Method for identification and quantitative analysis of protein lysine methylation using matrix-assisted laser desorption/ionization--time-of-flight mass spectrometry and amino acid analysis.
Protein methylation is a post-translational modification that might have important functional roles in cell regulation. We present a new technique with sufficient sensitivity (sub-pmol level) for analysis of methylation of proteins in abundances typically found on proteome maps produced by two-dimensional (2-D) gel electrophoresis. The method involves the identification and quantitation of lysine (Lys) methylation using Fmoc (9-fluorenylmethyl chloroformate)-based amino acid analysis (AAA). Tri- and monomethyl-Lys were baseline-separated from other amino acids using a modified buffer system. Trimethyl-Lys was quantitatively recovered after acid hydrolysis and AAA of two known methylated proteins - yeast cytochome c and human calmodulin. The methylated peptides from tryptic digestion of those two proteins were identified by high sensitivity matrix-assisted laser desorption/ionization - time-of-flight (MALDI-TOF) mass spectrometry (MS). An automated mass-screening approach is proposed for the study of various post-translational modifications to understand the distribution of those protein isoforms separated by two-dimensional polyacrylamide gel electrophoresis. It is concluded that the combination of AAA and MALDI-TOF-MS provides a high sensitivity quantitative tool for the analysis of protein post-translational methylation in the context of proteome studies.
Yan
J X
JX
Australian Proteome Analysis Facility, Macquarie University, Sydney, NSW. jun.yan@harefield.nthames.nhs.uk
Sanchez
J C
JC
Binz
P A
PA
Williams
K L
KL
Hochstrasser
D F
DF
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D002147:Calmodulin / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D008745:Methylation; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000596:Amino Acids; D002147:Calmodulin; D003574:Cytochrome c Group; D011506:Proteins; D008239:Lysine