Abstract:Histone-based chromatin organization enabled eukaryotic genome complexity. This epigenetic control mechanism allowed for the differentiation of stable gene-expression and thus the very existence of multicellular organisms. This existential role in biology makes histones one of the most complexly modified molecules in the biotic world, which makes these key regulators notoriously hard to analyze. We here provide a roadmap to enable fast and informed selection of a bottom-up mass spectrometry sample preparation … Show more
“…S2 (ESI †) displays the coverage plots of the hPTMs on H3.1 and H4 detected by the three Mascot searches, color coded according to the number of spectra covering this residue, as presented recently elsewhere. 34 In the standard search, 1309 PSMs were found with an Nterminal fixed propionylation, comprising a total of 596 PSMs with acetylation. From the targeted precursor ion population (171 precursor ions), 18 precursor ions got annotated in this standard search (Fig.…”
Section: Resultsmentioning
confidence: 99%
“…First, the search parameters were adapted to search for chemical noise derived from derivatization using propionic anhydride. 29,30,34,[36][37][38] Therefore, the data was re-searched by setting propionylation at N-terminus and at K as variable modifications (to assess underpropionylation) and by including variable formylation at STYK to detect (spontaneously) induced formylation (sample preparation search; Table 1). 34,39 596 spectra were annotated with a formylation, giving rise to one additional annotation of the declining ion population because they also carried an acetylation (Fig.…”
Section: Resultsmentioning
confidence: 99%
“…S4 (ESI †) that this peptide is in fact annotated in a chimeric spectrum and that because of its size, had not passed the scoring threshold in the previous search. Thus, avoiding formylation by omitting the propionylation 34 or using a different acid for reversing overpropionylation would minimize the This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.…”
Section: Resultsmentioning
confidence: 99%
“…A significant amount of relevant histone signal is in fact singly charged, as our mobile phase contains DMSO, which improves ionization, but also causes charge state reduction. 43 Because +1 ions are not measured by DDA, 34 they were excluded from the Progenesis QIP 4.2 analysis in the first place. The analysis was therefore redone without filtering the 13874 singly charged ions, of which 2062 turned out to have an ANOVA q-value o 0.01 and 322 showed an expression profile like the deacetylated ion population from Fig.…”
Section: View Article Onlinementioning
confidence: 99%
“…3C and D. Using instruments that can sample the precursor ion space to greater depth, by adjusting the LC gradient, by reducing 1+ ion formation using alternative enzyme approaches, 34 or by including them during acquisition could mediate both issues. An alternative solution is to apply DIA, where precursors ions are continuously fragmented in an unbiased fashion, as suggested earlier by us and others with the hSWATH acquisition.…”
Histone-based chromatin organization paved the way for eukaryotic genome complexity. Because of their key role in information management, the histone posttranslational modifications (hPTM), which mediate their function, have evolved into...
“…S2 (ESI †) displays the coverage plots of the hPTMs on H3.1 and H4 detected by the three Mascot searches, color coded according to the number of spectra covering this residue, as presented recently elsewhere. 34 In the standard search, 1309 PSMs were found with an Nterminal fixed propionylation, comprising a total of 596 PSMs with acetylation. From the targeted precursor ion population (171 precursor ions), 18 precursor ions got annotated in this standard search (Fig.…”
Section: Resultsmentioning
confidence: 99%
“…First, the search parameters were adapted to search for chemical noise derived from derivatization using propionic anhydride. 29,30,34,[36][37][38] Therefore, the data was re-searched by setting propionylation at N-terminus and at K as variable modifications (to assess underpropionylation) and by including variable formylation at STYK to detect (spontaneously) induced formylation (sample preparation search; Table 1). 34,39 596 spectra were annotated with a formylation, giving rise to one additional annotation of the declining ion population because they also carried an acetylation (Fig.…”
Section: Resultsmentioning
confidence: 99%
“…S4 (ESI †) that this peptide is in fact annotated in a chimeric spectrum and that because of its size, had not passed the scoring threshold in the previous search. Thus, avoiding formylation by omitting the propionylation 34 or using a different acid for reversing overpropionylation would minimize the This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.…”
Section: Resultsmentioning
confidence: 99%
“…A significant amount of relevant histone signal is in fact singly charged, as our mobile phase contains DMSO, which improves ionization, but also causes charge state reduction. 43 Because +1 ions are not measured by DDA, 34 they were excluded from the Progenesis QIP 4.2 analysis in the first place. The analysis was therefore redone without filtering the 13874 singly charged ions, of which 2062 turned out to have an ANOVA q-value o 0.01 and 322 showed an expression profile like the deacetylated ion population from Fig.…”
Section: View Article Onlinementioning
confidence: 99%
“…3C and D. Using instruments that can sample the precursor ion space to greater depth, by adjusting the LC gradient, by reducing 1+ ion formation using alternative enzyme approaches, 34 or by including them during acquisition could mediate both issues. An alternative solution is to apply DIA, where precursors ions are continuously fragmented in an unbiased fashion, as suggested earlier by us and others with the hSWATH acquisition.…”
Histone-based chromatin organization paved the way for eukaryotic genome complexity. Because of their key role in information management, the histone posttranslational modifications (hPTM), which mediate their function, have evolved into...
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