Alkylation kinetics of proteins in preparation for two-dimensional maps: a matrix assisted laser desorption/ionization-mass spectrometry investigation.
All existing protocols for protein separation by two-dimensional (2-D) gel electrophoresis require the full reduction, denaturation, and alkylation as a precondition for an efficient and meaningful separation of such proteins. Existing literature provides a strong evidence to suggest that full reduction and denaturation can be achieved in a relatively short time; the same thing, however, can not be said for the alkylation process, which the present study shows that more than 6 h are required for a complete alkylation. We have used matrix assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) to monitor protein alkylation by iodoacetamide over the period 0-24 h at pH 9. The present, fast and specific MS method provided clear indication on the extent and speed of alkylation which reached approximately 70% in the first 2 min, yet the remaining 30% resisted complete alkylation up to 6 h. The use of sodium dodecyl sulfate (SDS) during the alkylation step resulted in a strong quenching of this reaction, whereas 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) exerted a much reduced inhibition. The implications of the present measurements on 2-D gel analysis in particular and proteomics in general are discussed.
Galvani
M
M
University of Verona, Department of Agricultural and Industrial Biotechnologies, Verona, Italy. righetti@mailserver.unimi.it
Hamdan
M
M
Herbert
B
B
Righetti
P G
PG
D000478:Alkylation; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002645:Chickens; D002793:Cholic Acids; D003545:Cysteine / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D007460:Iodoacetamide; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry / Q000302:isolation & purification; D008239:Lysine / Q000737:chemistry; D009113:Muramidase / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D002793:Cholic Acids; D011506:Proteins; D012967:Sodium Dodecyl Sulfate; D007768:Lactalbumin; D009113:Muramidase; D008239:Lysine; D003545:Cysteine; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D007460:Iodoacetamide