Dataset Information

Long read proteogenomics to characterize protein isoform diversity in human umbilical vein endothelial cells (HUVECs)

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Gloria Sheynkman

PROVIDER: MSV000089326 | MassIVE | Thu Apr 28 09:33:00 BST 2022

REPOSITORIES: MassIVE

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Publications

Characterization of protein isoform diversity in human umbilical vein endothelial cells via long-read proteogenomics.

Mehlferber Madison M MM Jeffery Erin D ED Saquing Jamie J Jordan Ben T BT Sheynkman Leon L Murali Mayank M Genet Gael G Acharya Bipul R BR Hirschi Karen K KK Sheynkman Gloria M GM

RNA biology 20220101 1

Endothelial cells (ECs) comprise the lumenal lining of all blood vessels and are critical for the functioning of the cardiovascular system. Their phenotypes can be modulated by alternative splicing of RNA to produce distinct protein isoforms. To characterize the RNA and protein isoform landscape within ECs, we applied a long read proteogenomics approach to analyse human umbilical vein endothelial cells (HUVECs). Transcripts delineated from PacBio sequencing serve as the basis for a sample-specif ...[more]

PMID: 36457147

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Project description:Proteomic analysis Epilepsy was induced using perforant pathway stimulation (PPS) in rats as described (Norwood BA, et al. (2010) Classic hippocampal sclerosis and hippocampal-onset epilepsy produced by a single "cryptic" episode of focal hippocampal excitation in awake rats. J Comp 803 Neurol 518(16):3381-3407). Rats were killed under deep anesthesia (xylazine+ketamine) by transcardial perfusion with ice-cold 0.9 % NaCl solution at the following time points: 1 h, 24 h, 72 h, 10 d and 16 d after PPS (epileptogenesis); within 1 d of first spontaneous seizure (early epilepsy); 1 month after first spontaneous seizure (chronic epilepsy). Control rats were killed on day 17 after surgery (corresponding to day 10 after PPS). Hippocampi were removed and snap frozen at -80°C. Frozen rat hippocampi were resuspended in 200 ul lysis buffer (6M GdmCl, 10mM TCEP, Complete protease inhibitor cocktail, PhosSTOP inhibitor, 100 mM TEAB, benzonase) and dounced 30 times on ice. Samples were boiled for 5 min, sonicated on ice for 2min and centrifuged at 13000 rpm for 3 min. The supernatant was added chloroacetamide to a final concentration of 20 mM followed by incubation in the dark for 30 min at room temperature (RT). Proteins were digested with LysC for 30 min at RT and then diluting 10x— using 100 mM TEAB followed by adding trypsin and incubating overnight at 37°C with shaking. LysC and trypsin were added in a LysC/trypsin:protein ratio of 1:100. Samples were centrifuged at 13000 rpm for 5 min and from the supernatant a volume corresponding to 50 ug peptide was transferred to a new eppendorff tube. Sets of 6 samples were labelled using the TMTsixplex isobaric label reagent. (ThermoFisher Scientific). The labeling reagent was prepared using the manufactures instructions to first equilibrate to RT, then dissolving the labeling reagent in 41 ul anhydrous acetonitrile. The labeling reagent was added to each sample and incubated for 1 hour at RT. The reaction was quenched by incubating with 0.76 M lysine for 15 min. A small amount was taken from each sample, mixed and tested by mass spectrometry for labeling efficiency and mixing ratio. Remaining samples were mixed in a 1:1 ratio. The Stage-tips for the high-pH reverse phase fractionation were prepared by placing first two C18 disks in a p200 pipette tip, then adding a slurry of C18 beads (ReproSil-Pur, 3 um) in methanol and adding one additional C18 disk in the top. The column was activated and equilibrated by washing one time in 150 ul buffer B (50% buffer A, 50% ACN) and then two times in 150 ul buffer A (20mM Ammonium hydroxide, pH 10). The labeled peptide sample was adjusted to pH 10 using buffer A and then loaded onto the activated and equilibrated stage-tip by spinning the sample through the column and collecting 26 the flow-through. The column was washed 2 times with 150 ul buffer 585 A and the washes were collected and combined with the flow-through. Next, peptides were eluted stepwise in 17 fractions by spinning 150 ul of buffer A containing increasing amounts of ACN for each step (3.5%, 4.5%, 6%, 8%, 10%, 10.5%, 11.5%, 13.5%, 15%, 16%, 17.5%, 18.5%, 19.5%, 21%, 27%, 50%, 80%) through the column. After collection, the liquid was evaporated completely in a Concentrator Plus (Eppendorf) and prepared for MS by resuspending in buffer A* (2% ACN, 0.1% TFA). Samples were analyzed using an Easy-nLC system coupled online to a Q Exactive HF mass spectrometer (Thermo Scientific) equipped with a nanoelectrospray ion source (Proxeon/Thermo Scientific). The peptides were eluted into the mass spectrometer during a chromatographic separation from a fused silica column packed in-house with 3 um C18 beads (Reprosil, Dr. Maisch) using a 120 min gradient of buffer B (80% ACN, 0.5% acetic acid) with a flow rate of 250 nL/min. The Q Exactive HF was operated in positive ion mode with a top 12 data-dependent acquisition method where ions were fragmented by higher-energy collisional dissociation (HCD) using a normalized collisional energy (NCE)/ stepped NCE of 28 and 32. The resolution was set to 60,000 (at 400m/z), with a scan range of 300-1700 m/z and an AGC target of 3e6 for the MS survey. MS/MS was performed at a scan range of 100 - 2000 m/z using a resolution of 30,000 (at 400 m/z), an AGC target of 1e5, an intensity threshold of 1e5 and an isolation window of 1.2 m/z. Further parameters included an exclusion time of 45 sec and a maximum injection time for survey and MS/MS of 15 ms and 45 ms respectively.

