Peptide mapping of complex proteins at the low-picomole level with capillary electrophoretic separations.
A variety of different peptide-mapping schemes are presented, with emphasis on the development of procedures which can be done with limited quantities (i.e. 5 pmol) of protein. Results are obtained from model proteins which contain disulfide bonds, which must be broken prior to fragmentation of the protein. A reaction involving the simultaneous use of tributylphosphine and 2-methylaziridine to reduce and alkylate the disulfide bonds is employed, due to favorable attributes of these reagents for the scaled-down procedure. The traditional performic acid oxidation reaction to cleave cystine groups is also successfully used with low-picomole quantities of protein. Three different protein digestion reagents are used: trypsin, chymotrypsin, and cyanogen bromide. Each reagent produces a unique mixture of peptides. Capillary electrophoresis is used to separate the peptides, offering high separation efficiencies, short analysis times, and compatibility with small sample sizes. In addition to the conventional use of UV detection for underivatized peptides, laser-induced fluorescence detection is employed in conjunction with an arginine-selective derivatization reaction. This latter procedure for derivatization and detection offers an alternative peptide-mapping mode, in which only the arginine-containing peptides are detected, and is useful in simplifying the peptide maps of large proteins.
Cobb
K A
KA
Department of Chemistry, Indiana University, Bloomington 47405.
Novotny
M V
MV
D000478:Alkylation; D001388:Aziridines / Q000737:chemistry; D004586:Electrophoresis / Q000379:methods*; D010084:Oxidation-Reduction; D010449:Peptide Mapping / Q000379:methods*; D010720:Phosphines / Q000737:chemistry; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet
D001388:Aziridines; D010720:Phosphines; D011506:Proteins; C043255:tri-n-butylphosphine; C031132:propyleneimine