A Non-Hazardous Deparaffinization Protocol Enables Quantitative Proteomics of Core Needle Biopsy-Sized Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Specimens.

Autor: Mitsa G; Division of Experimental Medicine, McGill University, Montreal, QC H4A 3J1, Canada.; Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC H3T 1E2, Canada., Guo Q; Division of Experimental Medicine, McGill University, Montreal, QC H4A 3J1, Canada.; Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montreal, QC H4A 3T2, Canada., Goncalves C; Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montreal, QC H4A 3T2, Canada., Preston SEJ; Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montreal, QC H4A 3T2, Canada., Lacasse V; Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC H3T 1E2, Canada.; Research Pathology Core Facility, Department of Pathology, Segal Cancer Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC H3T 1E2, Canada., Aguilar-Mahecha A; Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montreal, QC H4A 3T2, Canada., Benlimame N; Research Pathology Core Facility, Department of Pathology, Segal Cancer Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC H3T 1E2, Canada., Basik M; Division of Experimental Medicine, McGill University, Montreal, QC H4A 3J1, Canada.; Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montreal, QC H4A 3T2, Canada., Spatz A; Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montreal, QC H4A 3T2, Canada.; Research Pathology Core Facility, Department of Pathology, Segal Cancer Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC H3T 1E2, Canada., Batist G; Division of Experimental Medicine, McGill University, Montreal, QC H4A 3J1, Canada.; Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montreal, QC H4A 3T2, Canada.; Exactis Innovation, 5450 Cote-des-Neiges, Suite 522, Montreal, QC H3T 1Y6, Canada., Miller WH Jr; Division of Experimental Medicine, McGill University, Montreal, QC H4A 3J1, Canada.; Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montreal, QC H4A 3T2, Canada.; Rossy Cancer Network, McGill University, Montreal, QC H3H 1E8, Canada., Del Rincon SV; Division of Experimental Medicine, McGill University, Montreal, QC H4A 3J1, Canada.; Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montreal, QC H4A 3T2, Canada., Zahedi RP; Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC H3T 1E2, Canada., Borchers CH; Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC H3T 1E2, Canada.; Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montreal, QC H4A 3T2, Canada.
Jazyk: angličtina
Zdroj: International journal of molecular sciences [Int J Mol Sci] 2022 Apr 18; Vol. 23 (8). Date of Electronic Publication: 2022 Apr 18.
DOI: 10.3390/ijms23084443
Abstrakt: Most human tumor tissues that are obtained for pathology and diagnostic purposes are formalin-fixed and paraffin-embedded (FFPE). To perform quantitative proteomics of FFPE samples, paraffin has to be removed and formalin-induced crosslinks have to be reversed prior to proteolytic digestion. A central component of almost all deparaffinization protocols is xylene, a toxic and highly flammable solvent that has been reported to negatively affect protein extraction and quantitative proteome analysis. Here, we present a 'green' xylene-free protocol for accelerated sample preparation of FFPE tissues based on paraffin-removal with hot water. Combined with tissue homogenization using disposable micropestles and a modified protein aggregation capture (PAC) digestion protocol, our workflow enables streamlined and reproducible quantitative proteomic profiling of FFPE tissue. Label-free quantitation of FFPE cores from human ductal breast carcinoma in situ (DCIS) xenografts with a volume of only 0.79 mm 3 showed a high correlation between replicates (r 2 = 0.992) with a median %CV of 16.9%. Importantly, this small volume is already compatible with tissue micro array (TMA) cores and core needle biopsies, while our results and its ease-of-use indicate that further downsizing is feasible. Finally, our FFPE workflow does not require costly equipment and can be established in every standard clinical laboratory.
Databáze: MEDLINE
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