Internal protein sequence analysis: enzymatic digestion for less than 10 micrograms of protein bound to polyvinylidene difluoride or nitrocellulose membranes.
A procedure for the generation and isolation of internal peptide fragments for less than 10 micrograms of protein bound to either polyvinylidene difluoride (PVDF) or nitrocellulose membranes after electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) is presented. This technique has produced internal sequence data for 120 peptides, with an average initial yield of 20 pmol. Membrane-bound proteins were enzymatically digested with either trypsin or endoproteinase Lys-C in the presence of 1% hydrogenated Triton X-100/10% acetonitrile/100 mM Tris-HCl, pH 8.0, for 24 h at 37 degrees C. The eluted peptides were then directly isolated by microbore HPLC for subsequent sequence analysis. One percent hydrogenated Triton X-100 did not inhibit enzymatic activity, distort HPLC resolution of peptides, or contain uv-absorbing contaminants that could interfere with peptide identification. Reproducible peptide maps and consistent recoveries are presented for standard proteins (3.5-8.0 micrograms) bound to either membrane, with higher recoveries for PVDF-bound proteins. Ninety percent of the proteins analyzed by this technique have produced results; representative peptide maps and sequence data are presented. This technique has a wide range of applications, particularly for proteins with blocked amino termini or those that can only be purified by SDS-PAGE or 2D isoelectric focusing SDS-PAGE.
Fernandez
J
J
Rockefeller University, Protein Sequencing Facility, New York, New York 10021.
DeMott
M
M
Atherton
D
D
Mische
S M
SM
D003101:Collodion; D003902:Detergents / Q000494:pharmacology; D004798:Enzymes / Q000378:metabolism; D008567:Membranes, Artificial; D008722:Methods; D010446:Peptide Fragments / Q000378:metabolism; D010449:Peptide Mapping; D011092:Polyethylene Glycols / Q000494:pharmacology; D011145:Polyvinyls; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D015203:Reproducibility of Results; D005465:Fluorocarbon Polymers
D003101:Collodion; D003902:Detergents; D004798:Enzymes; D008567:Membranes, Artificial; D010446:Peptide Fragments; D011092:Polyethylene Glycols; D011145:Polyvinyls; D011506:Proteins; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C043504:RTX 100