Mass Determination by ESI-MS-TOF

To determine molecular mass of proteins, ESI-Q-TOF is a method which offers a good mass accuracy (100 ppm) and is suitable for a wide range of masses (600 Da – 70 kDa) in routine. For higher mass proteins, please contact us.

The protein placed in appropriate buffer is infused in the ESI-Q-TOF mass spectrometer. The obtained spectrum shows a distribution of multiple charge states of the protein is visible (Raw spectrum in the figure). This spectrum will be deconvoluted to obtain a reconstructed spectrum showing the neutral average mass of the protein(s) (Deconvoluted spectrum in the figure).

With this type of analyses, the homogeneity of a protein can be assessed and if present modifications (i.e. glycosylations) could be detected (see bottom spectrum in the figure).

What is delivered?

The mass of the protein is given with a 100 ppm measure uncertainty. Report will be sent by email. Results are usually available in 10 working days.

What is needed for these analyses?

Protein concentration in the sample must be at least 40 µM and a minimum volume of 20 µL is required (or larger volume of less concentrated solution but a concentration step is needed prior to analysis).

Suitable buffer conditions for MS injection are water, ammonium acetate up to 100 mM or ammonium bicarbonate up to 100 mM.

Sample purity is critical and contaminants not compatible with MS, especially like salts, detergents, chaotrops, etc. must be eliminated before MS analysis. Therefore, preprocessing steps (usually Amicon Ultra with a cut-off membrane of 3 kDa) are somehow mandatory before analysis. While performing these extra steps, sample must be stable (no degradation, neither aggregation nor precipitation). Protein MS analysis classically involves that protein has to be charged (positively). Hence, acidic conditions by addition of 0.1-0.5% final FA is used to protonate sample before MS analysis.

Finally, it is important to have a protein that have at least 80% purity and that does not show too many isoforms, so that the major part of the MS signal will arise from the protein of interest. Indeed the MS signal is spread on different charge states for each compound present in the sample. If there are too much different compounds, the signal will be spread on too many peaks, so the signal-to-noise ratio will drop and the spectrum will become too noisy to be further treated.

Analysis Request Form for Mass Determination by ESI-Q-TOF