Inter-Regional Proteomic Profiling of the Human Brain Using an Optimized Protein Extraction Method from Formalin-Fixed Tissue to Identify Signaling Pathways
Proteomics offers vast potential for studying the molecular regulation of the human brain. Formalin fixation is a common method for preserving human tissue; however, it presents challenges for proteomic analysis. In this study, we compared the efficiency of two different protein-extraction buffers o...
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| Format: | Article |
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MDPI AG
2023-02-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/5/4283 |
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| author | Jennilee M. Davidson Stephanie L. Rayner Sidong Liu Flora Cheng Antonio Di Ieva Roger S. Chung Albert Lee |
| author_facet | Jennilee M. Davidson Stephanie L. Rayner Sidong Liu Flora Cheng Antonio Di Ieva Roger S. Chung Albert Lee |
| author_sort | Jennilee M. Davidson |
| collection | DOAJ |
| description | Proteomics offers vast potential for studying the molecular regulation of the human brain. Formalin fixation is a common method for preserving human tissue; however, it presents challenges for proteomic analysis. In this study, we compared the efficiency of two different protein-extraction buffers on three post-mortem, formalin-fixed human brains. Equal amounts of extracted proteins were subjected to in-gel tryptic digestion and LC-MS/MS. Protein, peptide sequence, and peptide group identifications; protein abundance; and gene ontology pathways were analyzed. Protein extraction was superior using lysis buffer containing tris(hydroxymethyl)aminomethane hydrochloride, sodium dodecyl sulfate, sodium deoxycholate, and Triton X-100 (TrisHCl, SDS, SDC, Triton X-100), which was then used for inter-regional analysis. Pre-frontal, motor, temporal, and occipital cortex tissues were analyzed by label free quantification (LFQ) proteomics, Ingenuity Pathway Analysis and PANTHERdb. Inter-regional analysis revealed differential enrichment of proteins. We found similarly activated cellular signaling pathways in different brain regions, suggesting commonalities in the molecular regulation of neuroanatomically-linked brain functions. Overall, we developed an optimized, robust, and efficient method for protein extraction from formalin-fixed human brain tissue for in-depth LFQ proteomics. We also demonstrate herein that this method is suitable for rapid and routine analysis to uncover molecular signaling pathways in the human brain. |
| first_indexed | 2024-03-11T07:23:25Z |
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| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-11T07:23:25Z |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-3fd12b2efb2949c0a9035dfd3554f0f92023-11-17T07:46:16ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-02-01245428310.3390/ijms24054283Inter-Regional Proteomic Profiling of the Human Brain Using an Optimized Protein Extraction Method from Formalin-Fixed Tissue to Identify Signaling PathwaysJennilee M. Davidson0Stephanie L. Rayner1Sidong Liu2Flora Cheng3Antonio Di Ieva4Roger S. Chung5Albert Lee6Centre for Motor Neuron Disease Research, Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, Level 1, 75 Talavera Road, Sydney, NSW 2109, AustraliaCentre for Motor Neuron Disease Research, Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, Level 1, 75 Talavera Road, Sydney, NSW 2109, AustraliaCentre for Health Informatics, Faculty of Medicine, Health and Human Sciences, Macquarie University, 75 Talavera Road, Sydney, NSW 2109, AustraliaCentre for Motor Neuron Disease Research, Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, Level 1, 75 Talavera Road, Sydney, NSW 2109, AustraliaComputational NeuroSurgery (CNS) Lab, Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, Level 1, 75 Talavera Road, Sydney, NSW 2109, AustraliaCentre for Motor Neuron Disease Research, Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, Level 1, 75 Talavera Road, Sydney, NSW 2109, AustraliaCentre for Motor Neuron Disease Research, Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, Level 1, 75 Talavera Road, Sydney, NSW 2109, AustraliaProteomics offers vast potential for studying the molecular regulation of the human brain. Formalin fixation is a common method for preserving human tissue; however, it presents challenges for proteomic analysis. In this study, we compared the efficiency of two different protein-extraction buffers on three post-mortem, formalin-fixed human brains. Equal amounts of extracted proteins were subjected to in-gel tryptic digestion and LC-MS/MS. Protein, peptide sequence, and peptide group identifications; protein abundance; and gene ontology pathways were analyzed. Protein extraction was superior using lysis buffer containing tris(hydroxymethyl)aminomethane hydrochloride, sodium dodecyl sulfate, sodium deoxycholate, and Triton X-100 (TrisHCl, SDS, SDC, Triton X-100), which was then used for inter-regional analysis. Pre-frontal, motor, temporal, and occipital cortex tissues were analyzed by label free quantification (LFQ) proteomics, Ingenuity Pathway Analysis and PANTHERdb. Inter-regional analysis revealed differential enrichment of proteins. We found similarly activated cellular signaling pathways in different brain regions, suggesting commonalities in the molecular regulation of neuroanatomically-linked brain functions. Overall, we developed an optimized, robust, and efficient method for protein extraction from formalin-fixed human brain tissue for in-depth LFQ proteomics. We also demonstrate herein that this method is suitable for rapid and routine analysis to uncover molecular signaling pathways in the human brain.https://www.mdpi.com/1422-0067/24/5/4283human braintissueneuroanatomic regionformalin-fixedproteomicsmethod |
| spellingShingle | Jennilee M. Davidson Stephanie L. Rayner Sidong Liu Flora Cheng Antonio Di Ieva Roger S. Chung Albert Lee Inter-Regional Proteomic Profiling of the Human Brain Using an Optimized Protein Extraction Method from Formalin-Fixed Tissue to Identify Signaling Pathways International Journal of Molecular Sciences human brain tissue neuroanatomic region formalin-fixed proteomics method |
| title | Inter-Regional Proteomic Profiling of the Human Brain Using an Optimized Protein Extraction Method from Formalin-Fixed Tissue to Identify Signaling Pathways |
| title_full | Inter-Regional Proteomic Profiling of the Human Brain Using an Optimized Protein Extraction Method from Formalin-Fixed Tissue to Identify Signaling Pathways |
| title_fullStr | Inter-Regional Proteomic Profiling of the Human Brain Using an Optimized Protein Extraction Method from Formalin-Fixed Tissue to Identify Signaling Pathways |
| title_full_unstemmed | Inter-Regional Proteomic Profiling of the Human Brain Using an Optimized Protein Extraction Method from Formalin-Fixed Tissue to Identify Signaling Pathways |
| title_short | Inter-Regional Proteomic Profiling of the Human Brain Using an Optimized Protein Extraction Method from Formalin-Fixed Tissue to Identify Signaling Pathways |
| title_sort | inter regional proteomic profiling of the human brain using an optimized protein extraction method from formalin fixed tissue to identify signaling pathways |
| topic | human brain tissue neuroanatomic region formalin-fixed proteomics method |
| url | https://www.mdpi.com/1422-0067/24/5/4283 |
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