A Practical and Analytical Comparative Study of Gel-Based Top-Down and Gel-Free Bottom-Up Proteomics Including Unbiased Proteoform Detection
Proteomics is an indispensable analytical technique to study the dynamic functioning of biological systems via different proteins and their proteoforms. In recent years, bottom-up shotgun has become more popular than gel-based top-down proteomics. The current study examined the qualitative and quant...
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| Format: | Article |
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MDPI AG
2023-02-01
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| Online Access: | https://www.mdpi.com/2073-4409/12/5/747 |
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| author | Huriye Ercan Ulrike Resch Felicia Hsu Goran Mitulovic Andrea Bileck Christopher Gerner Jae-Won Yang Margarethe Geiger Ingrid Miller Maria Zellner |
| author_facet | Huriye Ercan Ulrike Resch Felicia Hsu Goran Mitulovic Andrea Bileck Christopher Gerner Jae-Won Yang Margarethe Geiger Ingrid Miller Maria Zellner |
| author_sort | Huriye Ercan |
| collection | DOAJ |
| description | Proteomics is an indispensable analytical technique to study the dynamic functioning of biological systems via different proteins and their proteoforms. In recent years, bottom-up shotgun has become more popular than gel-based top-down proteomics. The current study examined the qualitative and quantitative performance of these two fundamentally different methodologies by the parallel measurement of six technical and three biological replicates of the human prostate carcinoma cell line DU145 using its two most common standard techniques, label-free shotgun and two-dimensional differential gel electrophoresis (2D-DIGE). The analytical strengths and limitations were explored, finally focusing on the unbiased detection of proteoforms, exemplified by discovering a prostate cancer-related cleavage product of pyruvate kinase M2. Label-free shotgun proteomics quickly yields an annotated proteome but with reduced robustness, as determined by three times higher technical variation compared to 2D-DIGE. At a glance, only 2D-DIGE top-down analysis provided valuable, direct stoichiometric qualitative and quantitative information from proteins to their proteoforms, even with unexpected post-translational modifications, such as proteolytic cleavage and phosphorylation. However, the 2D-DIGE technology required almost 20 times as much time per protein/proteoform characterization with more manual work. Ultimately, this work should expose both techniques’ orthogonality with their different contents of data output to elucidate biological questions. |
| first_indexed | 2024-03-11T07:29:00Z |
| format | Article |
| id | doaj.art-aa1ab9f5de674fefb4f04f283ad0a704 |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-11T07:29:00Z |
| publishDate | 2023-02-01 |
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| record_format | Article |
| series | Cells |
| spelling | doaj.art-aa1ab9f5de674fefb4f04f283ad0a7042023-11-17T07:27:37ZengMDPI AGCells2073-44092023-02-0112574710.3390/cells12050747A Practical and Analytical Comparative Study of Gel-Based Top-Down and Gel-Free Bottom-Up Proteomics Including Unbiased Proteoform DetectionHuriye Ercan0Ulrike Resch1Felicia Hsu2Goran Mitulovic3Andrea Bileck4Christopher Gerner5Jae-Won Yang6Margarethe Geiger7Ingrid Miller8Maria Zellner9Centre for Physiology and Pharmacology, Medical University of Vienna, 1090 Vienna, AustriaCentre for Physiology and Pharmacology, Medical University of Vienna, 1090 Vienna, AustriaCentre for Physiology and Pharmacology, Medical University of Vienna, 1090 Vienna, AustriaProteomics Core Facility, Clinical Department of Laboratory Medicine, Medical University of Vienna, 1090 Vienna, AustriaDepartment of Analytical Chemistry, Faculty of Chemistry, University of Vienna, 1090 Vienna, AustriaDepartment of Analytical Chemistry, Faculty of Chemistry, University of Vienna, 1090 Vienna, AustriaCentre for Physiology and Pharmacology, Medical University of Vienna, 1090 Vienna, AustriaCentre for Physiology and Pharmacology, Medical University of Vienna, 1090 Vienna, AustriaInstitute of Medical Biochemistry, University of Veterinary Medicine Vienna, 1210 Vienna, AustriaCentre for Physiology and Pharmacology, Medical University of Vienna, 1090 Vienna, AustriaProteomics is an indispensable analytical technique to study the dynamic functioning of biological systems via different proteins and their proteoforms. In recent years, bottom-up shotgun has become more popular than gel-based top-down proteomics. The current study examined the qualitative and quantitative performance of these two fundamentally different methodologies by the parallel measurement of six technical and three biological replicates of the human prostate carcinoma cell line DU145 using its two most common standard techniques, label-free shotgun and two-dimensional differential gel electrophoresis (2D-DIGE). The analytical strengths and limitations were explored, finally focusing on the unbiased detection of proteoforms, exemplified by discovering a prostate cancer-related cleavage product of pyruvate kinase M2. Label-free shotgun proteomics quickly yields an annotated proteome but with reduced robustness, as determined by three times higher technical variation compared to 2D-DIGE. At a glance, only 2D-DIGE top-down analysis provided valuable, direct stoichiometric qualitative and quantitative information from proteins to their proteoforms, even with unexpected post-translational modifications, such as proteolytic cleavage and phosphorylation. However, the 2D-DIGE technology required almost 20 times as much time per protein/proteoform characterization with more manual work. Ultimately, this work should expose both techniques’ orthogonality with their different contents of data output to elucidate biological questions.https://www.mdpi.com/2073-4409/12/5/747top-down proteomics2D-DIGEbottom-up proteomicsshotgun proteomicsproteoformspost-translational modification (PTM) |
| spellingShingle | Huriye Ercan Ulrike Resch Felicia Hsu Goran Mitulovic Andrea Bileck Christopher Gerner Jae-Won Yang Margarethe Geiger Ingrid Miller Maria Zellner A Practical and Analytical Comparative Study of Gel-Based Top-Down and Gel-Free Bottom-Up Proteomics Including Unbiased Proteoform Detection Cells top-down proteomics 2D-DIGE bottom-up proteomics shotgun proteomics proteoforms post-translational modification (PTM) |
| title | A Practical and Analytical Comparative Study of Gel-Based Top-Down and Gel-Free Bottom-Up Proteomics Including Unbiased Proteoform Detection |
| title_full | A Practical and Analytical Comparative Study of Gel-Based Top-Down and Gel-Free Bottom-Up Proteomics Including Unbiased Proteoform Detection |
| title_fullStr | A Practical and Analytical Comparative Study of Gel-Based Top-Down and Gel-Free Bottom-Up Proteomics Including Unbiased Proteoform Detection |
| title_full_unstemmed | A Practical and Analytical Comparative Study of Gel-Based Top-Down and Gel-Free Bottom-Up Proteomics Including Unbiased Proteoform Detection |
| title_short | A Practical and Analytical Comparative Study of Gel-Based Top-Down and Gel-Free Bottom-Up Proteomics Including Unbiased Proteoform Detection |
| title_sort | practical and analytical comparative study of gel based top down and gel free bottom up proteomics including unbiased proteoform detection |
| topic | top-down proteomics 2D-DIGE bottom-up proteomics shotgun proteomics proteoforms post-translational modification (PTM) |
| url | https://www.mdpi.com/2073-4409/12/5/747 |
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