Flagging False Positives Following Untargeted LC-MS Characterization of Histone Post-Translational Modification Combinations.
Epigenetic changes can be studied with an untargeted characterization of histone post-translational modifications (PTMs) by liquid chromatography-mass spectrometry (LC-MS). While prior information about more than 20 types of histone PTMs exists, little is known about histone PTM combinations (PTMCs). Because of the combinatorial explosion it is intrinsically impossible to consider all potential PTMCs in a database search. Consequentially, high-scoring false positives with unconsidered but correct alternative isobaric PTMCs can occur. Current quality controls can neither estimate the amount of unconsidered alternatives nor flag potential false positives. Here, we propose a conceptual workflow that provides such options. In this workflow, an in silico modeling of all candidate isoforms with known-to-exist PTMs is made. The most frequently occurring PTM sets of these candidate isoforms are determined and used in several database searches. This suppresses the combinatorial explosion while considering as many candidate isoforms as possible. Finally, annotations can be classified as unique or ambiguous, the latter implying false positives. This workflow was evaluated on an LC-MS data set containing 44 histone extracts. We were able to consider 60% of all candidate isoforms. Importantly, 40% of all annotations were classified as ambiguous. This highlights the need for a more thorough evaluation of modified peptide annotations.
Laboratory of Pharmaceutical Biotechnology, Ghent University , Ghent, 9000, Belgium.
Sander
0000-0002-7124-610X
S
Willems
Laboratory of Pharmaceutical Biotechnology, Ghent University , Ghent, 9000, Belgium.
Maarten
M
Dhaenens
Laboratory of Pharmaceutical Biotechnology, Ghent University , Ghent, 9000, Belgium.
Elisabeth
E
Govaert
Laboratory of Pharmaceutical Biotechnology, Ghent University , Ghent, 9000, Belgium.
Laura
L
De Clerck
Laboratory of Pharmaceutical Biotechnology, Ghent University , Ghent, 9000, Belgium.
Paulien
P
Meert
Laboratory of Pharmaceutical Biotechnology, Ghent University , Ghent, 9000, Belgium.|Bioinformatics Institute Ghent, Ghent University , Ghent, 9052, Belgium.|Center for Medical Genetics Ghent, Ghent University , Ghent, 9000, Belgium.
Christophe
C
Van Neste
Laboratory of Pharmaceutical Biotechnology, Ghent University , Ghent, 9000, Belgium.
Filip
F
Van Nieuwerburgh
Laboratory of Pharmaceutical Biotechnology, Ghent University , Ghent, 9000, Belgium.
Dieter
D
Deforce
D000595:Amino Acid Sequence / Q000235:genetics; D002853:Chromatography, Liquid; D003198:Computer Simulation; D044127:Epigenesis, Genetic / Q000235:genetics; D006657:Histones / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D058977:Molecular Sequence Annotation; D020033:Protein Isoforms / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D040901:Proteomics; D053719:Tandem Mass Spectrometry
D006657:Histones; D020033:Protein Isoforms
combinatorial explosion; histone; mass spectrometry (MS); post-translational modification (PTM); quality control