139 A novel liquid nitrogen-free snap freezer vials with shortest vs longest (2 vs 25 seconds) freezing time, polypropylene vials frozen in LN2 vs isopentane were selected for molecular analysis by MS-based global phosphoproteomics. This was successfully performed in five out of six samples. One LN2-cooled sample was lost due to a technical error in the mass spectrometer. A total of 8597 unique peptides were identified of which 5726 (66.6 %) were phosphorylated, reflecting adequate enrichment for phosphopeptides. The median number of identified phosphopeptides per sample (500 μg protein input/sample) was 4668 (range 4035 - 4780). A total of 5643 unique phosphosites were identified (phosphorylated in 87% at serine residues, 12% threonine and 1% tyrosine), with a median of 4127 (range 3765 - 4251) phosphosites per sample. Unsupervised clustering of the global phosphoproteome did not separate HCT116 samples frozen in polypropylene vials of 2 seconds versus 25 seconds freezing rates (Figure 4A). Fifty-one percent of all identified phosphopeptides were present in all 5 samples and only ≤ 1.6% were uniquely identified in one of the samples; 47-48% of identified phosphopeptides per sample were present in at least one other sample (Figure 4B). The overlap between workflow replicates (47% for LN2 and 51% for isopentane, data not shown) was comparable to the overlap between the different conditions (51% as per the Venn diagram in Figure 4B). The correlation between all samples was high (Pearson’s r 0.93 – 0.99, Figure 4C). Table 2. Three different types of vials containing HCT116 lysate were immersed in either liquid nitrogen (-196 °C) or pre-cooled isopentane (-80 °C) (Figure 2). The time in seconds (s) elapsed from the point of room temperature until the vials reached a temperature of -80 °C. Three technical replicates per freezing condition were used. Vial type Liquid nitrogen Precooled isopentane Aluminum 4 s 10 s Polypropylene 2 s 25 s Aluminum covered in paper tape < 2 s N/A 5
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