A simple and effective method to analyze membrane proteins by SDS-PAGE and MALDI mass spectrometry.
OBJECTIVE
Identification and characterization of membrane proteins is a crucial challenge in proteomics research. Thus, we designed a novel method to prepare proteins possessing extensive hydrophobic stretches for mass spectrometry studies, without sacrificing other classes of proteins.


METHODS
This method uses sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) separation and relies solely on a matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) instrument, the most common and easiest to use mass spectrometer.


RESULTS
Using this analytical procedure, a significant number of hydrophobic peptides were recovered, with no reduction in overall sequence coverage and with a good identification of transmembrane proteins sequence. Applying this method to the systematic identification of proteins located in lipid rafts, up to 47% of identified proteins were obtained with an improvement of sequence coverage.


CONCLUSIONS
The procedure presented here is suitable for both identifying purified hydrophobic proteins and systematically investigating hydrophobic protein mixtures. It can be easily applied even in non-dedicated laboratories, such as those mostly devoted to clinical chemistry.
DI Girolamo
Francesco
F
Department of Laboratory Medicine and Advanced Biotechnologies, IRCCS San Raffaele, Via della Pisana, Rome, Italy.
Ponzi
Marta
M
Crescenzi
Marco
M
Alessandroni
Jhessica
J
Guadagni
Fiorella
F
D000199:Actins / Q000032:analysis; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D008565:Membrane Proteins / Q000032:analysis*; D051379:Mice; D018994:Myosin Light Chains / Q000032:analysis; D040901:Proteomics / Q000379:methods; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000199:Actins; D001457:Anion Exchange Protein 1, Erythrocyte; D008565:Membrane Proteins; D018994:Myosin Light Chains; C076659:myosin light chain I