2f21bba8fb6207f47ac20288397f1567 biomolecules-14-01221-v2.pdf d428123a43c6d2058af5ce6a2dc0ad77e24b0c82 biomolecules-14-01221-v2.pdf f3349f64d4047899bd0c6d1d37233fc154e6a69d3774f0dfc2d36489e2d8936f biomolecules-14-01221-v2.pdf Title: Quantification of Histone H1 Subtypes Using Targeted Proteomics Subject: Histone H1 is involved in the regulation of chromatin structure. Human somatic cells express up to seven subtypes. The variability in the proportions of somatic H1s (H1 complement) is one piece of evidence supporting their functional specificity. Alterations in the protein levels of different H1 subtypes have been observed in cancer, suggesting their potential as biomarkers and that they might play a role in disease development. We have developed a mass spectrometry-based (MS) parallel reaction monitoring (PRM) assay suitable for the quantification of H1 subtypes. Our PRM method is based on the quantification of unique peptides for each subtype, providing high specificity. Evaluation of the PRM performance on three human cell lines, HeLa, K562, and T47D, showed high reproducibility and sensitivity. Quantification values agreed with the electrophoretic and Western blot data, indicating the accuracy of the method. We used PRM to quantify the H1 complement in peripheral blood samples of healthy individuals and chronic myeloid leukemia (CML) patients. In CML, the first line of therapy is a tyrosine kinase inhibitor, imatinib. Our preliminary data revealed differences in the H1 complement in CML patients between imatinib responders and non-responders. These results support further research to determine if the H1 content or subtype composition could help predict imatinib response. Keywords: histone H1; functional differentiation; cancer biomarker; chronic myeloid leukemia; imatinib resistance; parallel reaction monitoring Author: Jordi López-Gómez, Laura Villarreal, Marta Andrés, Inma Ponte, Blanca Xicoy, Lurdes Zamora, Marta Vilaseca and Alicia Roque Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Sun Oct 6 16:57:43 2024 CEST ModDate: Sun Oct 6 17:00:31 2024 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 18 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1904297 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- HJOMHV+VnURWPalladioL-Bold Type 1 Custom yes yes yes 10 0 KIHUGJ+URWPalladioL-Bold Type 1 Custom yes yes yes 16 0 HPOTEX+URWPalladioL-Roma Type 1 Custom yes yes yes 22 0 QGRSZE+URWPalladioL-Ital Type 1 Custom yes yes yes 27 0 AZMWTB+VnURWPalladioL Type 1 Custom yes yes yes 32 0 IVVSWS+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 50 0 QHDCGG+CMSY10 Type 1 Builtin yes yes yes 78 0 OVNEXW+EURM10 Type 1 Builtin yes yes yes 83 0 NNFDBB+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 110 0 NNFDBD+PalatinoLinotype TrueType WinAnsi yes yes no 113 0 NNFDBF+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 116 0 NNFDGF+PalatinoLinotype TrueType MacRoman yes yes no 119 0 XIRVNA+EURB10 Type 1 Builtin yes yes yes 126 0 NNFDBB+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 141 0 NNFDBC+PalatinoLinotype CID TrueType Identity-H yes yes yes 144 0 NNFDBD+PalatinoLinotype TrueType WinAnsi yes yes no 150 0 NNFDBF+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 153 0 NNFDGF+PalatinoLinotype TrueType MacRoman yes yes no 156 0 NNFDBB+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 170 0 NNFDBC+PalatinoLinotype CID TrueType Identity-H yes yes yes 173 0 NNFDBD+PalatinoLinotype TrueType WinAnsi yes yes no 179 0 NNFDBF+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 182 0 NNFDGF+PalatinoLinotype TrueType MacRoman yes yes no 185 0 NNFDBB+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 201 0 NNFDBC+PalatinoLinotype CID TrueType Identity-H yes yes yes 204 0 NNFDBD+PalatinoLinotype TrueType WinAnsi yes yes no 210 0 NNFDBF+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 213 0 NNFDGF+PalatinoLinotype TrueType MacRoman yes yes no 216 0 NNFFIJ+PalatinoLinotype,Italic TrueType MacRoman yes yes no 219 0 NNFDBB+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 233 0 NNFDBC+PalatinoLinotype CID TrueType Identity-H yes yes yes 236 0 NNFDBD+PalatinoLinotype TrueType WinAnsi yes yes no 242 0 NNFDBF+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 245 0 NNFDGF+PalatinoLinotype TrueType MacRoman yes yes no 248 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-12-10 02:44:01 CET RepresentationInformation: biomolecules-14-01221-v2.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-12-05 07:04:36 CET Size: 1904297 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 452 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Synthetic Peptides Destination: subsection.2.1 Item: Title: Cell Culture, Transfection, and Treatments Destination: subsection.2.2 Item: Title: Peripheral Blood Samples Destination: subsection.2.3 Item: Title: In Silico Analysis of Peptide Candidates Destination: subsection.2.4 Item: Title: Preparation of Protein Extracts Destination: subsection.2.5 Item: Title: Western Blot Destination: subsection.2.6 Item: Title: Sample Preparation for Proteomic Analyses Destination: subsection.2.7 Item: Title: Internal and External Curve Calibration Preparation Destination: subsection.2.8 Item: Title: nanoLC-MS/MS Destination: subsection.2.9 Children: Item: Title: Database Searches for MS Method Optimization Destination: subsubsection.2.9.1 Item: Title: Database Searches and Data Processing for Absolute Protein Quantification Destination: subsubsection.2.9.2 Item: Title: RT-qPCR Destination: subsection.2.10 Item: Title: Data Analysis Destination: subsection.2.11 Item: Title: Results Destination: section.3 Children: Item: Title: Peptide Selection and PRM Setup Destination: subsection.3.1 Item: Title: Quantification of H1 Subtypes in Human Cell Lines by PRM Destination: subsection.3.2 Item: Title: Quantification of H1 Subtypes from Human Samples Destination: subsection.3.3 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: appendix.A. Info: Title: Quantification of Histone H1 Subtypes Using Targeted Proteomics Author: Jordi López-Gómez, Laura Villarreal, Marta Andrés, Inma Ponte, Blanca Xicoy, Lurdes Zamora, Marta Vilaseca and Alicia Roque Subject: Histone H1 is involved in the regulation of chromatin structure. Human somatic cells express up to seven subtypes. The variability in the proportions of somatic H1s (H1 complement) is one piece of evidence supporting their functional specificity. Alterations in the protein levels of different H1 subtypes have been observed in cancer, suggesting their potential as biomarkers and that they might play a role in disease development. We have developed a mass spectrometry-based (MS) parallel reaction monitoring (PRM) assay suitable for the quantification of H1 subtypes. Our PRM method is based on the quantification of unique peptides for each subtype, providing high specificity. Evaluation of the PRM performance on three human cell lines, HeLa, K562, and T47D, showed high reproducibility and sensitivity. Quantification values agreed with the electrophoretic and Western blot data, indicating the accuracy of the method. We used PRM to quantify the H1 complement in peripheral blood samples of healthy individuals and chronic myeloid leukemia (CML) patients. In CML, the first line of therapy is a tyrosine kinase inhibitor, imatinib. Our preliminary data revealed differences in the H1 complement in CML patients between imatinib responders and non-responders. These results support further research to determine if the H1 content or subtype composition could help predict imatinib response. 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