DOI: 10.5281/zenodo.16731901
title: Proteomic sequencing data of human pancreatic cancer cell lines

description: This dataset originates from the proteomic sequencing component of a multi-omics analysis of human pancreatic cancer cell lines, aimed at identifying key proteins that influence pancreatic cancer metastasis.

 

Protein Extraction and Digestion

Samples were suspended in lysis buffer (1% sodium deoxycholate, 8 M urea, 1x Protease Inhibitor Cocktail (Roche Ltd. Basel, Switzerland)). The mixture was allowed to vortex to mix well and treated by ultrasound for 2 min. Then the mixture was allowed to settle at 4 ℃ for 30 min during which the sample was vortexed at every 10 min. After centrifugation at 12,000 g at 4 °C for 20 min, the concentration of protein supernatant was determined by Bicinchoninic acid (BCA) method by BCA Protein Assay Kit. The   concentrations of the configured standard were 0, 0.2, 0.4, 0.8, 1.2, 1.6, 2 mg / ml, and 150 μL BCA working solution was added respectively. Electrophoresis and Coomassie brilliant blue staining were performed after configuring SDS-PAGE glue.100 μg protein was taken from each sample and adjusted to 100 μL constant volume with 8 M urea. Then, 2 μL 0.5 M Tris (2-carboxyethyl) phosphine (TCEP) was added and reacted at 37 ℃ for 1 h and alkylated with 4 μL 1 M iodoacetamide (IAM) at room temperature in the dark. According to the ratio of 1:5, cold acetone was added to precipitate overnight at -20 ℃. After high-speed centrifugation (12000 g, 20 min, 4 ℃), the supernatant was collected. This step was repeated twice. Samples were then resolubilized in 100 μL 100 mM TEAB and trypsin (Promega, Madison, WI) was added, followed by overnight digestion. At last, samples were desalted using a C18 Zip Tip, and the peptides were quantified by Pierce TM Quantitative Colorimetric Peptide Assay (23275) and lyophilized by SpeedVac.

High PH Reverse Phase Separation

The peptide mixture was redissolved in buffer A (buffer A: 20 mM ammonium formate in water, adjusted to pH 10.0 with ammonium hydroxide), and then fractionated using Ultimate 3000 system (ThermoFisher Scientific, MA, USA) connected to a reverse-phase column (XBridge C18 column, 4.6mm x 250 mm, 5μm, (Waters Corporation, MA, USA)). High pH separation was performed using a linear gradient from 5% B to 45% B in 40 min (B: 80% ACN with 20 mM ammonium formate, adjusted to pH 10.0 with ammonium hydroxide). The column was equilibrated for 15 min under the initial conditions, the flow rate was maintained at 1 mL/min and the column temperature was maintained at 30 ℃. Ten fractions were collected. The fractions were dried in a vacuum concentrator for use.Data-dependent Acquisition (DDA): nano-HPLC-MS/MS AnalysisThe desalted lyophilized peptides were redissolved in solvent A (A: 0.1% formic acid in water) and analyzed by LC-MS/MS with an online nanojet ion source. The complete system is an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, MA, USA) in tandem with an EASY-nLC 1200 system. The Orbitrap Fusion Lumos mass spectrometer was operated under data-dependent acquisition mode, automatically switching between MS and MS/MS mode. The MS parameters were set as follows: (1) MS: Scan range (m/z)=350–1500; Resolution=120,000; AGC target=400,000; Maximum injection time=50 ms; Dynamic exclusion duration=30 s; (2) HCD-MS/MS: Resolution=15,000; AGC target=50,000; Maximum injection time=35 ms; Collision energy=32.

Database Search

Raw data were processed through Spectronaut X (Biognosys AG, Switzerland) with default parameters. The database was set up for Trypsin digestion using Uniprot. Library search parameters fixed modification: Carbamidomethyl (C), variable modification: methionine oxidation (M)  . The false positive rate (FDR) was set to 1% for both parent ion and peptide levels.

Data-independent Acquisition (DIA): nano-HPLC-MS/MS Analysis

Each sample was suspended by adding 30 μL solvent A (A: 0.1% formic acid in water) and 10 x iRT peptide, then separated by nano-LC and analyzed by on-line nanospray LC-MS/MS on an Orbitrap Fusion Lumos coupled to EASY-nLC 1200 system (Thermo Fisher Scientific, MA, USA). A total of 3 μL peptide sample was loaded onto the analytical column (Acclaim PepMap C18, 75 μm x 25 cm) and separated in a 120 min gradient: 5% B to 35% B (B: 0.1% formic acid in ACN). The column flow rate was controlled at 200 nL/min at 40 °C and the electrospray voltage was 2 kV. The MS spectrometry parameters were set as follows: (1) MS: Scan range (m/z)=350–1500; Resolution=120,000; AGC target=4,000,000; Maximum injection time=50 ms; (2) HCD-MS/MS: Resolution=30,000; AGC target=1,000,000; Collision energy=32; Energy increase=5%. (3) Set up 60 variable windows and each window overlapped by 1 m/z.

Data Analysis

The DIA data were processed by Spectronaut X (Biognosys AG, Switzerland) with default parameters. Protein qualitative standards: Precursor Threshold 1.0% FDR, Protein Threshold 1.0% FDR. Decoy database was generated using a mutated strategy, which was similar to the one applied to disrupt a random number of amino acid sequences (min=2, max=length/2). The automatic calibration was performed by Spectronaut, and all selected precursors passing the filters were used to calculate the expression level. The average top 3 filtered peptides with a Q-value less than 1% were selected for protein quantification.