132 CHAPTER 5 TISSUE BIOPSY COLLECTION, LYSIS AND PROTEIN DIGESTION Normal liver tissue biopsies were collected from five patients with cancer who underwent liver metastasectomy at Amsterdam UMC location VUmc in September 2019. Since the Dutch Medical Research Involving Human Subjects Act does not apply to normal adjacent tissue that is removed, this tissue could be used for research purposes; patients have the possibility to opt-out of the use of their residual tissue for future research. For each patient and immediately after resection, six 14 gauge core needle biopsies of adjacent normal liver tissue were taken from the resection specimen by the surgeon, placed into separate aluminum vials and snap frozen within 5 minutes. After below mentioned freezing procedures, biopsies were longitudinally cut in 10 μm sections (cryomicrotome, Leica CM1850) and processed to tumor lysates for mass spectrometry (MS)-based global phopshoproteomics as described elsewhere14,15. Lysates were stored at -80 °C. BENCHMARKING PERFORMANCE SNAP FREEZER VERSUS LIQUID NITROGEN QUENCHING Three triplicates of 5-10 ml K562 suspension cell line, each corresponding to 500 µg of protein, and 3-9 normal liver tissue biopsies per patient were snap frozen in aluminum vials by one of the following three methods: (i) cooling to -196 °C by immersion in LN2 (golden standard), (ii) cooling to -73°C in the snap freezer, and (iii) storage at room temperature for 2 hours, followed by immersion in LN2 to -196 °C (+2hr positive control). -73° C (200K) is in general accepted as an adequate temperature to preserve stability of biospecimens for storage16,17. Before start of the experiments, a vessel filled with LN2 was placed in the laboratory and the electrically powered snap freezer was pre-cooled to -73°C (200K). In each experiment, one vial was placed into the snap freezer and simultaneously another vial was immersed in LN2, alternatingly performed for the 2 tissue replicates or 3 cell suspension workflow replicates (Figure 1). After cooling of the vials, all vials in the experiment were transported in LN2 and stored in a freezer at -80 °C until further use.
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