One-dimensional proteomic mapping of human liver cytochromes p450.
A method for constructing one-dimensional proteomic maps (1D-PM) based on mass spectrometric identification of proteins from adjacent slices of one-dimensional electrophoregram has been developed. For the proteomic mapping, gel lanes were sectioned into slices less than 0.2 mm thick and each slice was subjected to enzymatic hydrolysis. The resultant mixture of peptide fragments was analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF) and liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). Proteins were identified by the mass spectra obtained. Data on peptide fragments and corresponding identified proteins were presented as a 1D-PM. Proteomic maps were constructed by assigning individual proteins to gel slices based on number of matching peptides in a corresponding MS-data. On 1D-PM of human liver microsomal fraction, 18 proteins were identified in the region of 40-65 kDa. These included 12 membrane proteins belonging to the superfamily of cytochromes P450. Pooling of mass spectrometric data, obtained from several adjacent gel slices (molecular zooming) increased sequence coverage of CYP2A (cytochrome P450 family 2A). The maximal coverage of 66% significantly exceeded the level of 48% that could be obtained using one (even the most informative) slice. This method can be applied to the proteomic profiling of membrane-bound proteins.
Lisitsa
A V
AV
Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Moscow, Russia.
Petushkova
N A
NA
Nikitin
I P
IP
Zgoda
V G
VG
Karuzina
I I
II
Moshkovskii
S A
SA
Larina
O V
OV
Skipenko
O G
OG
Polyschuk
L O
LO
Thiele
H
H
Archakov
A I
AI
D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D002853:Chromatography, Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology*; D020543:Proteome / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013250:Steroid Hydroxylases / Q000378:metabolism; D053719:Tandem Mass Spectrometry
D008565:Membrane Proteins; D020543:Proteome; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C029009:steroid hormone 7-alpha-hydroxylase