Molecular mass determination

Molecular mass determination is the simplest analysis that can be achieved by mass spectrometry. We perform it routinely on several kinds of samples:

  • Small molecules (Mw < 1000 Da): by ESI-FT-ICR (with molecular formula determination)
  • Purified homogeneous proteins with Mw up to 70 kDa : by ESI-Q-TOF
  • Other cases: by MALDI-TOF analysis

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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 – 70000 Da) in routine. For higher mass proteins, please contact us.

 

What is delivered?

 

The mass of the protein is given with a 100 ppm measure uncertainty.

 

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[DB1] ) 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.

 

What is the time to get results?

 

Results are generally available in 10 working days

 

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MALDI-MS

Mass determination by MALDI-TOF is recommended when the available amount is lower than 2 nmol or if the protein is very heterogeneously modified (for example: pegylated proteins).

 

What is delivered?

 

The mass of the protein is given with a 500 ppm measure uncertainty.

 

What is needed for these analyses?

 

Protein amount must be at least 100 pmol in the sample (minimum concentration of 50 μM is required, otherwise a pre-concentration step will be needed).

Suitable buffer conditions 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. In this case, a ZipTip C4 is performed and proteins are resuspended in the matrix solution. While performing these extra steps, sample must be stable (no degradation, neither aggregation nor precipitation).

 

What is the time to get results?

 

Results are generally available in 10 working days

 

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FT

Thanks to its high resolution and high mass precision, FT-ICR is the technique of choice to determine raw formula of small compounds and facilitate structural elucidation by MS/MS. The samples are infused on a Bruker Solarix FT-ICR MS 9.4T. With this instrument, accurate mass determination (< 2 ppm) of the molecule (with mass < 1000 Da) will be achieved and its molecular formula will be determined.

 

What is delivered?

 

The exact mass of the compound as well as its potential molecular formulas is given with a < 2 ppm measure uncertainty.

 

What is needed for these analyses?

 

Amount: at least 50 µL of 50 µM solution (or 2.5 nmoles) of sample is needed. If possible, a more concentrated solution is preferred.

The sample must be provided lyophilized or in solution, but the solvent used to dissolve the molecule must be MS compatible (a.o. water and ammonium acetate or ammonium bicarbonate solutions, acetonitrile, methanol, dichloroethane).

If you have a doubt concerning MS compatibility of your solution, please contact us before sending the sample.

Information to provide:

- If lyophilized sample, nature of solvent(s) to be used to dissolve the product. Please mention if samples are not stable in acidic or basic conditions as a small amount of formic acid (positive ion mode) or trimethylamine (negative ion mode) can be used to enhance the signal.

- Expected raw formula if known or a masse range of interest and atoms potentially present.

 

What is the time to get results?

 

Results are generally available in 10 working days

 

Form

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