Development and applications of in-gel CNBr/tryptic digestion combined with mass spectrometry for the analysis of membrane proteins.
Hydrophobic membrane proteins often have complex functions and are thus of great interest. However, their analysis presents a challenge because they are not readily soluble in polar solvents and often undergo aggregation. We present a sequential CNBr and trypsin in-gel digestion method combined with mass spectrometry for membrane protein analysis. CNBr selectively cleaves methionine residues. But due to the low number of methionines in proteins, CNBr cleavage produces a small number of large peptide fragments with MWs typically >2000, which are difficult to extract from gel pieces. To produce a larger number of smaller peptides than that obtained by using CNBr alone, we demonstrate that trypsin can be used to further digest the sample in gel. The use of n-octyl glucoside (n-OG) to enhance the digestion efficiency and peptide recovery was also studied. We demonstrate that the sensitivity of this membrane protein identification method is in the tens of picomole regime, which is compatible to the Coomassie staining gel-spot visualization method, and is more sensitive than other techniques reported in the literature. This CNBr/trypsin in-gel digestion method is also found to be very reproducible and has been successfully applied for the analysis of complex protein mixtures extracted from biological samples. The results are presented from a study of the analysis of bacteriorhodopsin, nitrate reductase 1 gamma chain, and a complex protein mixture extracted from the endoplasmic recticulum membrane of mouse liver.
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
Thuy Tien T
TT
Quach
Nan
N
Li
Dawn P
DP
Richards
Jing
J
Zheng
Bernd O
BO
Keller
Liang
L
Li
D000818:Animals; D001436:Bacteriorhodopsins / Q000032:analysis; D003488:Cyanogen Bromide / Q000378:metabolism*; D030562:Databases, Protein; D004721:Endoplasmic Reticulum / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry; D005782:Gels; D057927:Hydrophobic and Hydrophilic Interactions; D008099:Liver / Q000166:cytology; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry*; D051379:Mice; D008970:Molecular Weight; D009565:Nitrate Reductases / Q000032:analysis; D010446:Peptide Fragments / Q000737:chemistry; D017421:Sequence Analysis; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism*
D005782:Gels; D008565:Membrane Proteins; D010446:Peptide Fragments; D001436:Bacteriorhodopsins; D009565:Nitrate Reductases; D014357:Trypsin; D003488:Cyanogen Bromide