Protein analysis by MALDI-TOF (Matrix Assisted Laser Desorption Ionization – Time Of Fly) mass spectrometry is a method particularly suitable for the analysis of low-complexity proteins to:
- identify proteins and their partners obtained by pull-down
- characterize them (mutations, post-translational modifications)
- map interaction sites between cross-linked proteins
The technique used is the peptide mass fingerprinting, which consists in digesting a protein with a protease of known specificity (mainly trypsin) and determining the mass of the peptides resulting from this digestion using a mass spectrometer. The comparison of the experimental masses of the peptides with the theoretical masses resulting from in silico digestions of protein sequences from databases allows the identification of the proteins.
MALDI-TOF analysis
After protease digestion, the peptide mixture obtained is co-crystallized in the presence of a specific matrix and deposited on a metal plate. The mass spectrometer used is the MALDI AB SCIEX TOF/TOF 5800 located at IPBS. Pulsed laser shots desorb the matrix which ionizes the sample by charge transfer. Under an electric field, the ionized molecules are accelerated and the ions will fly freely in a flight tube for a fixed and determined distance. The device measures the time of flight which will be proportional to the m/z ratio (mass/charge) of the ion and thus measures the mass of the ionized peptide. All the ions obtained form the characteristic mass spectrum of the sample.
In practice
The analysis is performed on bands excised from SDS-PAGE after Coomassie staining. The gel, staining and cutting of the bands are done by the users.
Peptide digestion, MALDI-TOF analysis, and data processing are performed by the facility. The results are returned in the form of lists of identified proteins and their peptide mass fingerprints.
Cost of analysis
22 euros per sample.
Contact: Anne CAUMONT-SARCOS – Email – 05 61 33 58 16 (LMGM) & Régine CAPEYROU – Email – 05 61 33 58 75 (MCD)