Molecular weight determination proteins

Be familiar with methods for protein molecular weight determination and the limitations of such methods gel filtration, gel electrophoresis, and hydrodynamic methods. 

Capillary gel electrophoresis (CGE), Size determination Protein molecular weight determination, aggregates, oligonucleotides, DNA fragments, polysaccharides... 

Accuracy of protein molecular weight determinations In different eluant systems. 

Detergents and salts interfere with protein molecular weight determinations, so proteins should be isolated to homogeneity in a volatile buffer system. Ideally, proteins are desalted by reverse-phase HPLC in aqueous acetonitrile or methanol solutions. These solvents can be infused directly into the mass spectrometer. Alternatively, proteins can be lyophilized from ammonium bicarbonate, ammonium acetate, or N-ethyl morpholine solutions. 

Effective hydrodynamic radius, R, dictates solute partitioning in a gel. This can be related to the number of units in a flexible polymer. It is then a corollary that partitioning of a linear polymer is a function of Its molecular weight. This has been proved experimentally leading to popularization of gel chromatography in 6M guanidinium chloride as a useful method of protein molecular weight determination. 

Strong denaturants such as sodium dodecyl sulfate (SDS) and guanidine are responsible for the conversion of native protein structures into random coil conformations. In many cases, this induces a imiform protein conformation, which increases the resolution and leads to an improvement in the accuracy of the protein molecular weight determination. It has also been observed that the... 

The molecular mass of the protein was redetermined by infusing a 5-10 pmolp.l solution of the protein in 50% aqueous acetonitrile containing 0.2% formic acid at a flow rate of 6 p,lmin into an electrospray source. The scan rate employed on the mass spectrometer was from m/z 60 to m/z 1800 in 12 s. This is a relatively slow scan speed which will lead to a more precise molecular weight determination. Scan speeds of this order may be, and indeed should be, utilized for infusion experiments if sufficient sample is available but it is unlikely to be feasible when chromatographic separations, particularly those involving capillary columns, are employed because of the restriction imposed by the chromatographic peak width (see Section 3.5.2.1 above). 

The most rigorous evidence that proteins had defined structures was probably the molecular weight determinations of Adair and Svedberg. From 1900, however the crystallization of increasing numbers of proteins, while not a very reliable indication of purity, suggested to Schulz that proteins were not colloidal aggregates but large molecules with definite structures. 

For accurate determination of protein molecular weight, mass spectrometry and LC-MS have largely displaced SDS-PAGE. However, SDS-PAGE will still be used where estimates of molecular weight suffice or where MS instrument time is limited. 

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