Evaluation and optimization of ZIC-HILIC-RP as an alternative MudPIT strategy
Evaluation and optimization of ZIC-HILIC-RP as an alternative MudPIT strategy
In proteomics, a digested cell lysate is often too complex for direct comprehensive mass spectrometric analysis. To reduce complexity, several peptide separation techniques have been introduced including very successful two-dimensional liquid chromatography (2D-LC) approaches. Here, we assess the potential of zwitterionic Hydrophilic Interaction Liquid Chromatography (ZIC-HILIC) as a first dimension for the analysis of complex peptide mixtures. We show that ZIC-HILIC separation is dramatically dependent on buffer pH in the range from 3 to 8, due to deprotonation of acidic amino acids. ZIC-HILIC exhibits a mixed-mode effect consisting of electrostatic and polar interactions. We developed a 2D-LC system that hyphenates ZIC-HILIC off-line with reversed-phase (RP). The two dimensions are fairly orthogonal, and the system performs very well in the analysis of minute amounts of complex peptide mixtures. Applying this method to the analysis of 10 mug of a cellular nuclear lysate, we were able to confidently identify over 1000 proteins. Compared to strong cation exchange chromatography (SCX), ZIC-HILIC shows better chromatographic resolution and absence of clustering of prevalent +2 and +3 charged peptides. At pH 3, ZIC-HILIC separation allows best orthogonality with RP and resembles conventional SCX separation. A significant enrichment of N-acetylated peptides in the first fractions is observed at these conditions. ZIC-HILIC separation at high pH (6.8 and 8), however, enables better chromatography, resulting in more comprehensive data acquisition. With this extended flexibility, we conclude that ZIC-HILIC is a very good alternative for the more conventional SCX in multidimensional peptide separation strategies.
Chromatography, Liquid/*methods/standards Hydrogen-Ion Concentration Nuclear Proteins/isolation & purification Peptides/*isolation & purification Proteomics/methods Static Electricity
937-946
Boersema, P. J.
e54b08c9-a5ff-4733-b976-a2e98dd20188
Divecha, N.
5c2ad0f8-4ce7-405f-8a15-2fc4ab96d787
Heck, A. J.
f15e1fc0-2c5a-4232-83b6-dcc9b042a228
Mohammed, S.
9902085d-913b-4a0e-b215-ddb8b56f9d95
27 January 2007
Boersema, P. J.
e54b08c9-a5ff-4733-b976-a2e98dd20188
Divecha, N.
5c2ad0f8-4ce7-405f-8a15-2fc4ab96d787
Heck, A. J.
f15e1fc0-2c5a-4232-83b6-dcc9b042a228
Mohammed, S.
9902085d-913b-4a0e-b215-ddb8b56f9d95
Boersema, P. J., Divecha, N., Heck, A. J. and Mohammed, S.
(2007)
Evaluation and optimization of ZIC-HILIC-RP as an alternative MudPIT strategy.
Journal of Proteome Research, 6 (3), .
(doi:10.1021/pr060589m).
Abstract
In proteomics, a digested cell lysate is often too complex for direct comprehensive mass spectrometric analysis. To reduce complexity, several peptide separation techniques have been introduced including very successful two-dimensional liquid chromatography (2D-LC) approaches. Here, we assess the potential of zwitterionic Hydrophilic Interaction Liquid Chromatography (ZIC-HILIC) as a first dimension for the analysis of complex peptide mixtures. We show that ZIC-HILIC separation is dramatically dependent on buffer pH in the range from 3 to 8, due to deprotonation of acidic amino acids. ZIC-HILIC exhibits a mixed-mode effect consisting of electrostatic and polar interactions. We developed a 2D-LC system that hyphenates ZIC-HILIC off-line with reversed-phase (RP). The two dimensions are fairly orthogonal, and the system performs very well in the analysis of minute amounts of complex peptide mixtures. Applying this method to the analysis of 10 mug of a cellular nuclear lysate, we were able to confidently identify over 1000 proteins. Compared to strong cation exchange chromatography (SCX), ZIC-HILIC shows better chromatographic resolution and absence of clustering of prevalent +2 and +3 charged peptides. At pH 3, ZIC-HILIC separation allows best orthogonality with RP and resembles conventional SCX separation. A significant enrichment of N-acetylated peptides in the first fractions is observed at these conditions. ZIC-HILIC separation at high pH (6.8 and 8), however, enables better chromatography, resulting in more comprehensive data acquisition. With this extended flexibility, we conclude that ZIC-HILIC is a very good alternative for the more conventional SCX in multidimensional peptide separation strategies.
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Published date: 27 January 2007
Additional Information:
Boersema, Paul J Divecha, Nullin Heck, Albert J R Mohammed, Shabaz eng Evaluation Study Research Support, Non-U.S. Gov't 2007/01/30 J Proteome Res. 2007 Mar;6(3):937-46. doi: 10.1021/pr060589m. Epub 2007 Jan 27.
Keywords:
Chromatography, Liquid/*methods/standards Hydrogen-Ion Concentration Nuclear Proteins/isolation & purification Peptides/*isolation & purification Proteomics/methods Static Electricity
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Local EPrints ID: 480174
URI: http://eprints.soton.ac.uk/id/eprint/480174
ISSN: 1535-3893
PURE UUID: 2710fbb5-e7e7-4c6f-9d38-52caf6b962cf
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Date deposited: 01 Aug 2023 16:57
Last modified: 17 Mar 2024 02:59
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Author:
P. J. Boersema
Author:
A. J. Heck
Author:
S. Mohammed
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