Mass measurements of biological molecules

- General information

- Sample preparation notes

General information What can you expect from mass measurements of biological molecules?

For mass measurements, molecules have to be transferred from solution to the gaseous phase and ionised. At present two techniques of ionisation are mostly used in biological sciences - electrospray (ESI) and laser desorption (MALDI). In ESI molecules are transferred to gas phase directly from solvent via fast solvent evaporation in a high electric field. In MALDI the solution is first mixed with matrix which facilitates the transfer to gaseous phase by a laser pulse. The above two techniques allow the transfer of even very large, megaDalton molecules to the gas phase.

The parameter that is actually measured in mass spectrometers is the ratio of mass to charge (m/z) and not the mass itself.
Molecules that bear no charge are not detected and their mass cannot be measured.
MALDI ionised molecules basically acquire a single charge whereas ESI ionised molecules can be multiply charged.

For singly charged molecules (MALDI techniqe mainly) the mass is obtained in a straightforward manner. For multiply charged molecules (ESI technique) the obtained data has to be deconvoluted from m/z domain. An m/z value of 1000 may come from a molecule of mass 1000 Da and a single charge or mass 2000 Da and charge 2 etc. Moreover, fractions of a pool of protein or peptide molecules may bear different charges, and the mass spectrum coming from a homogeneous species in the m/z domain is split into several signals at different m/z values (See figure 1 ).

Moreover, the signal at each m/z value is further split due to the natural abundance of 13C and other isotopes (See figure 2 ). The presence of a 13C atom shifts the mass of the molecule by 1 Da.

If the molecules are singly charged a series of m/z values is obtained with an envelope of peaks separated by 1. For +2 charged molecule peaks within the isotope envelope differ by 0.5 (See figure 3a ), for +3 the difference is 0.33 (See Figure 3b ). Visual inspection is usually sufficient for proper assignment of peaks to a single envelope.

In today's commercially available instruments the resolution (the ratio between m/z value and the peak's width at half height) is better than 10000 (See figure 4 ). At this resolution the 1000 m/z peak's half width is 0.1 Dalton and even the isotope peaks for molecules charged +10 can be resolved (See figure 5 ).

For these peaks it is possible to measure the distance between isotope peaks which determines charge and calculate the mass in a straightforward manner (See figure 6a). Alternatuely a software procedure can be applied to calculate the mass distribution in the spectrum (See figure 6b). For mass calculation a simple formula is used m=n*(m/z -n*1.008 where n denotes assigned charge). 6 ).

However, proteins in electrospray experiment can bear much more charge and isotope peaks cannot be resolved (See figure 7 ). Instead, the m/z value can be deconvoluted to mass value by analysis of the masses of at least three subsequent m/z peaks corresponding to charges n-1, n and n+1. This analysis sometimes needs support of a sophisticated software but for uniform and not contaminated samples it is straightforward. In result very precise mass measurements of even large proteins and their complexes are possible.

Measurement accuracy

The m/z value (and thus the mass) can be measured in today's commercially available instruments with the accuracy approaching ten part per million values (10 ppm accuracy means 0.01 Dalton for a molecule of mass 1000 Da, and 1 Da for a protein of 100 kDa). The best accuracy is obtained when the unknown sample is mixed with some internal standard of known mass. To put this short, the expected mass accuracy for a peptide should be ca. 0.05 Da and for proteins from 0.1 to 10 Da depending on protein mass. One should not expect to obtain the mass of the entire protein by submitting the stained gel slice.

Instrument sensitivity

Sensitivity depends strongly on often unpredictable efficiency of ionisation of a given molecule. See figure 9 , which presents the results of the mass measurement carried out using 10 attomoles of a peptide.

Sample quantity requested

Routine measurements are carried out at the concentration of 1 pmole/ul (1 uM) and the volume of 50 ul, so the sample consumption is 50 pmoles or 1ug of a protein of 20 kDa. However, good results can be obtained for protein concentrations even orders of magnitude lower.

Sample preparation notes

For best results the mass measurements of peptides or proteins are carried out at low pH in the presence of an organic solvent (preferably 50% H2O, 50% acetonitrile, 0.05% formic acid) Oligonucleotide masses are best obtained from volatile buffers at neutral pH.
For ESI measurements sample should be carefully desalted, MALDI technique tolerates salts better.
Solubility in one of the following solvents is required (H20, methanol, ethanol, methylene chloride, tetrahydrofuran, formic acid, acetic acid, NH4OH, TEA (triethanoloamine).
High ionic strength and presence of polymeric detergents (Triton X-100, Tween etc.) or TCA significantly lowers sensitivity or even precludes measurements.