An optimized strategy for ICAT quantification of membrane proteins.
The work presented here focuses on the development of a method adapting isotope labeling of proteins with ICAT to the study of highly hydrophobic proteins. Conditions for the labeling of proteins were first established using two standard soluble proteins and iodoacetamidyl-3,6-dioxaoctanediamine biotin (PEO-iodoacetyl biotin). Results demonstrated the efficiency of the labeling in the presence of high concentrations of both SDS and urea. These conditions were then used to label a highly hydrophobic mitochondrial membrane protein, the adenine nucleotide translocator ANT-1, with PEO-iodoacetyl biotin and then with the cleavable ICAT reagent. The results presented here show that labeling of proteins with cleavable ICAT is possible and may even be improved in strong denaturing buffers containing both SDS at a concentration higher than 0.5% (w/v) and urea. These results open the possibility of applying the ICAT strategy to complex samples containing very hydrophobic proteins solubilized in urea-SDS buffers. The adaptability of the developed method is demonstrated here with preliminary results obtained during the study of membrane-enriched fractions prepared from murine embryonic stem cells.
Ramus
Claire
C
Laboratoire de Chimie des Protéines ERM-0201, INSERM/Commissariat à l'Energie Atomique, CEA/Université Joseph Fourier, UJF, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble-cedex 9, France.
Gonzalez de Peredo
Anne
A
Dahout
Cécile
C
Gallagher
Maighread
M
Garin
Jérôme
J
D000345:Affinity Labels; D000818:Animals; D001710:Biotin / Q000737:chemistry; D002417:Cattle; D002645:Chickens; D003545:Cysteine / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D004622:Embryo, Mammalian / Q000166:cytology* / Q000378:metabolism*; D004625:Embryo, Nonmammalian; D007553:Isotope Labeling; D007782:Lactoglobulins / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D010047:Ovalbumin / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013234:Stem Cells / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism; D014508:Urea / Q000378:metabolism
D000345:Affinity Labels; D007782:Lactoglobulins; D008565:Membrane Proteins; D001710:Biotin; D014508:Urea; D010047:Ovalbumin; D014357:Trypsin; D003545:Cysteine