MassIVE MSV000094028

Description

Description Cells were cultured as described above and synchronized in G2/M phase by incubating them for 24 hours with 2mM thymidine and, subsequently, for 12 hours with STLC 10uM. From each culture, 106 cells were collected by mitotic shake off, centrifuged and pellets were washed twice with PBS. Cells were lysed in 200 ul lysis buffer (2% sodium deoxycholate (SDC), 0.1 M ammoniumbicarbonate) using strong ultra-sonication (two cycles of sonication S3 for 10 seconds, Hielscher Ultrasonicator). Protein concentration was determined by BCA assay (Thermo Fisher Scientific) using a small sample aliquot. 50ug of proteins were digested as described previously (Ahrne et al., 2016), reduced with 5 mM TCEP for 15 min at 95 C and alkylated with 10 mM iodoacetamide for 30 min in the dark at 25 C. After diluting samples with 100 mM ammonium bicarbonate buffer to a final DOC concentration of 1%, proteins were digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, Wisconsin) overnight at 37C. Then, the samples were acidified with 2 M HCl to a final concentration of 50 mM, incubated for 15 min at 37 C and the precipitated detergent removed by centrifugation at 10,000xg for 15 min. Subsequently, peptides were desalted on C18 reversed-phase spin columns according to the manufacturers instructions (Microspin, Harvard Apparatus) and dried under vacuum. The dried peptide samples were subsequently labeled with isobaric tags (TMT 10plex, Thermo Fisher Scientific) according to the manufacturers instructions. To control for ratio distortion during quantification, a peptide calibration mixture consisting of six digested standard proteins mixed in different amounts was added to each sample before TMT labeling as recently described (Ahrne et al., 2016). After pooling the TMT labeled peptide samples, peptides were again desalted on C18 reversed-phase spin columns according to the manufacturers instructions (Macrospin, Harvard Apparatus) and dried under vacuum. TMT-labeled peptides were fractionated by high-pH reversed phase separation using a XBridge Peptide BEH C18 column (3,5 um, 130 A, 1 mm x 150 mm, Waters) on an Agilent 1260 Infinity HPLC system. Peptides were loaded onto the column in buffer A (ammonium formate (20 mM, pH 10) in water) and eluted using a two-step linear gradient starting from 2% to 10% in 5 minutes and then to 50% (v/v) buffer B (90% acetonitrile / 10% ammonium formate (20 mM, pH 10) over 55 minutes at a flow rate of 42 ul/min. Elution of peptides was monitored with a UV detector (215 nm, 254 nm). A total of 36 fractions were collected, pooled into 12 fractions using a post-concatenation strategy as previously described (Wang et al., 2011), dried under vacuum and subjected to LC-MS/MS analysis. [doi:10.25345/C5NC5SP9X] [dataset license: CC0 1.0 Universal (CC0 1.0)] [doi:10.25345/C5SB3X89B] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: aneuploid cells ; TMT

Contact