TY - JOUR
T1 - Reduction of alkylation of proteins in preparation of two-dimensional map analysis
T2 - Why, when, and how?
AU - Herbert, Ben
AU - Galvani, Marina
AU - Hamdan, Mahmoud
AU - Olivieri, Erna
AU - MacCarthy, John
AU - Pedersen, Sanne
AU - Righetti, Pier Giorgio
PY - 2001
Y1 - 2001
N2 - The standard procedure adopted up to the present in proteome analysis calls for just reduction prior to the isoelectric focusing/immobilized pH gradient (IEF/IPG) step, followed by a second reduction/alkylation step in between the first and second dimension, in preparation for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) step. This protocol is far from being optimal. It is here demonstrated, by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry, that failure to reduce and alkylate proteins prior to any electrophoretic step (including the first dimension) results in a large number of spurious spots in the alkaline pH region, due to "scrambled" disulfide bridges among like and unlike chains. This series of artefactual spots comprises not only dimers, but an impressive series of oligomers (up to nonamers) in the case of simple polypeptides such as the human α- and β-globin chains, which possess only one (α-) or two (β-) -SH groups. As a result, misplaced spots are to be found in the resulting two-dimensional (2-D) map, if performed with the wrong protocol. The number of such artefactual spots can be impressively large. In the case of analysis of complex samples, such as human plasma, it is additionally shown that failure to alkylate proteins results in a substantial loss of spots in the alkaline gel region, possibly due to the fact that these proteins, at their p/, regenerate their disulfide bridges with concomitant formation of macroaggregates which become entangled with and trapped within the polyacrylamide gel fibers. This strongly quenches their transfer in the subsequent SDS-PAGE step.
AB - The standard procedure adopted up to the present in proteome analysis calls for just reduction prior to the isoelectric focusing/immobilized pH gradient (IEF/IPG) step, followed by a second reduction/alkylation step in between the first and second dimension, in preparation for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) step. This protocol is far from being optimal. It is here demonstrated, by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry, that failure to reduce and alkylate proteins prior to any electrophoretic step (including the first dimension) results in a large number of spurious spots in the alkaline pH region, due to "scrambled" disulfide bridges among like and unlike chains. This series of artefactual spots comprises not only dimers, but an impressive series of oligomers (up to nonamers) in the case of simple polypeptides such as the human α- and β-globin chains, which possess only one (α-) or two (β-) -SH groups. As a result, misplaced spots are to be found in the resulting two-dimensional (2-D) map, if performed with the wrong protocol. The number of such artefactual spots can be impressively large. In the case of analysis of complex samples, such as human plasma, it is additionally shown that failure to alkylate proteins results in a substantial loss of spots in the alkaline gel region, possibly due to the fact that these proteins, at their p/, regenerate their disulfide bridges with concomitant formation of macroaggregates which become entangled with and trapped within the polyacrylamide gel fibers. This strongly quenches their transfer in the subsequent SDS-PAGE step.
KW - Alkylation
KW - Globin chains
KW - Ionization-time of flight-mass spectrometry
KW - Matrix assisted laser desorption
KW - Reduction
KW - Two-dimentional maps
UR - http://www.scopus.com/inward/record.url?scp=0034940957&partnerID=8YFLogxK
U2 - 10.1002/1522-2683(200106)22:10<2046::AID-ELPS2046>3.0.CO;2-C
DO - 10.1002/1522-2683(200106)22:10<2046::AID-ELPS2046>3.0.CO;2-C
M3 - Article
C2 - 11465505
AN - SCOPUS:0034940957
SN - 0173-0835
VL - 22
SP - 2046
EP - 2057
JO - Electrophoresis
JF - Electrophoresis
IS - 10
ER -