Chemical C-terminal protein sequence analysis: improved sensitivity, length of degradation, proline passage, and combination with edman degradation.
Use of a C-terminal sequencer with modified solvents, reagent concentrations, chromatographic parameters, temperatures, and reaction cartridge geometry yields four sets of improvements in chemical degradations. They are increased sensitivity, longer runs, passage of Pro residues, and practical combination with N-terminal degradation. Over 200 proteins and protein fragments with sizes between 20 and 600 residues were analyzed. C-terminal sequences could be interpreted for more than 10 residues at high picomole sample levels, while the 10-pmol level gave 4-5 residues. The average initial yield was 15% but up to 30% could be achieved. The improved performance allowed combination of C- and N-terminal degradations from the same sample application. After initial Edman degradation, the sample is moved to the C-terminal instrument for continued sequencing. Proteins available in limited amount are thereby efficiently analyzed. Lys, modified from the N-terminal degradation, may be detected as the alkylated thiohydantoin-phenylthiocarbamyl-Lys derivative in the C-terminal degradation. Notably, C-terminal sequence analysis could be proceeded through Pro residues which unexpectedly were no absolute hindrance. The improved technique provides characterization of truncation patterns and microheterogeneities in proteins down to the 10-pmol level and is a useful approach for analysis of N-terminally blocked polypeptides.
Bergman
T
T
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, SE-171 77, Sweden.
Cederlund
E
E
Jörnvall
H
H
D013058:Mass Spectrometry; D009943:Organophosphorus Compounds / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D011392:Proline / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D012680:Sensitivity and Specificity; D020539:Sequence Analysis, Protein / Q000379:methods*; D013997:Time Factors
C043517:2-(4-isothiocyanatophenoxy)-1,3,2-dioxaphosphinene 2-oxide; D009943:Organophosphorus Compounds; D010446:Peptide Fragments; D010455:Peptides; D011506:Proteins; D011392:Proline