For the determination of molecular weights in 2-D electrophoresis gels two different procedures are used.

 

Co-running molecular weight standard proteins.

Molecular weight marker proteins can be applied as a separate track to the 2-D gel. The molecular weights of the proteins in the 2-D map are then interpolated with the molecular weight curve obtained from the positions of the marker proteins. The problem with this method is its limited accuracy. The markers have to be applied at the lateral sides of the gels. Often the space is very limited and the bands are curved because of the edge effects in SDS PAGE.

Interpolation between identified sample proteins with known Mr values.

Prominent spots showing up in each 2-D map of a sample type can be analyzed for identification and amino acid sequence information. The theoretical Mr values can then be used as key stones for interpolating the Mr values of the other proteins. This method is much more exact than the method described above.

The mass spectrum is a graph of ion intensity as a function of the mass - to - charge ratio and it is used to determine the molecular weight and structure of the compounds being analyzed. These techniques enable fragmentation of large polypeptides and proteins to be analyzed and accurate measurement of the molecular weight and extensive sequence information can be acquired in one analysis.

Molecular weight distribution of peptides or proteins

The molecular weight distribution describes the relationship between the number of moles of the peptide and protein (Ni) and their molecular weight (Mi), and there is basically a distribution around the average value of all the peptides and proteins in the sample.

Two basic methods used for measuring molecular weight distributions are as follows:

Methods for estimating molecular weight distribution without fractionation include light scattering studies, electron microscopy, dilute solution viscosity measurements, gel permeation chromatography, ultracentrifugation and diffusion.

Methods for estimating molecular weight distribution with fractionation methods, which actually divide the specimen into fractions of various molecular weight ranges, include fraction precipitation, chromatography, liquid-liquid partition, ultra centrifugation. After fractionation by any of these methods, the weight of each fraction is plotted against the average molecular weight to obtain a curve of the distribution.

The most common technique for measuring molecular mass used in modern times is a variant of high-pressure liquid chromatography (HPLC) known by the interchangeable terms of size exclusion chromatography (SEC) and gel permeation chromatography (GPC), through the relationship between molar mass and the hydrodynamic volume.

Among the detectors used for size exclusion chromatography, the most common is the differential refractive index detector that measures the change in refractive index of the solvent. This detector is concentration-sensitive and very molecular-mass-insensitive, so it is ideal for a single-detector GPC system. Less common but more accurate and reliable is a molecular-mass-sensitive detector using multi-angle laser-light scattering - see Static Light Scattering.