High sensitivity identification of membrane proteins by MALDI TOF-MASS spectrometry using polystyrene beads.
Membrane proteins play a large variety of functions in life and represent 30% of all genomes sequenced. Due to their hydrophobic nature, they are tightly bound to their biological membrane, and detergents are always required to extract and isolate them before identification by mass spectrometry (MS). The latter, however remains difficult. Peptide mass fingerprinting methods using techniques such as MALDI-TOF MS, for example, have become an important analytical tool in the identification of proteins. However, PMF of membrane proteins is a real challenge for at least three reasons. First, membrane proteins are naturally present at low levels; second, most of the detergents strongly inhibit proteases and have deleterious effects on MALDI spectra; and third, despite the presence of detergent, membrane proteins are unstable and often aggregate. We took the mitochondrial uncoupling protein 1 (UCP1) as a model and showed that differential acetonitrile extraction of tryptic peptides combined with the use of polystirene Bio-Beads triggered high resolution of the MALDI-TOF identification of mitochondrial membrane proteins solubilized either with Triton-X100 or CHAPS detergents.
Bensalem
Noura
N
INSERM, U806, Paris, France.
Masscheleyn
Sandrine
S
Mozo
Julien
J
Vallée
Benoit
B
Brouillard
Franck
F
Trudel
Stéphanie
S
Ricquier
Daniel
D
Edelman
Aleksander
A
Guerrera
Ida Chiara
IC
Miroux
Bruno
B
D000097:Acetonitriles / Q000737:chemistry; D000818:Animals; D002793:Cholic Acids / Q000737:chemistry; D003902:Detergents / Q000737:chemistry; D007473:Ion Channels / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis*; D051379:Mice; D008863:Microspheres; D024101:Mitochondrial Proteins / Q000032:analysis; D017830:Octoxynol / Q000737:chemistry; D010449:Peptide Mapping / Q000379:methods*; D011137:Polystyrenes / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000737:chemistry; D000071256:Uncoupling Protein 1
D000097:Acetonitriles; D002793:Cholic Acids; D003902:Detergents; D007473:Ion Channels; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011137:Polystyrenes; C000604372:Ucp1 protein, mouse; D000071256:Uncoupling Protein 1; D017830:Octoxynol; D014357:Trypsin; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; C032159:acetonitrile