Project description:Bacteria were washed three times in NaCl (150 mM). Cells were resuspended in 1 ml TSU buffer (50 mM Tris pH 8.0, 0.1% SDS, 2.5 M urea) and lysed by two freeze-thaw cycles in liquid nitrogen. Lysate proteins (40 ug) were separated and digested. Digested peptides were separated by nano-LC using an Ultimate 3000 HPLC and autosampler system (Dionex; Amsterdam, Netherlands). Samples (1 ul) were concentrated and desalted onto a micro C18 pre-column (500 um×2 mm, Michrom Bioresources; Auburn, CA, USA) with H2O:CH3CN (98:2, 0.05% trifluoroacetic acid) at 15 ul min−1. After a 4 min wash the pre-column was switched (Valco 10 port valve; Dionex) into line with a fritless nano column (75 u×~10 cm) containing C18 media (5 u, 200 A Magic; Michrom) manufactured according to Gatlin. Peptides were eluted using a linear gradient of H2O:CH3CN (98:2, 0.1% formic acid) to H2O:CH3CN (64:36, 0.1% formic acid) at 250 nl min−1 over 30 min. High voltage (2000 V) was applied to low volume tee (Upchurch Scientific) and the column tip positioned ~0.5 cm from the heated capillary (T = 280 degrees C) of an Orbitrap Velos (Thermo Electron; Bremen, Germany) mass spectrometer. Positive ions were generated by electrospray and the Orbitrap operated in data dependent acquisition mode (DDA). A survey scan m/z 350–1750 was acquired in the Orbitrap (Resolution = 30,000 at m/z 400, with an accumulation target value of 1,000,000 ions) with lockmass enabled. Up to the 10 most abundant ions (>5,000 counts) with charge states >+2 were sequentially isolated and fragmented within the linear ion trap using collisionally induced dissociation with an activation q = 0.25 and activation time of 30 ms at a target value of 30,000 ions. M/z ratios selected for MS/MS were dynamically excluded for 30 s. Peak lists were generated using Mascot Daemon/extract_msn (Matrix Science, Thermo; London, England) using the default parameters, and submitted to the database search program Mascot (version 2.1, Matrix Science). Search parameters were: Precursor tolerance 4 ppm and product ion tolerances +/-0.4 Da; Oxidation (M) and Carbamidomethyl (C) specified as variable modifications, Enzyme specificity was trypsin, 1 missed cleavage was possible

Project description:Washed three times in NaCl (150 mM). Cells were resuspended in 1 ml TSU buffer (50 mM Tris pH 8.0, 0.1% SDS, 2.5 M urea) and lysed by two freeze-thaw cycles in liquid nitrogen. Lysate proteins (40 ug) were separated and digested. Digested peptides were separated by nano-LC using an Ultimate 3000 HPLC and autosampler system (Dionex; Amsterdam, Netherlands). Samples (1 ul) were concentrated and desalted onto a micro C18 pre-column (500 um×2 mm, Michrom Bioresources; Auburn, CA, USA) with H2O:CH3CN (98:2, 0.05% trifluoroacetic acid) at 15 ul min−1. After a 4 min wash the pre-column was switched (Valco 10 port valve; Dionex) into line with a fritless nano column (75 u×~10 cm) containing C18 media (5 u, 200 A Magic; Michrom) manufactured according to Gatlin. Peptides were eluted using a linear gradient of H2O:CH3CN (98:2, 0.1% formic acid) to H2O:CH3CN (64:36, 0.1% formic acid) at 250 nl min−1 over 30 min. High voltage (2000 V) was applied to low volume tee (Upchurch Scientific) and the column tip positioned ~0.5 cm from the heated capillary (T = 280 degrees C) of an Orbitrap Velos (Thermo Electron; Bremen, Germany) mass spectrometer. Positive ions were generated by electrospray and the Orbitrap operated in data dependent acquisition mode (DDA). A survey scan m/z 350–1750 was acquired in the Orbitrap (Resolution = 30,000 at m/z 400, with an accumulation target value of 1,000,000 ions) with lockmass enabled. Up to the 10 most abundant ions (>5,000 counts) with charge states >+2 were sequentially isolated and fragmented within the linear ion trap using collisionally induced dissociation with an activation q = 0.25 and activation time of 30 ms at a target value of 30,000 ions. M/z ratios selected for MS/MS were dynamically excluded for 30 s. Peak lists were generated using Mascot Daemon/extract_msn (Matrix Science, Thermo; London, England) using the default parameters, and submitted to the database search program Mascot (version 2.1, Matrix Science). Search parameters were: Precursor tolerance 4 ppm and product ion tolerances +/-0.4 Da Oxidation (M) and Carbamidomethyl (C) specified as variable modifications Enzyme specificity was trypsin, 1 missed cleavage was possible

Dataset Information

Long read proteogenomics to characterize protein isoform diversity in human umbilical vein endothelial cells (HUVECs)

Publications

Characterization of protein isoform diversity in human umbilical vein endothelial cells via long-read proteogenomics.

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