Abundance of H4 peptides used for normalization. the histone variant), if their length is within 6C23 amino acids, if they are not chemically altered, and if the monitored peptides have no neighboring K/R residue. 13072_2017_172_MOESM2_ESM.xlsx (14K) GUID:?1818A71D-FEC3-4547-B7FB-9D9D06F2941E Additional file 3. Abundance of H4 peptides used for normalization. The nucleosome contains two copies of each histone. No H4 variant has been described in mammals [8], so that H4 Rabbit Polyclonal to TRADD was logically chosen to normalize the abundance of H2A and H2B variants between samples. The MS signals for three H4 peptides (P50C52) are presented for the analysis of histones extracted from spermatocytes (Sc), round spermatids (R), and elongating and condensing spermatids (EC). They were brought to the same scale as for spermatocytes to allow easier inter-peptide signal comparisons. Three technical replicates of LCCMS/MS analyses were analyzed. The relative MS signals measured on peptides P50CP52 are comparable in the three cell types. 13072_2017_172_MOESM3_ESM.png (29K) GUID:?3A2D5609-97E2-4D32-82FB-2C98C3DC216B Additional file 4. Details of the SRM transitions for each signature peptide. SRM assay parameters including precursor and Citalopram Hydrobromide fragment ion type, charge state, elution time as well as natural data are provided in Suppl. data. (*) Indicates peptides monitored only in their endogenous form. 13072_2017_172_MOESM4_ESM.xlsx (39K) GUID:?A347ECDC-715B-406D-8708-754527F5B51D Additional file 5. Composition of the mixture of standard peptides. 13072_2017_172_MOESM5_ESM.docx (65K) GUID:?2A552609-2E6C-4477-AA2C-FB64612940E8 Additional file 6. Reproducibility of the LC-SRM analysis. R2 values are indicated for each technical replicate with increasing matrix amounts or increasing standard peptide amounts. Plotted data are presented in Fig.?5. 13072_2017_172_MOESM6_ESM.docx (57K) GUID:?E605A90B-547B-40B8-B010-523F7D63B3A4 Additional file 7. PRM transition results. List of transitions used to quantify each monitored peptide. Precursor charge, fragment ion, light precursor m/z ratio, light product m/z ratio, light retention time are indicated. 13072_2017_172_MOESM7_ESM.xlsx (14K) GUID:?04FCB167-24DE-4EF3-89C5-E82F2A97BFD3 Additional file 8. Abundances of altered and non-modified forms of H2B and H4 signature peptides in round spermatids extracted from WT and Sly-KD mice testis. Two biological replicates are presented. 13072_2017_172_MOESM8_ESM.png (73K) GUID:?2E61B1FE-A783-4C71-BB10-294C79175864 Additional file 9. Rules used to select or reject peptides using their transition profiles. The validation of the best transitions was performed using a signal-to-noise ratio ( ?5) and a perfect co-elution of the heavy standard peptide with the endogenous peptide. Three fragment ions (F1, F2, and F3) are represented for the heavy and the endogenous peptides. a All fragment Citalopram Hydrobromide ions can be integrated because the heavy and endogenous fragment ions co-elute in the same intensity order. b In that case, only F2 can be integrated because the ratio heavy/endogenous is different for F1 and F3. c The fragment F2 is usually contaminated by another analyte eluting at a slightly later time; it has to be excluded from the analysis. d Here, the signal-to-noise ratio is usually below five, no fragment ion can be integrated. e. The endogenous peptide traces do not co-elute with the heavy peptide traces. 13072_2017_172_MOESM9_ESM.png (173K) GUID:?671CE660-D222-4509-80E9-B17FA48C4D3A Data Availability StatementThe datasets supporting the conclusions of this article are included within the article and its additional files. SRM data have been deposited in PeptideAtlas under the accession number PASS01118 and PRM data under PASS01124. All other MS data have been deposited to the ProteomeXchange Consortium via the PRIDE [75] partner repository with the dataset identifier PXD008358 and 10.6019/pxd008358. Abstract Background Histones organize DNA into Citalopram Hydrobromide chromatin through a variety of processes. Among them, a vast diversity of histone variants can be incorporated into chromatin and finely modulate its organization and functionality. Classically, the study of histone variants has largely relied on antibody-based assays. However, antibodies have a limited efficiency to discriminate between highly similar histone variants. Results In this study, we established a mass spectrometry-based analysis to address this challenge. We developed a targeted proteomics method, using selected reaction monitoring or parallel reaction monitoring, to quantify a maximum number of histone variants in a single multiplexed assay, even when histones are present Citalopram Hydrobromide in a crude extract. This strategy was developed on H2A and H2B variants,.