TY - JOUR
T1 - Alkylation kinetics of proteins in preparation for two-dimensional maps
T2 - A matrix assisted laser desorption/ionization-mass spectrometry investigation
AU - Galvani, Marina
AU - Hamdan, Mahmoud
AU - Herbert, Ben
AU - Righetti, Pier Giorgio
PY - 2001
Y1 - 2001
N2 - 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 ∼ 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.
AB - 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 ∼ 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.
KW - Alkylation
KW - Ionization-time of flight-mass spectrometry
KW - Matrix assisted laser desorption
KW - Reduction
KW - Two-dimensional maps
UR - http://www.scopus.com/inward/record.url?scp=0034946628&partnerID=8YFLogxK
U2 - 10.1002/1522-2683(200106)22:10<2058::AID-ELPS2058>3.0.CO;2-Z
DO - 10.1002/1522-2683(200106)22:10<2058::AID-ELPS2058>3.0.CO;2-Z
M3 - Article
C2 - 11465506
AN - SCOPUS:0034946628
VL - 22
SP - 2058
EP - 2065
JO - Electrophoresis
JF - Electrophoresis
SN - 0173-0835
IS - 10
ER -