Analysis of histone post translational modifications in primary monocyte derived macrophages using reverse phase×reverse phase chromatography in conjunction with porous graphitic carbon stationary phase
Analysis of histone post translational modifications in primary monocyte derived macrophages using reverse phase×reverse phase chromatography in conjunction with porous graphitic carbon stationary phase
A two dimensional-liquid chromatography (2D-LC) based approach was developed for the identification and quantification of histone post translational modifications in conjunction with mass spectrometry analysis. Using a bottom-up strategy, offline 2D-LC was developed using reverse phase chromatography. A porous graphitic carbon stationary phase in the first dimension and a C18 stationary phase in the second dimension interfaced with mass spectrometry was used to analyse global levels of histone post translational modifications in human primary monocyte-derived macrophages. The results demonstrated that 84 different histone peptide proteoforms, with modifications at 18 different sites including combinatorial marks were identified, representing an increase in the identification of histone peptides by 65% and 51% compared to two different 1D-LC approaches on the same mass spectrometer. The use of the porous graphitic stationary phase in the first dimension resulted in efficient separation of histone peptides across the gradient, with good resolution and is orthogonal to the online C18 reverse phase chromatography. Overall, more histone peptides were identified using the 2D-LC approach compared to conventional 1D-LC approaches. In addition, a bioinformatic pipeline was developed in-house to enable the high throughput efficient and accurate quantification of fractionated histone peptides. The automation of a section of the downstream analysis pipeline increased the throughput of the 2D-LC-MS/MS approach for the quantification of histone post translational modifications.
Cells, Cultured, Chromatography, Liquid, Chromatography, Reverse-Phase, Computational Biology, Graphite, Histones, Humans, Macrophages, Peptides, Porosity, Protein Processing, Post-Translational, Tandem Mass Spectrometry, Journal Article
43-53
Minshull, Thomas C.
3d39be4a-4ce3-4db7-ad7b-a5ba945fbdf8
Cole, Joby
546f9b85-70c2-4f0b-9915-aa2b2016804f
Dockrell, David H.
a068c9bf-35b8-4c10-8f91-58639cfeca0b
Read, Robert C.
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Dickman, Mark J.
213ec03a-2bb2-4bcf-a4a9-2a140bad888e
1 July 2016
Minshull, Thomas C.
3d39be4a-4ce3-4db7-ad7b-a5ba945fbdf8
Cole, Joby
546f9b85-70c2-4f0b-9915-aa2b2016804f
Dockrell, David H.
a068c9bf-35b8-4c10-8f91-58639cfeca0b
Read, Robert C.
b5caca7b-0063-438a-b703-7ecbb6fc2b51
Dickman, Mark J.
213ec03a-2bb2-4bcf-a4a9-2a140bad888e
Minshull, Thomas C., Cole, Joby, Dockrell, David H., Read, Robert C. and Dickman, Mark J.
(2016)
Analysis of histone post translational modifications in primary monocyte derived macrophages using reverse phase×reverse phase chromatography in conjunction with porous graphitic carbon stationary phase.
Journal of Chromatography A, 1453, .
(doi:10.1016/j.chroma.2016.05.025).
Abstract
A two dimensional-liquid chromatography (2D-LC) based approach was developed for the identification and quantification of histone post translational modifications in conjunction with mass spectrometry analysis. Using a bottom-up strategy, offline 2D-LC was developed using reverse phase chromatography. A porous graphitic carbon stationary phase in the first dimension and a C18 stationary phase in the second dimension interfaced with mass spectrometry was used to analyse global levels of histone post translational modifications in human primary monocyte-derived macrophages. The results demonstrated that 84 different histone peptide proteoforms, with modifications at 18 different sites including combinatorial marks were identified, representing an increase in the identification of histone peptides by 65% and 51% compared to two different 1D-LC approaches on the same mass spectrometer. The use of the porous graphitic stationary phase in the first dimension resulted in efficient separation of histone peptides across the gradient, with good resolution and is orthogonal to the online C18 reverse phase chromatography. Overall, more histone peptides were identified using the 2D-LC approach compared to conventional 1D-LC approaches. In addition, a bioinformatic pipeline was developed in-house to enable the high throughput efficient and accurate quantification of fractionated histone peptides. The automation of a section of the downstream analysis pipeline increased the throughput of the 2D-LC-MS/MS approach for the quantification of histone post translational modifications.
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More information
Accepted/In Press date: 3 May 2016
e-pub ahead of print date: 7 May 2016
Published date: 1 July 2016
Keywords:
Cells, Cultured, Chromatography, Liquid, Chromatography, Reverse-Phase, Computational Biology, Graphite, Histones, Humans, Macrophages, Peptides, Porosity, Protein Processing, Post-Translational, Tandem Mass Spectrometry, Journal Article
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Local EPrints ID: 416148
URI: http://eprints.soton.ac.uk/id/eprint/416148
ISSN: 0021-9673
PURE UUID: 6127c67a-cbee-437d-bd43-197cf79e4e21
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Date deposited: 06 Dec 2017 17:30
Last modified: 16 Mar 2024 04:10
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Contributors
Author:
Thomas C. Minshull
Author:
Joby Cole
Author:
David H. Dockrell
Author:
Mark J. Dickman
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