Intact Mass

IMG_20120529_113409What do we use to measure the mass of intact proteins?

We do LC/MS analysis of intact proteins using a Q-TOF mass spectrometer with electrospray ionization. This means that samples must be free of detergents. An ideal solvent is 5% acetonitrile in water containing 1-5% formic acid or acetic acid, though many standard biological buffers and salts are acceptable (see below).

What solution you should prepare your protein sample into?

Samples must be reconstituted into an acceptable buffer prior to submission to the Proteomics Core facility.  Samples which are known to contain detergent or are not solubilized will not be accepted.

The most ideal solution for analysis would be a simple water and organic solvent solution containing 1-5% of formic acid or acetic acid. Organic solvents, such as acetonitrile, methanol and isopropanol, are all fine. In most cases, customers have their proteins dissolved in solutions containing buffers and salts.  The following components are acceptable:

Acceptable components: buffers (tris, ammonium buffer, HEPES, PBS), chaotropes (guanidine, urea), salt (NaCl, KCl), chelators (EDTA), reducing agents* (DTT, TCEP, 2-mercaptoethanol).

Components to avoid: detergents (SDS, Triton, CHAPS), stabilizers (PEG, glycerol >10%)

*reducing agents need to be added for less than 2 days. Prolonged contact of reducing agent may cause modifications on the protein cysteine residue.

What amount is needed?

The starting amount we recommend is 0.5 mg/mL, with 10ul minimum volume. The success of the intact mass measurement depends on many factors, including how pure the sample is (single protein vs. a complex mixture) and how well the protein ionizes during electrospray ionization.  To increase the chances of success, improved purity of the sample is the most important, followed by the concentration.

What else is required for intact mass sample submission?

When filling out the online form for intact mass sample submission, please include the following:

1)    all buffer components and concentrations
2)    protein concentration
3)    sample volume
4)    molecular weight of protein of interest

 

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