MALDI-TOF/TOF (5800, AB SCIEX)


Application domains

Identification of proteins in simple mixtures (purified by 1D or 2D acrylamide gels) or in complex mixtures after an LC separation and spotting on a MALDI plate by a Probot.

Analysis of whole proteins, characterization of the entire protein.

Post-translational modification research.

Quantification of peptides of an iTRAQ or SILAC labeling.

Measurement principle

Peptides produced by the hydrolysis of proteins are spotted on a MALDI plate and crystallized in the presence of chemical compound called matrix (CHCA, DHB). Laser shots on the crystallized peptide spots form peptidic ions in the source part of the instrument. In the case of a whole protein analysis, different matrices that are adapted to the sample are used (SA, ferulic acid), the principle of the analysis remains however the same with the difference of it being done in the linear mode.

These ions are accelerated and enter the TOF analyzer to be separated according to their mass/charge ratio. Indeed, in this part of this instrument that’s not submitted to an electric field, ions move at a velocity that is inversely proportional to the square root of their mass. Thus, the time of flight information is converted to mass information for each ion. All this information gathered with each laser shot is recorded as a spectrum. This spectrum is called a peptide mass fingerprint. Searching these spectra against a protein database allows to identify the protein that is at the origin of the measured spectrum.

In the TOF/TOF mode, the selected peptides are fragmented by collision with an inert gas, producing fragments whose masses are measured in the second analyzer. The analysis of the masses of these fragments allows to obtain the peptidic sequence.

Technical properties

Make : MALDI TOF/TOF 5800 Applied
Ionization : MALDI
Analyzer : Time of flight
Sensitivity : 0,1 to 0,2 fmoles in routine analyses
Precision : 50 ppm for external calibration / 5ppm for internal calibration
Resolution :
Mass range : 50 to 500000 Da
Control and data processing :Protein Pilot, GPS ;Mascot

Remarks

Our instruments is equipped with a delayed extraction system and an electrostatic reflector. The instrument is also equipped with a 1.3 m time of flight tube in the linear mode and 2.4 m in the reflectron mode. Its laser irradiation system, the "OptiBeam™ On-axis", is the sample’s axis. This provides the instrument with a better sensitivity in both MS and MS/MS analysis modes. Its optical deceleration system upstream of the collision cell controls the kinetic energy of the ions that enter the collision cell to allow for an optimal fragmentation.

Installation date:

December 2012

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