DOI: 10.25345/c5x63b88p
title: MassIVE MSV000089326 - Long read proteogenomics to characterize protein isoform diversity in human umbilical vein endothelial cells (HUVECs)
title: Mass spectrometry-based proteomics sample preparation Harvested HUVECs, approximately 5 million cells each, were pelleted and frozen at -80C. The sample pellet was lysed according to the Filter Aided Sample Preparation (FASP) protocol.59 Lysis buffer used in the FASP was changed to 6% SDS, 150 mM DTT, 75 mM Tris-HCl. To the 30 uL pellet of 5 million cells, an aliquot of 60 uL of lysis buffer was added and probe-sonicated to lyse the cells and shear the nucleotide material. Sonication continued for 1-5 minutes until the sample was clear and no longer viscous. The lysate was then incubated at 95C for 5 minutes. Protein quantitation was estimated by BCA assay to be approximately 500-600 ug. Quadruplicate aliquots of 20 uL each were subjected to FASP and trypsin digest (1 ug per aliquot) and allowed to incubate at 37C overnight. Nanodrop analysis estimated peptide content at 22 ug per trypsin digest (total of 88 ug). Offline HPLC Fractionation The tryptic digests were pooled and dried down to a volume of 40 uL and subjected to offline high pH RP-HPLC fractionation using an Agilent 1200 HPLC. Sample was were loaded onto a Thermo Scientific Hypersil Gold C18 column (150 mm x 3 mm x 3 um C18), equilibrated with 95% solvent A (20 mM NH4 formate, pH 10) and 5% solvent B (70% acetonitrile/30% solvent A), and eluted at a flow rate of 400 uL/min, with fractions collected every 1 minute from RT 38-63 min. The following gradient was used: 5% B from 0-30 min, 5-65% B from 30-63 min, 65-100% B from 64-69 min, 100-5% B from 69-70 min, 5% B from 70-73 min. Samples containing peptide, according to UV 214 nm corresponding to the HUVEC pellet were digested with trypsin. Collected fractions 4-20 were selected for LC-MS/MS analysis. NanoLC-MS/MS analysis The resulting peptides were dried to 12 uL and analyzed by nanoLC-MS/MS using a Dionex Ultimate 3000 (Thermo Fisher Scientific, Bremen, Germany) coupled to an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Three microliters of each peptide-containing sample were loaded onto an Acclaim PepMap 100 trap column (300 um x 5 mm x 5 um C18) and gradient-eluted from an Acclaim PepMap 100 analytical column (75 um x 25 cm, 3 um C18) equilibrated in 96% solvent A (0.1% formic acid in water) and 4% solvent B (80% acetonitrile in 0.1% formic acid). The peptides were eluted at 300 nL/min using the following gradient: 4% B from 0-5 min, 4 to 28% B from 5-210 min, 28-40% B from 210-240 min, 40-95% B from 240-250 min and 95%B from 250-260 min. The Orbitrap Eclipse was operated in positive ion mode with 1.9 kV at the spray source, RF lens at 30% and data dependent MS/MS acquisition with XCalibur version 4.3.73.11. Positive ion Full MS scans were acquired in the Orbitrap from 375-1500 m/z with 120,000 resolution. Data dependent selection of precursor ions was performed in Cycle Time mode, with 3 seconds in between Master Scans, using an intensity threshold of 2 x 104 ion counts and applying dynamic exclusion (n=1 scans within 30 seconds for an exclusion duration of 60 seconds and +/- 10 ppm mass tolerance). Monoisotopic peak determination was applied and charge states 2-6 were included for HCD scans (quadrupole isolation mode; 1.6 m/z isolation window). The resulting fragments were detected in the Orbitrap at 15,000 resolution with standard AGC target.