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.
Department of Chemical and Biological Engineering, ChELSI Institute, University of Sheffield, Mappin Street, Sheffield S1 3JD, United Kingdom; Department of Infection, Immunity & Cardiovascular Disease, University of Sheffield Medical School, United Kingdom; Sheffield Teaching Hospitals, United Kingdom.
Thomas C
TC
Minshull
Department of Chemical and Biological Engineering, ChELSI Institute, University of Sheffield, Mappin Street, Sheffield S1 3JD, United Kingdom; Department of Infection, Immunity & Cardiovascular Disease, University of Sheffield Medical School, United Kingdom; Sheffield Teaching Hospitals, United Kingdom.
Joby
J
Cole
Department of Infection, Immunity & Cardiovascular Disease, University of Sheffield Medical School, United Kingdom; Sheffield Teaching Hospitals, United Kingdom.
David H
DH
Dockrell
Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine and Institute for Life Sciences, University of Southampton, NIHR Respiratory Biomedical Research Unit, University Hospital Southampton, Southampton SO166YD, United Kingdom.
Robert C
RC
Read
Department of Chemical and Biological Engineering, ChELSI Institute, University of Sheffield, Mappin Street, Sheffield S1 3JD, United Kingdom. Electronic address: m.dickman@sheffield.ac.uk.
Mark J
MJ
Dickman
D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods; D019295:Computational Biology; D006108:Graphite / Q000737:chemistry; D006657:Histones / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008264:Macrophages / Q000378:metabolism; D010455:Peptides / Q000032:analysis / Q000302:isolation & purification; D016062:Porosity; D011499:Protein Processing, Post-Translational; D053719:Tandem Mass Spectrometry / Q000379:methods*
D006657:Histones; D010455:Peptides; D006108:Graphite
Histone post translational modifications; Mass spectrometry; Porous graphitic stationary phase; Two dimensional chromatography