Background/Aim: 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. Materials and 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. Conclusion: 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.
A Simple and Effective Method to Analyze Membrane Proteins by SDS-PAGE and MALDI Mass Spectrometry
Guadagni F
2010-01-01
Abstract
Background/Aim: 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. Materials and 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. Conclusion: 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
