Effective Protein Separation by Coupling Hydrophobic Interaction and Reverse Phase Chromatography for Top-down Proteomics

One of the challenges in proteomics is the proteome’s complexity, which necessitates the fractionation of proteins prior to the mass spectrometry (MS) analysis. Despite recent advances in top-down proteomics, separation of intact proteins remains challenging. Hydrophobic interaction chromatography (...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2014-08, Vol.86 (15), p.7899-7906
Main Authors: Xiu, Lichen, Valeja, Santosh G, Alpert, Andrew J, Jin, Song, Ge, Ying
Format: Article
Language:English
Subjects:
Amino Acid Sequence
ammonium sulfate
Biochemistry
Chromatography
Chromatography, Reverse-Phase - methods
Fractionation
Hydrophobic and Hydrophilic Interactions
hydrophobic bonding
hydrophobic interaction chromatography
Hydrophobic surfaces
Joining
Mass Spectrometry
Molecular Sequence Data
Proteins
Proteins - chemistry
Proteins - isolation & purification
proteome
Proteomics
reversed-phase liquid chromatography
Separation
Two dimensional
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Summary:One of the challenges in proteomics is the proteome’s complexity, which necessitates the fractionation of proteins prior to the mass spectrometry (MS) analysis. Despite recent advances in top-down proteomics, separation of intact proteins remains challenging. Hydrophobic interaction chromatography (HIC) appears to be a promising method that provides high-resolution separation of intact proteins, but unfortunately the salts conventionally used for HIC are incompatible with MS. In this study, we have identified ammonium tartrate as a MS-compatible salt for HIC with comparable separation performance as the conventionally used ammonium sulfate. Furthermore, we found that the selectivity obtained with ammonium tartrate in the HIC mobile phases is orthogonal to that of reverse phase chromatography (RPC). By coupling HIC and RPC as a novel two-dimensional chromatographic method, we have achieved effective high-resolution intact protein separation as demonstrated with standard protein mixtures and a complex cell lysate. Subsequently, the separated intact proteins were identified by high-resolution top-down MS. For the first time, these results have shown the high potential of HIC as a high-resolution protein separation method for top-down proteomics.
ISSN:0003-2700
1520-6882
1520-6882
DOI:10.1021/ac501836k