Combined chemical and enzymatic stable isotope labeling for quantitative profiling of detergent-insoluble membrane proteins isolated using Triton X-100 and Brij-96.
Effective quantitative profiling of detergent-insoluble membrane proteins using high-throughput mass spectrometry (MS)-based proteomics would allow a better understanding of physiological and pathological processes that take place at the cell surface. To increase the coverage of proteins present in detergent-resistant membrane microdomains (DRMMs), a combination of 16O/18O and isotope coded affinity tags (ICAT) labeling was used in a comparative analysis of detergent-insoluble membrane proteins isolated from rat basophilic leukemia cells (RBL-2H3), with either Triton X-100 or Brij-96. The analysis resulted in the quantification of 738 unique proteins from Triton X-100 and Brij-96 isolated DRMMs, significantly exceeding the number of proteins quantified from either single labeling technique. Twenty-five noncysteine-containing proteins were quantified, as well as 32 cysteine-containing proteins that would have been missed if either 16O/18O or ICAT labeling had been used exclusively, which illustrate better proteome coverage and enhanced ability to quantitate. The comparative analysis revealed that proteins were more readily extracted using Triton X-100 than Brij-96; however, Triton X-100 also extracted larger quantities of non-DRMMs-associated proteins. This result confirms previous, targeted studies suggesting that DRMMs isolated using Triton X-100 and Brij-96 differ in their protein content.
Blonder
Josip
J
Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland 21702-1201, USA. blonder@ncifcrf.gov
Yu
Li-Rong
LR
Radeva
Galina
G
Chan
King C
KC
Lucas
David A
DA
Waybright
Timothy J
TJ
Issaq
Haleem J
HJ
Sharom
Frances J
FJ
Veenstra
Timothy D
TD
D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin / Q000737:chemistry; D002250:Carbon Radioisotopes / Q000737:chemistry; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D003902:Detergents / Q000737:chemistry; D003903:Deuterium / Q000737:chemistry; D007553:Isotope Labeling; D021962:Membrane Microdomains / Q000737:chemistry; D008565:Membrane Proteins / Q000032:analysis*; D008969:Molecular Sequence Data; D017830:Octoxynol / Q000737:chemistry*; D010104:Oxygen Radioisotopes / Q000737:chemistry; D010938:Plant Oils / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D040901:Proteomics; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization; D014357:Trypsin / Q000737:chemistry*
D002250:Carbon Radioisotopes; D003902:Detergents; D008565:Membrane Proteins; D010104:Oxygen Radioisotopes; D010938:Plant Oils; D011092:Polyethylene Glycols; D001710:Biotin; D017830:Octoxynol; C033084:polyethylene glycol oleyl ether; D003903:Deuterium; D014357:Trypsin