PolyLC Switzerland, Europe, Germany, Austria | HPLC Columns Purification and Analysis of Proteins and Peptides

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Top-Down Proteomics

The reversed-phase columns used for bottom-up proteomics of peptides don't work with many proteins. Some don't elute at all while others elute in peaks 15 minutes wide. Progress in top-down proteomics requires alternative modes of chromatography. Examples:

 WCX-HILIC of Histone isoforms: Histone H3(2-51)

Over 700 variants of H3 have been identified with various combinations of 17 types of post-translational modifications (PTM's). Most of the PTM's are in residues 2-51. H3 is typically analyzed via that fragment from Glu-C digestion ("middle-down" proteomics). Success in this identification requires good separation of the variants by chromatography prior to MS. This is accomplished with capillaries of our WCX material, PolyCAT A. Inclusion of 60-70& ACN superimposes hydrophilic interaction and hence sensitivity to variations in polarity (e.g. methylation) as well as charge. WCX material lose their (-) charge below pH 4. WCX-HILIC can then be performed with a totally volatile mobile phase with a decreasing gradient of ACN (tuning down the hydrophilic interaction) and pH (uncharging the PolyCAT A). The proteins can then be eluted directly to MS.

Full MS spectra (= intact mass) of histone H3 fragments: The two peak clusters represent light and heavy (= isotopically heavy Arg & Lys residues) SILAC histone H3 tails differing by 1 methyl-group (= 14 Da). The 2-μm PolyCAT A material [RIGHT] affords sharp peaks with half as many unresolved variants complicating the mass spectrum as with the 3-μm material's results [LEFT].

Size-Exclusion Chromatography: Examples with antibodies

SEC can be performed using volatile solvents. Here, antibody light and heavy chains all elute in the total exclusion volume peak (Vo) from a PolyHYDROXYETHYL A column with a pore diameter of 200 Å and a mobile phase of 50 mM formic acid [LEFT]. The Vo peak is sent to a mass spectrometer and deconvoluted mass spectra are obtained for the light and heavy chains [RIGHT].

from: L.J. Brady et al. J. Am. Soc. Mass Spectrom. 19 (2008) 502

3-D Protein Fractionation: An IEX-HIC-RPC Sequence

Sample: HEK 293 cell lysate

 RESULT:

Starting with all 35 HIC fractions from IEX Fraction #3, 640 intact proteins were identified. 201 of these id's were nonredundant (some proteins were identified in more than one HIC fraction).

A 3D analysis of all 35 IEX fractions would have required 35 x 35 = 1225 RPC runs. Many more proteins would have been identified, at the cost of more time and effort. There is a tradeoff involved. It is still worthwhile ; omitting the HIC step (with an IEX-RPC sequence only) resulted in just 47 nonredundant id's.

(data adapted from S.G. Valeja, L.Xiu, Z.R. Gegorich, H. Guner, S. Jin and Y. Ge. Anal. Chem. online 4/13/2015 [doi: 10.1021/acs.analchem.5b00657])