MS results for identifying SPINK1-interacting partners
Description
Raw data of mass spectrometry (MS) experiments in "SPINK1-COL18A1 Crosstalk Shapes Epigenome and Drives Cancer Stemness in Pancreatic Ductal Adenocarcinoma" project. A method of proximity dependent biotinylation with UltraID was used in PANC-1 cell line. PANC-1 cells overexpressing SPINK1-UltraID (plasmid plentiSFFV-hSPINK1-UltraID) or Ultra Ctrl (plasmid plentiSFFV-hSPINK1SP-UltraID-ALFA) were allowed to grow to ~90% confluency in 150 mm Petri dish. On the day of the experiment, cells were incubated in 50 µM biotin for 1h at 37℃. Then the biotinylated proteins were pull-down and digested on beads before MS. Species: Homo sapiens Tissue: Cell culture Cell type: Permanent cell line cell (PANC-1 cell line) Disease: Pancreatic ductal adenocarcinoma Subcellular: Whole cell lysate Method: On-beads digestion Digestion Enzyme: Trypsin Instrument Type: Affinity purification (AP-MS) Instrument: Q Executive HF-X Data was analyzed using Proteome Discoverer (version 2.5)(Thermo Scientific) software and searched in-house using the Mascot algorithm (version 2.8.3) (Matrix Science). Scaffold v5.1.2 (Proteome Software Inc.) was used to validate MS/MS based peptide and protein identifications. Ultra Ctrl (control group) associated file names: HFX24-1103_Shang_20240313_Control_Digestion.raw; HFX24-1103_Shang_20240313_Control_Digestion.mzid_HFX24-1103_Shang_20240313_Control_Digestion.MGF; HFX24-1103_Shang_20240313_Control_Digestion.mzid SPINK1-UltraID (experiment group) associated file names: HFX24-1108_Shang_20240313_SPINK1-UltraID_Digestion; HFX24-1108_Shang_20240313_SPINK1-UltraID_Digestion.mzid_HFX24-1108_Shang_20240313_SPINK1-UltraID_Digestion.MGF; HFX24-1108_Shang_20240313_SPINK1-UltraID_Digestion.mzid
Files
Steps to reproduce
All micro centrifuge tubes used in this section were low-protein binding (Thermo Fisher, Cat# 90140). PANC-1 cells overexpressing SPINK1-UltraID (plasmid plentiSFFV-hSPINK1-UltraID) or Ultra Ctrl (plasmid plentiSFFV-hSPINK1SP-UltraID-ALFA) were allowed to grow to ~90% confluency in 150 mm Petri dish. On the day of the experiment, cells were incubated in 50 µM biotin for 1h at 37℃. After incubation, cells were washed three times with 10 mL DPBS. 1mL ice-cold lysis buffer (RIPA + 1X HaltTM protease inhibitor cocktail (Thermo Fisher, Cat# 78429)) was then added for each 150 mm Petri dish and spread by a cell scraper. The resulting lysate from each plate was then transferred into a micro centrifuge tube and incubated on ice for 30 min with periodic vortexing every 10 min. The lysate was then centrifuged at 13,000g for 10 min at 4℃ and the supernatant was transferred into a new tube. The protein concentration of each sample was then determined by BCA assay and adjusted to the same. The lysate from each Petri dish was then incubated with 50 µL trypsin-resistant streptavidin beads (MagReSyn, Cat# MR-STP002) at 4℃ with gentle shaking overnight. The beads were then pulled down from the lysate and washed at room temperature twice with 1mL lysis buffer, once with 1mL 1M KCl, once with 0.9 mL 0.1M Na2CO3, once with 0.9 mL 2 M urea in 50 mM ammonium bicarbonate (ABC) buffer and twice with 1mL lysis buffer. After the final wash, beads were transferred to a new centrifuge tube with 0.5 mL lysis buffer. Beads were then washed three times with 1 mL 50 mM ABC buffer and resuspended in 200 µL ABC buffer. 10 µL of the beads were aliquoted for quality control. 2 µL 0.5 M TCEP (Thermo Fisher, Cat# 77720) was added to the remaining beads in each centrifuge and vortexed to mix. 10 µL freshly prepared 200 mM 2-chloroacetamide (Sigma-Aldrich, Cat# C0267-100G) in 50 mM ABC buffer was then added to each centrifuge tube and vortexed to mix. The reaction mixture was shaken in dark at room temperature for 1 h. Beads were then pulled down and washed once with 1 mL 50 mM ABC buffer and resuspended in 200 µL 50 mM ABC buffer containing 250 ng LysC/Trypsin (Promega, Cat# V5071). The reaction mixture was then incubated with gentle sharking overnight. Beads were the pulled down and the supernatant was collected and mixed with 25 µL 10% formic acid to quench trypsin digestion. All supernatants were then sent on ice to the Mass Spectrometry (MS) & Proteomics Resource of the W.M. Keck Foundation Biotechnology Resource Laboratory at Yale University for analysis by mass spectrometry.
Institutions
- Yale University