HPLC enrichment/isolation of proteins for post-translational modification studies from complex mixtures

Eszter Tóth; Olivér Ozohanics; Balázs Bobály; Ágnes Gömöry; Anita Jekő; László Drahos; Károly Vékey

doi:10.1016/j.jpba.2014.06.025

HPLC enrichment/isolation of proteins for post-translational modification studies from complex mixtures

J Pharm Biomed Anal. 2014 Sep:98:393-400. doi: 10.1016/j.jpba.2014.06.025. Epub 2014 Jun 21.

Authors

Eszter Tóth¹, Olivér Ozohanics¹, Balázs Bobály², Ágnes Gömöry¹, Anita Jekő¹, László Drahos¹, Károly Vékey³

Affiliations

¹ Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok körútja 2., Budapest 1117, Hungary.
² Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok körútja 2., Budapest 1117, Hungary; Budapest University of Technology and Economics, Department of Inorganic and Analytical Chemistry, Szt. Gellért tér 4., Budapest 1111, Hungary.
³ Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok körútja 2., Budapest 1117, Hungary. Electronic address: [email protected].

PMID: 25005889
DOI: 10.1016/j.jpba.2014.06.025

Abstract

The paper describes a macroporous RP-HPLC method for separation and isolation/enrichment of proteins from complex mixtures. The method is robust and efficient; using 2.1 or 4.6mm diameter columns provides sufficient material for subsequent proteomic analysis. The main advantage of the method is that most protein variants are isolated in the same fraction, as separation is not based on differences in isoelectric point. This is highly advantageous for studying complex mixtures and post-translational modifications. Examples related to glycosylation analysis are discussed in detail.

Keywords: Glycosylation; HPLC; Mass spectrometry; Proteins; Sample preparation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Chromatography, High Pressure Liquid / methods
Complex Mixtures / chemistry*
Glycosylation
Humans
Plasma / chemistry
Protein Processing, Post-Translational / genetics*
Proteins / chemistry*
Proteomics / methods

Substances

Complex Mixtures
Proteins