135 A novel liquid nitrogen-free snap freezer RESULTS BENCHMARKING OF SNAP FREEZER VERSUS LIQUID NITROGEN QUENCHING IN MOLECULAR PROFILING Cancer cell line samples Using a snap freezer at -73°C and the cold sink temperature of LN2 13, a comparative analysis of the phosphoproteome and transcriptome of suspension cell line K562 was performed (Figure 1A). Mass spectrometry-based global phosphoproteomics was successfully performed on all nine (3 triplicates) K562 cell suspension lysate samples. A total of 16,341 unique peptides were identified of which 14,835 (90.8%) were phosphorylated. The median number of phosphopeptides per sample was 10,357 (range 9,317 – 10,735). The number of identified phosphopeptides did not differ significantly between both freezing methods (p = 0.44 by students’ t-test). A total of 14,812 unique phosphosites were identified (83.4% serine, 15.2% threonine and 1.4% tyrosine), with a median of 9,502 (range 8,306 – 9,871) per sample. Unsupervised cluster analysis of phosphosites did not show separation of samples processed in LN2 from samples processed in the snap freezer (Figure 2A). Comparison of the nine study samples with each other showed high Pearson correlations (median r 0.96 (range 0.92-0.98) for either direct freezing method) while the positive control samples with 2 additional hours of bench-time did cluster separately. (Supplementary Figure 1A); 4,789 phosphopeptides (29% of total number of identified phosphopeptides) were shared between all samples (Figure 2B). Next, a read-out at the transcriptomic level was used to compare LN2- versus snap freezer-based biospecimen freezing. No significant difference was observed in RNA integrity between cell line samples processed using the two freezing methods, including the +2hr positive controls, indicating that integrity of the RNA molecules is preserved by the snap freezer (Table 1). Also, RNA molecules were shown to be stable after 2 hours at room temperature (Table 1). Unsupervised cluster analysis of the 100 most variably expressed genes showed two main clusters, one smaller consisting of the three positive control samples; the second cluster was a mixed cluster of samples processed using either method (Figure 2C). The two snap freezing methods could not be distinguished based on the RNA expression profiles, even when selecting only the top 100 varying genes between the samples for clustering analysis. Again, comparison of all separate samples with each other showed very high correlation (Pearson’s r >0.99, Supplementary Figure 1B). 5
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