Abstract
Histone posttranslational modifications (hPTMs) play a key role in regulating chromatin dynamics and fine-tuning DNA-based processes. Mass spectrometry (MS) has emerged as a versatile technology for the analysis of histones, contributing to the dissection of hPTMs, with special strength in the identification of novel marks and in the assessment of modification cross talks. Stable isotope labeling by amino acid in cell culture (SILAC), when adapted to histones, permits the accurate quantification of PTM changes among distinct functional states; however, its application has been mainly confined to actively dividing cell lines. A spike-in strategy based on SILAC can be used to overcome this limitation and profile hPTMs across multiple samples. We describe here the adaptation of SILAC to the analysis of histones, in both standard and spike-in setups. We also illustrate its coupling to an implemented “shotgun” workfl ow, by which heavy arginine-labeled histone peptides, produced upon Arg-C digestion, are qualitatively and quantitatively analyzed in an LC-MS/MS system that combines ultrahigh-pressure liquid chromatography (UHPLC) with new-generation Orbitrap high-resolution instrument.
| Original language | English |
|---|---|
| Title of host publication | Methods in Molecular Biology |
| Publisher | Humana Press Inc. |
| Pages | 97-119 |
| Number of pages | 23 |
| Volume | 1528 |
| DOIs | |
| Publication status | Published - 2017 |
Publication series
| Name | Methods in Molecular Biology |
|---|---|
| Volume | 1528 |
| ISSN (Print) | 10643745 |
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Keywords
- Epigenetics
- High-resolution mass spectrometry
- Histone posttranslational modifications
- SILAC
- SILAC spike-in
- Ultrahigh-pressure liquid chromatography
ASJC Scopus subject areas
- Molecular Biology
- Genetics
Cite this
SILAC-based quantitative strategies for accurate histone posttranslational modification profiling across multiple biological samples. / Cuomo, Alessandro; Soldi, Monica; Bonaldi, Tiziana.
Methods in Molecular Biology. Vol. 1528 Humana Press Inc., 2017. p. 97-119 (Methods in Molecular Biology; Vol. 1528).Research output: Chapter in Book/Report/Conference proceeding › Chapter
}
TY - CHAP
T1 - SILAC-based quantitative strategies for accurate histone posttranslational modification profiling across multiple biological samples
AU - Cuomo, Alessandro
AU - Soldi, Monica
AU - Bonaldi, Tiziana
PY - 2017
Y1 - 2017
N2 - Histone posttranslational modifications (hPTMs) play a key role in regulating chromatin dynamics and fine-tuning DNA-based processes. Mass spectrometry (MS) has emerged as a versatile technology for the analysis of histones, contributing to the dissection of hPTMs, with special strength in the identification of novel marks and in the assessment of modification cross talks. Stable isotope labeling by amino acid in cell culture (SILAC), when adapted to histones, permits the accurate quantification of PTM changes among distinct functional states; however, its application has been mainly confined to actively dividing cell lines. A spike-in strategy based on SILAC can be used to overcome this limitation and profile hPTMs across multiple samples. We describe here the adaptation of SILAC to the analysis of histones, in both standard and spike-in setups. We also illustrate its coupling to an implemented “shotgun” workfl ow, by which heavy arginine-labeled histone peptides, produced upon Arg-C digestion, are qualitatively and quantitatively analyzed in an LC-MS/MS system that combines ultrahigh-pressure liquid chromatography (UHPLC) with new-generation Orbitrap high-resolution instrument.
AB - Histone posttranslational modifications (hPTMs) play a key role in regulating chromatin dynamics and fine-tuning DNA-based processes. Mass spectrometry (MS) has emerged as a versatile technology for the analysis of histones, contributing to the dissection of hPTMs, with special strength in the identification of novel marks and in the assessment of modification cross talks. Stable isotope labeling by amino acid in cell culture (SILAC), when adapted to histones, permits the accurate quantification of PTM changes among distinct functional states; however, its application has been mainly confined to actively dividing cell lines. A spike-in strategy based on SILAC can be used to overcome this limitation and profile hPTMs across multiple samples. We describe here the adaptation of SILAC to the analysis of histones, in both standard and spike-in setups. We also illustrate its coupling to an implemented “shotgun” workfl ow, by which heavy arginine-labeled histone peptides, produced upon Arg-C digestion, are qualitatively and quantitatively analyzed in an LC-MS/MS system that combines ultrahigh-pressure liquid chromatography (UHPLC) with new-generation Orbitrap high-resolution instrument.
KW - Epigenetics
KW - High-resolution mass spectrometry
KW - Histone posttranslational modifications
KW - SILAC
KW - SILAC spike-in
KW - Ultrahigh-pressure liquid chromatography
UR - http://www.scopus.com/inward/record.url?scp=84995902667&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84995902667&partnerID=8YFLogxK
U2 - 10.1007/978-1-4939-6630-1_7
DO - 10.1007/978-1-4939-6630-1_7
M3 - Chapter
AN - SCOPUS:84995902667
VL - 1528
T3 - Methods in Molecular Biology
SP - 97
EP - 119
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -