DIA label-free proteomic analysis of murine bone-marrow-derived macrophages
Summary: Here, we describe an optimized protocol to analyze murine bone-marrow-derived macrophages using label-free data-independent acquisition (DIA) proteomics. We provide a complete step-by-step protocol describing sample preparation utilizing the S-Trap approach for on-column digestion and pepti...
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2022-12-01
|
| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166722006050 |
| _version_ | 1828165832075640832 |
|---|---|
| author | Christa P. Baker Iain R. Phair Alejandro J. Brenes Abdelmadjid Atrih Dylan G. Ryan Roland Bruderer Albena T. Dinkova-Kostova Douglas J. Lamont J. Simon C. Arthur Andrew J.M. Howden |
| author_facet | Christa P. Baker Iain R. Phair Alejandro J. Brenes Abdelmadjid Atrih Dylan G. Ryan Roland Bruderer Albena T. Dinkova-Kostova Douglas J. Lamont J. Simon C. Arthur Andrew J.M. Howden |
| author_sort | Christa P. Baker |
| collection | DOAJ |
| description | Summary: Here, we describe an optimized protocol to analyze murine bone-marrow-derived macrophages using label-free data-independent acquisition (DIA) proteomics. We provide a complete step-by-step protocol describing sample preparation utilizing the S-Trap approach for on-column digestion and peptide purification. We then detail mass spectrometry data acquisition and approaches for data analysis. Single-shot DIA protocols achieve comparable proteomic depth with data-dependent MS approaches without the need for fractionation. This allows for better scaling for large sample numbers with high inter-experimental reproducibility.For complete details on the use and execution of this protocol, please refer to Ryan et al. (2022). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| first_indexed | 2024-04-12T01:51:34Z |
| format | Article |
| id | doaj.art-55dbb36cd7934934bcf60bd53c5f3bf4 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-04-12T01:51:34Z |
| publishDate | 2022-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-55dbb36cd7934934bcf60bd53c5f3bf42022-12-22T03:52:56ZengElsevierSTAR Protocols2666-16672022-12-0134101725DIA label-free proteomic analysis of murine bone-marrow-derived macrophagesChrista P. Baker0Iain R. Phair1Alejandro J. Brenes2Abdelmadjid Atrih3Dylan G. Ryan4Roland Bruderer5Albena T. Dinkova-Kostova6Douglas J. Lamont7J. Simon C. Arthur8Andrew J.M. Howden9School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, ScotlandDivision of Cellular and Systems Medicine, School of Medicine, University of Dundee, Ninewells Hospital and Medical School, James Arrott Drive, Dundee DD1 9SY, ScotlandSchool of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, ScotlandSchool of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, ScotlandMRC Mitochondrial Biology Unit, University of Cambridge, Cambridge CB2 0XY, ScotlandBiognosys, Schlieren, Zurich 8952, SwitzerlandDivision of Cellular and Systems Medicine, School of Medicine, University of Dundee, Ninewells Hospital and Medical School, James Arrott Drive, Dundee DD1 9SY, ScotlandSchool of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, ScotlandSchool of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland; Corresponding authorSchool of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland; Corresponding authorSummary: Here, we describe an optimized protocol to analyze murine bone-marrow-derived macrophages using label-free data-independent acquisition (DIA) proteomics. We provide a complete step-by-step protocol describing sample preparation utilizing the S-Trap approach for on-column digestion and peptide purification. We then detail mass spectrometry data acquisition and approaches for data analysis. Single-shot DIA protocols achieve comparable proteomic depth with data-dependent MS approaches without the need for fractionation. This allows for better scaling for large sample numbers with high inter-experimental reproducibility.For complete details on the use and execution of this protocol, please refer to Ryan et al. (2022). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166722006050BioinformaticsImmunologyProtein biochemistryProteomicsMass spectrometry |
| spellingShingle | Christa P. Baker Iain R. Phair Alejandro J. Brenes Abdelmadjid Atrih Dylan G. Ryan Roland Bruderer Albena T. Dinkova-Kostova Douglas J. Lamont J. Simon C. Arthur Andrew J.M. Howden DIA label-free proteomic analysis of murine bone-marrow-derived macrophages STAR Protocols Bioinformatics Immunology Protein biochemistry Proteomics Mass spectrometry |
| title | DIA label-free proteomic analysis of murine bone-marrow-derived macrophages |
| title_full | DIA label-free proteomic analysis of murine bone-marrow-derived macrophages |
| title_fullStr | DIA label-free proteomic analysis of murine bone-marrow-derived macrophages |
| title_full_unstemmed | DIA label-free proteomic analysis of murine bone-marrow-derived macrophages |
| title_short | DIA label-free proteomic analysis of murine bone-marrow-derived macrophages |
| title_sort | dia label free proteomic analysis of murine bone marrow derived macrophages |
| topic | Bioinformatics Immunology Protein biochemistry Proteomics Mass spectrometry |
| url | http://www.sciencedirect.com/science/article/pii/S2666166722006050 |
| work_keys_str_mv | AT christapbaker dialabelfreeproteomicanalysisofmurinebonemarrowderivedmacrophages AT iainrphair dialabelfreeproteomicanalysisofmurinebonemarrowderivedmacrophages AT alejandrojbrenes dialabelfreeproteomicanalysisofmurinebonemarrowderivedmacrophages AT abdelmadjidatrih dialabelfreeproteomicanalysisofmurinebonemarrowderivedmacrophages AT dylangryan dialabelfreeproteomicanalysisofmurinebonemarrowderivedmacrophages AT rolandbruderer dialabelfreeproteomicanalysisofmurinebonemarrowderivedmacrophages AT albenatdinkovakostova dialabelfreeproteomicanalysisofmurinebonemarrowderivedmacrophages AT douglasjlamont dialabelfreeproteomicanalysisofmurinebonemarrowderivedmacrophages AT jsimoncarthur dialabelfreeproteomicanalysisofmurinebonemarrowderivedmacrophages AT andrewjmhowden dialabelfreeproteomicanalysisofmurinebonemarrowderivedmacrophages |