Question

What are the three most popular 2D chromatography methods for shotgun proteomics? Example, “size exclusion then normal phase,” etc?

Answer 1

2D fractionation commonly used tools to increase the dynamic range & proteome coverage for bottom-up shotgun proteomics.2D methodologies used low micrograms of samples.

1)the identification of protein from a complex biological matrix has traditionally been performed using 2D gel electrophoresis. 2DE separate proteins in both by their isoelectric point & molecular weight producing a high-resolution protein map from which individual protein spot is picked excised & sequenced

2)in a typical shotgun proteomics experiment a complex protein sample is enzymatically digested into peptide which is then separated by HPLC introduced into a mass spectrometer, fragmented sequenced to identify the parent protein via database searching.

3) LC-MS/MS shotgun proteomics, 2DE employee a divide and conquer strategy by resolving proteins into an individual spot which can then be selectively excised & sequenced.

size exclusion chromatography (gel filtration) - size exclusion chromatography is a method where the separation of different compounds occurs according to their size (hydrodynamic volume ) measured by how efficiently then penetrate the pores of stationery phase.