Label Free relative protein quantification by nLC MS/MS
Lately we settled in our lab a gel-free label-free method for differential protein expression analysis. The protein extracts coming from cells and/or tissues are digested with trypsin, using digestion methods that have been improved in order to gain reproducibility and sensitivity.
The peptides resulting from the digestion are loaded onto a nano Acquity UPLC chromatograph and analysed in an LTQ-Orbitrap XL (Thermo) or Synapt G2Si (waters) mass spectrometer. The data obtained is then processed and loaded onto the Progenesis LC MS software. This software allows the alignment of chromatographic runs, increasing analysis reliability and reproducibility. Then it performs a intensity-based relative quantification of peptides in order to get the information about deregulated peptides.
Then, this information is converted to deregulation paterns at protein level, and the relative quantification is finally done.
Different studies have shown us that the method is a robust and straightforward approach for differential protein expression analysis. As an example of this, this method was applied in a project leaded by our colleague Dr. Ashwin Woodhoo and the results where integrated in the paper by Gomez-Sanchez et al. J Cell Biol. 2015 Jul 6; 210(1):153-68.