134 CHAPTER 5 PHOSPHOPROTEOMICS: PHOSPHOPEPTIDE ENRICHMENT, LC-MS/ MS MEASUREMENT, PROTEIN IDENTIFICATION AND LABEL-FREE PHOSPHOPEPTIDE QUANTIFICATION K562 and HCT116 cell lysate aliquots and tissue lysates were reduced, alkylated and digested as described previously14. Desalted peptides were enriched for phosphopeptides using titanium oxide (TiOx) beads based using aliphatic hydroxy-acid modified metal oxide chromatography21,22. Further sample preparation details are provided in Supplementary Methods. Phosphopeptides were separated by nanoLC and detected as described previously 21,23,24 on a Q exactive HF mass spectrometer (Thermo Fisher, Bremen, Germany). Protein identification and phosphopeptide quantification were performed as previously described14. In short, LC-MS/ MS spectra were searched against the Uniprot human reference proteome FASTA file (release February 2019, 42417 entries, no fragments) using MaxQuant 1.6.4.025. (Phospho)peptide identifications were propagated across samples using the match-between-runs option checked. Searches were performed as previously described in detail with the label-free quantification option selected24. Phosphopeptides were quantified by their extracted ion chromatograms (‘Intensity’ in MaxQuant). For each sample the phosphopeptide intensities were normalized on the median intensity of all identified peptides in the sample (‘normalized intensity’ from the MaxQuant Evidence table). Further details are provided in the Supplementary Methods. RNA EXTRACTION AND INTEGRITY, RNA SEQUENCING Tissue: Dissection of fresh frozen biopsies was performed at −25°C in a cryotome. Twenty micrometer sections were cut and snap frozen in liquid nitrogen and stored at −80°C until RNA extraction. RNA was isolated from the tissue specimens and the surplus of K562 cell suspension samples used for the phosphoproteomics analysis, using the RNeasy Plus Mini K (Qiagen) according to the manufacturers protocol, eluted in 30 µl nuclease free water and quantified using a NanoDrop One UV-Vis Spectrophotometer (Thermo Scientific). To analyze differences in RNA integrity between samples processed in different freezing conditions, the RNA Integrity Number (RIN) was determined using the RNA 6000 Picochip (Bioanalyzer 2100, Agilent). The Bioanalyzer 2100 quality and quantity measures were collected from the automatically generated Bioanalyzer result reports using default settings. Next generation sequencing (NGS) using Illumina’s TruSeq Small RNA Sample Preparation protocol and data filtering were performed as previously described26. Illumina’s TruSeq Small RNA Sample Preparation protocol was used for the generation of cDNA libraries. These libraries were amplified on the flow cells with Illumina’s cluster station (Illumina Inc, San Diego, CA, USA) and sequenced using Illumina’s HiSeq 2000 (Illumina Inc, San Diego, CA, USA). Further details are provided in Supplementary Methods.
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