In situ alkylation with acrylamide for identification of cysteinyl residues in proteins during one- and two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis.
Cysteinyl residues in proteins were alkylated with acrylamide during sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to yield a thioether derivative, cys-S-beta-propionamide (PAM cys). The process was termed in situ alkylation with acrylamide. Using this method, the recovery of PAM-cys peptides from bovine serum albumin (BSA) was 88.6% at 10 picomol in one-dimensional (1-D) SDS-PAGE and 97.1% at 50 picomol in two-dimensional (2-D) SDS-PAGE. The coverage of tryptic peptide of BSA in 1-D and 2-D SDS-PAGE was 83.7% and 81.1%, respectively. The advantages of in situ alkylation with acrylamide were the following: (i) cysteinyl peptides were effectively derived in a single PAM cys and then proteins were precisely identified using databases; (ii) marked reduction of salts compared with post alkylation, e.g., using carboxymethylamide (CAM), resulting in higher signal intensity and wider coverage of cysteinyl peptides from PAM cys, compared with those of CAM derivatives, in mass spectrometry peptide mapping; and (iii) shorter duration by excluding the processes of post alkylation and desalting before peptide mapping.
Mineki
Reiko
R
Division of Biochemical Analysis, Central Laboratory of Medical Sciences, Juntendo University School of Medicine, Tokyo, Japan.
Taka
Hikari
H
Fujimura
Tsutomu
T
Kikkawa
Mika
M
Shindo
Noriko
N
Murayama
Kimie
K
D020106:Acrylamide / Q000737:chemistry*; D000478:Alkylation; D000818:Animals; D002417:Cattle; D002458:Cell Fractionation; D003545:Cysteine / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D008239:Lysine / Q000737:chemistry; D051379:Mice; D008930:Mitochondria, Liver / Q000737:chemistry; D009113:Muramidase / Q000737:chemistry / Q000235:genetics; D011506:Proteins / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry
D011506:Proteins; D020106:Acrylamide; D012710:Serum Albumin, Bovine; D009113:Muramidase; D008239:Lysine; D003545:Cysteine