcription/translation of the VDE cDNA which produced a 55 kDa polypeptide when analyzed by SDS-PAGE. The deduced preprotein contained a 125 amino acid bipartite transit peptide for transport into the chloroplast and thylakoid lumen. Based on the N-terminal sequence of the purified VDE from lettuce, which indicates the transit peptide cleavage site, the mature VDE protein is encoded by 348 amino acids with a calculated molecular mass of... 

Mass spectrometry may be used to determine primary sequences. Partial sequential chemical or enzymic degradation of peptides or proteins can yield a mixture of peptides with progressively shorter sequences. For example, consider the following (hypothetical) polypeptide sequence that has been partially digested with a proteolytic enzyme (carboxypeptidase) to yield the following ladder" of fragments ... 

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