title: Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) Analysis for Characterization and Quantification of Histone Post-translational Modifications.
abstract: Histone post-translational modifications (PTMs) have a fundamental function in chromatin biology, as they model chromatin structure and recruit enzymes involved in gene regulation, DNA repair, and chromosome condensation. High throughput characterization of histone PTMs is mostly performed by using nano-liquid chromatography coupled to mass spectrometry. However, limitations in speed and stochastic sampling of data dependent acquisition methods in MS lead to incomplete discrimination of isobaric peptides and loss of low abundant species. In this work, we analyzed histone PTMs with a data-independent acquisition method, namely SWATH™ analysis. This approach allows for MS/MS-based quantification of all analytes without upfront assay development and no issues of biased and incomplete sampling. We purified histone proteins from human embryonic stem cells and mouse trophoblast stem cells before and after differentiation, and prepared them for MS analysis using the propionic anhydride protocol. Results on histone H3 peptides verified that sequential window acquisition of all theoretical mass spectra could accurately quantify peptides (<9% average coefficient of variation, CV) over four orders of magnitude, and we could discriminate isobaric and co-eluting peptides (e.g. H3K18ac and H3K23ac) using MS/MS-based quantification. This method provided high sensitivity and precision, supported by the fact that we could find significant differences for remarkably low abundance PTMs such as H3K9me2S10ph (relative abundance <0.02%). We performed relative quantification for few sample peptides using different fragment ions and observed high consistency (CV <15%) between the fragments. This indicated that different fragment ions can be used independently to achieve the same peptide relative quantification. Taken together, sequential window acquisition of all theoretical mass spectra proved to be an easy-to-use MS acquisition method to perform high quality MS/MS-based quantification of histone-modified peptides.
authors: From the ‡Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104;
authors: Simone
authors: S
authors: Sidoli
authors: From the ‡Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104;
authors: Shu
authors: S
authors: Lin
authors: §AB SCIEX, Redwood City, California 94065.
authors: Lei
authors: L
authors: Xiong
authors: From the ‡Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104;
authors: Natarajan V
authors: NV
authors: Bhanu
authors: From the ‡Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104;
authors: Kelly R
authors: KR
authors: Karch
authors: §AB SCIEX, Redwood City, California 94065.
authors: Eric
authors: E
authors: Johansen
authors: §AB SCIEX, Redwood City, California 94065.
authors: Christie
authors: C
authors: Hunter
authors: §AB SCIEX, Redwood City, California 94065.
authors: Sahana
authors: S
authors: Mollah
authors: From the ‡Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104; bgarci@mail.med.upenn.edu.
authors: Benjamin A
authors: BA
authors: Garcia
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D006657:Histones / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D051379:Mice; D010455:Peptides / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods; D014327:Trophoblasts / Q000166:cytology / Q000378:metabolism
chemical_list: D006657:Histones; D010455:Peptides
references: Methods Enzymol. 2012;512:3-28
references: 22910200
references: Mol Cell Proteomics. 2012 Jun;11(6):O111.016717
references: 22261725
references: Nat Biotechnol. 2014 Mar;32(3):219-23
references: 24727770
references: Genome Biol. 2012;13(10):R86
references: 23034525
references: Proteomics. 2014 Oct;14(19):2200-11
references: 25073878
references: J Proteome Res. 2014 Oct 3;13(10):4470-8
references: 25167464
references: Anal Chem. 2003 Feb 1;75(3):663-70
references: 12585499
references: Cell. 2007 Feb 23;128(4):693-705
references: 17320507
references: Cell. 1991 Oct 4;67(1):89-104
references: 1680565
references: Nature. 2003 Mar 13;422(6928):198-207
references: 12634793
references: Nat Protoc. 2007;2(4):933-8
references: 17446892
references: J Proteome Res. 2009 Jul;8(7):3284-97
references: 19400588
references: Mol Cell Proteomics. 2009 Oct;8(10):2266-84
references: 19654425
references: J Proteome Res. 2009 Nov;8(11):5367-74
references: 19764812
references: Cell Cycle. 2009 Nov 15;8(22):3688-94
references: 19855177
references: Anal Biochem. 2010 Jan 15;396(2):180-7
references: 19761750
references: Bioinformatics. 2010 Apr 1;26(7):966-8
references: 20147306
references: Cell Stem Cell. 2010 May 7;6(5):457-67
references: 20452320
references: Nat Rev Cancer. 2010 Jul;10(7):457-69
references: 20574448
references: Nat Biotechnol. 2010 Jul;28(7):710-21
references: 20622845
references: Nat Biotechnol. 2010 Oct;28(10):1057-68
references: 20944598
references: Cell Mol Life Sci. 2010 Dec;67(23):3983-4000
references: 20683756
references: Nat Methods. 2011 May;8(5):430-5
references: 21423193
references: Nat Rev Mol Cell Biol. 2012 Mar;13(3):153-67
references: 22358331
references: Anal Bioanal Chem. 2012 Mar;402(8):2587-96
references: 22203371
references: Nat Methods. 2012 Jun;9(6):555-66
references: 22669653
references: J Proteomics. 2012 Jun 27;75(12):3419-33
references: 22234360
references: Proteomics. 2013 Apr;13(8):1247-56
references: 23322582