HPLC size exclusion chromatography/laser light scattering determines the native molecular weights and sizes of proteins and their protein protein complexes in solution. Why do we use this technique when moleculare weights can be determined via mass spectrometry and analytical centrifugation?

Roshan said:

HPLC size exclusion chromatography/laser light scattering determines the native molecular weights and sizes of proteins and their protein protein complexes in solution. Why do we use this technique when moleculare weights can be determined via mass spectrometry and analytical centrifugation?

Although molecular weights can be determined via mass spectrometry and analytical centrifugation, only light scattering and analytical centrifugation monitor the properties of macromolecules in solution and provide information about the oligometric state of the protein.

Also, SEC is a lot easier and less expensive than analytical ultracentrifugation (although it is susceptible to shape artifacts, so will not always tell you the true size of your molecule).

Also, there are times when you want to see the dynamics of a complex at work?

currently, I measure the molecular weight of my macromolecule by dynamic light scattering method. If I measure it through mass spectroscopy, will it be different? what inferance can be diduced from the difference?

It is hard to make too many general statements, but each method for determining molecular weights has it's own advantages and disadvantages. Since all are based on different physical and chemical principles, you would expect each to be susceptible to different sources of variation. So, expect different answers from each technique. However, knowing these susceptibilities can help you understand your material, at least in a qualitative sense.

In theory, mass spectroscopy should be one of the most accurate techniques, and that is certainly true for small molecules. However, proteins present many difficulties for analysis, including the fact that the charge on the protein is sensitive to environmental conditions. Also, the mass resolution for very large molecules is not at the unit mass level. But you can still get much information.

Sorry to be so vague in my response, but there is no simple answer.