DOI: 10.25345/c5cz32f5s
title: MassIVE MSV000091168 - Proteogenomic Identification and Validation of Key Differentially Expressed Molecular Targets for Keloid Disease
title: The study incorporated 5 keloid disease (KD) and 5 normal skin (NS) samples, and due to challenges in obtaining primary samples, we had a sample size of 3 biological replicates for FKN, 3 for DFSP made up of 2 primary cells and 1 cell line, and 2 Fibrosarcoma cell lines FS-CL-1 (HT-1080 [HT1080], CCL-121, ATCC, Manassas, VA, USA) and FS-CL-2 (HT-1080-Luc2 CCL-121-LUC2). Fibroblast subcellular proteins were isolated by employing the Qproteome Cell Compartment Kit (QIAGEN Pharmaceutical). Also, different cell lysis or cell extraction (CE) buffers specific for the different subcellular compartments, including CE1 for cytosolic proteins, and CE2, CE3 and CE4 for membrane, nuclear and cytoskeletal proteins respectively. Proteomics service took place at the Centre for Proteomic and Genomic Research (CPGR) in Cape Town, South Africa. Extracted proteins were transported on dry ice and samples were logged and stored at -80 degrees C until further processing. Briefly, HILIC beads (ReSyn Biosciences, HLC010) were aliquoted into a new tube and the shipping solution removed. Beads were then washed with 250 uL wash buffer [15% Acetonitrile, 100 mM Ammonium acetate (Sigma 14267) pH 4.5] for one minute. This was repeated once. The beads were then resuspended in loading buffer (30% ACN, 200 mM Ammonium acetate pH 4.5) to a concentration of 2.5 mg/mL. A total of 25 ug of protein (or the entire sample) from each tube was transferred to a 96-well protein LoBind plate (Merck, 0030504.100). Protein was reduced with tris (2-carboxyethyl) phosphine (TCEP; Sigma 646547) which was added to a final concentration of 10 mM TCEP and incubated at 60 degrees C for one hour. Samples were cooled to room temperature (RT) and then alkylated with methylmethanethiosulphonate (MMTS; Sigma 208795) which was added to a final concentration of 10 mM MMTS and incubated at RT for 15 minutes. HILIC magnetic beads were added at an equal volume to that of the sample and a ratio of 5:1 total protein. The plate was then incubated at RT on the shaker at 900 RPM for 30 minutes for binding of protein to beads. After binding, the beads were washed four times with 500 uL of 95% ACN for one minute. For digestion, Trypsin (Promega PRV5111), was made up in 50 mM Triethylammonium bicarbonate buffer (TEAB) and added at a ratio of 1:12.5 total protein and the plate was then incubated at 37 degrees C on the shaker for 4 hours. After digestion, the plate was placed on a magnet to separate the beads from the supernatant. The supernatant containing peptides was removed and dried down. Samples were then resuspended in LC loading buffer: 0.1% formic acid (FA; Sigma 56302), 2.5% CAN. LCMS analysis was conducted using a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, USA) coupled to a Dionex Ultimate RS3000 nano-UPLC system. Data was acquired using: Xcalibur v4.1.31.9, Chromeleon v6.8 (SR13), Orbitrap MS v2.9 (build 2926) and Thermo Foundations 3.1 (SP4). Peptides were dissolved in 0.1% Formic Acid (FA; Sigma 56302), 2% Acetonitrile (ACN) and loaded on a C18 trap column (PepMap100, 300um x 5mm x 5um). An estimate of approximately 400 ng of peptide was injected for each sample. Samples were trapped onto the pre-analytic column and washed for 3 minutes before the valve was switched and peptides eluted onto the analytical column. Chromatographic separation was performed with a Waters nanoEase (Zenfit) M/Z Peptide CSH C18 analytic column (186008810, 75 um x 25 cm x 1.7 um). The solvent system employed was solvent A: LC water (Burdick and Jackson BJLC365), 0.1% FA and solvent B: ACN, 0.1% FA. All data acquisition employed Proxeon stainless steel emitters (Thermo Fisher TFES523). The multi-step gradient for peptide separation was generated at 300 nL/min as follows: time change 5 min, gradient change: 2 to 5% Solvent B, time change 40 min, gradient change 5 to 18% Solvent B, time change 10 min, gradient change 18 to 30% Solvent B, time change 2 min, gradient change 30 to 80% Solvent B. The gradient was then held at 80% solvent B for 10 min before returning it to 2% solvent B and conditioning the column for 15 min. The mass spectrometry was operated in positive ion mode with a capillary temperature of 320 degrees C with the applied electrospray voltage of 1.95 kV. Based on the study design, each fraction was run as a block randomised set with a fraction specific pool run after, at the start, middle and end of each block. The fractions were run in clusters to minimise carry over of proteins across sub-cellular locations during data acquisition. In between each fraction block, a pool produced from each fraction pool to represent a complete sample pool was run to monitor system performance during the sequence of acquisition. Blanks were utilised to minimise carry over from QCs throughout the sequence of acquisition.