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Comparison of different digestion methods for proteomic analysis of isolated cells and FFPE tissue samples

A. Pirog, J. Faktor, Z. Urban-Wojciuk, S. Kote, E. Chruściel, Ł. Arcimowicz, N. Marek-Trzonkowska, B. Vojtesek, TR. Hupp, S. Al Shboul, PM. Brennan, RT. Smoleński, DR. Goodlett, I. Dapic

. 2021 ; 233 (-) : 122568. [pub] 20210528

Language English Country Netherlands

Document type Journal Article

Persistent link   https://www.medvik.cz/link/bmc21024937

Proteomics of human tissues and isolated cellular subpopulations create new opportunities for therapy and monitoring of a patients' treatment in the clinic. Important considerations in such analysis include recovery of adequate amounts of protein for analysis and reproducibility in sample collection. In this study we compared several protocols for proteomic sample preparation: i) filter-aided sample preparation (FASP), ii) in-solution digestion (ISD) and iii) a pressure-assisted digestion (PCT) method. PCT method is known for already a decade [1], however it is not widely used in proteomic research. We assessed protocols for proteome profiling of isolated immune cell subsets and formalin-fixed paraffin embedded (FFPE) tissue samples. Our results show that the ISD method has very good efficiency of protein and peptide identification from the whole proteome, while the FASP method is particularly effective in identification of membrane proteins. Pressure-assisted digestion methods generally provide lower numbers of protein/peptide identifications, but have gained in popularity due to their shorter digestion time making them considerably faster than for ISD or FASP. Furthermore, PCT does not result in substantial sample loss when applied to samples of 50 000 cells. Analysis of FFPE tissues shows comparable results. ISD method similarly yields the highest number of identifications. Furthermore, proteins isolated from FFPE samples show a significant reduction of cleavages at lysine sites due to chemical modifications with formaldehyde-such as methylation (+14 Da) being among the most common. The data we present will be helpful for making decisions about the robust preparation of clinical samples for biomarker discovery and studies on pathomechanisms of various diseases.

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$a Proteomics of human tissues and isolated cellular subpopulations create new opportunities for therapy and monitoring of a patients' treatment in the clinic. Important considerations in such analysis include recovery of adequate amounts of protein for analysis and reproducibility in sample collection. In this study we compared several protocols for proteomic sample preparation: i) filter-aided sample preparation (FASP), ii) in-solution digestion (ISD) and iii) a pressure-assisted digestion (PCT) method. PCT method is known for already a decade [1], however it is not widely used in proteomic research. We assessed protocols for proteome profiling of isolated immune cell subsets and formalin-fixed paraffin embedded (FFPE) tissue samples. Our results show that the ISD method has very good efficiency of protein and peptide identification from the whole proteome, while the FASP method is particularly effective in identification of membrane proteins. Pressure-assisted digestion methods generally provide lower numbers of protein/peptide identifications, but have gained in popularity due to their shorter digestion time making them considerably faster than for ISD or FASP. Furthermore, PCT does not result in substantial sample loss when applied to samples of 50 000 cells. Analysis of FFPE tissues shows comparable results. ISD method similarly yields the highest number of identifications. Furthermore, proteins isolated from FFPE samples show a significant reduction of cleavages at lysine sites due to chemical modifications with formaldehyde-such as methylation (+14 Da) being among the most common. The data we present will be helpful for making decisions about the robust preparation of clinical samples for biomarker discovery and studies on pathomechanisms of various diseases.
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$a Urban-Wojciuk, Zuzanna $u International Centre for Cancer Vaccine Science, University of Gdansk, Kładki 24, 80-822, Gdańsk, Poland
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$a Kote, Sachin $u International Centre for Cancer Vaccine Science, University of Gdansk, Kładki 24, 80-822, Gdańsk, Poland
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$a Chruściel, Elżbieta $u International Centre for Cancer Vaccine Science, University of Gdansk, Kładki 24, 80-822, Gdańsk, Poland
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$a Arcimowicz, Łukasz $u International Centre for Cancer Vaccine Science, University of Gdansk, Kładki 24, 80-822, Gdańsk, Poland
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$a Marek-Trzonkowska, Natalia $u International Centre for Cancer Vaccine Science, University of Gdansk, Kładki 24, 80-822, Gdańsk, Poland; Laboratory of Immunoregulation and Cellular Therapies, Department of Family Medicine, Medical University of Gdańsk, Dębinki 2, 80-210, Gdańsk, Poland
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$a Vojtesek, Borek $u Research Centre for Applied Molecular Oncology (RECAMO), Masaryk Memorial Cancer Institute, 656 53, Brno, Czech Republic
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$a Hupp, Ted R $u International Centre for Cancer Vaccine Science, University of Gdansk, Kładki 24, 80-822, Gdańsk, Poland; Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, Scotland, EH4 2XR, United Kingdom
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$a Al Shboul, Sofian $u Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, Scotland, EH4 2XR, United Kingdom; Department of Basic Medical Sciences, Faculty of Medicine, The Hashemite University, Zarqa, Jordan
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$a Brennan, Paul M $u Translational Neurosurgery, Centre for Clinical Brain Sciences, Bioquarter, University of Edinburgh, Edinburgh, UK
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$a Smoleński, Ryszard Tomasz $u Department of Biochemistry, Medical University of Gdansk, Dębinki 1, 80-210, Gdańsk, Poland
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$a Goodlett, David R $u International Centre for Cancer Vaccine Science, University of Gdansk, Kładki 24, 80-822, Gdańsk, Poland; Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, V8P 5C2, Canada
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$a Dapic, Irena $u International Centre for Cancer Vaccine Science, University of Gdansk, Kładki 24, 80-822, Gdańsk, Poland. Electronic address: irena.dapic@ug.edu.pl
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