Dataset Information

Label free quantification of LC-MS analysis of Dental pulp cells

ABSTRACT: LC/MS analysis of secretion of dental pulp cell. Methanol/chloroform precipitation was performed for protein purification. Resulting pellets were resolved by MS buffer (8 M urea and 50 mM Tris–HCl pH 8.0), followed by reduction in 5 mM DTT (FUJIFILM WAKO) and alkylation in 27.5 mM iodoacetamide (FUJIFILM WAKO) for 30 min in the dark at room temperature. After being diluted eight times in 50 mM Tris–HCl pH 8.0, proteins were digested overnight with 50 ng Lys-C (FUJIFILM WAKO) and trypsin (Promega, Madison, WI) at 37°C. Peptides were purified using GL-Tips SDB (GL Science, Tokyo, Japan) based on the manufacturer's protocol. The concentration of peptides was measured using a Pierce™ quantitative colorimetric peptide assay kit (Thermo Fisher Scientific). Subsequently, peptides (268 ng each) were injected into an EASY-nLC™ connected to a Q-Exactive PLUSTM (Thermo Fisher Scientific) mass spectrometry system. Protein identification and label-free quantification were performed using Proteome Discoverer 2.2™ (Thermo Fisher Scientific).

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Akihito Yamamoto

PROVIDER: PXD032142 | JPOST Repository | Thu Mar 09 00:00:00 GMT 2023

REPOSITORIES: jPOST

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	200115_Hashimoto_CM_LFQ.pdResult	Other
	387_Hashimoto_Fibro_CM_1.raw	Raw
	389_Hashimoto_Fibro_CM_2.raw	Raw
	391_Hashimoto_Fibro_CM_3.raw	Raw
	393_Hashimoto_SHED_CM_1.raw	Raw

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Project description:Sample preparation: P-AMPK+AMP, P-AMPK+ATPγS, and AMPK+ATPγS protein samples were crosslinked in HBST buffer (25 mM HEPES pH 7.5 at room temperature, 200 mM NaCL, and 1 mM TCEP). Non-crosslinked negative controls were generated using the same procedure without the addition of DSSO. Reactions were initiated by spiking 1 uL of 75 mM DSSO (Thermo-Fisher) in DMSO into a 50 uL solution containing 10 uM protein and mixing. The final molar ratio of crosslinker:protein was 150:1. The reactions were incubated at 25°C for 45 minutes prior to being quenched by the addition of Tris pH 8.0 to a final concentration of 50 mM. Each crosslink reaction was done in triplicate and the reaction replicates were pooled after quenching. 10 μL samples of the pooled samples were loaded for SDS-PAGE analysis with Coomassie staining to confirm the presence of crosslinked protein. The remaining 140 μL of crosslinked and non-crosslinked samples were then acetone precipitated at -20°C overnight and the protein was pelleted by centrifugation at 16,000 rcf for 5 minutes at 4°C. After decanting, the pellets were dried for 15 minutes at room temperature before being resuspended in 12.5 uL of resuspension buffer (50 mM ammonium bicarbonate, 8M urea, pH 8.0). ProteaseMAX (Promega) was added to 0.02% and the solutions were mixed on an orbital shaker operating at 400 RPM for 5 minutes. After resuspension, 87.5 uL of digestion buffer (50 mM ammonium bicarbonate, pH 8.0) was added. Cysteines in the protein samples were reduced and akylated as follows. DTT was added to 5 mM final concentration and the protein solutions were incubated at 56°C in an orbital shaker operating at 400 RPM and for 20 minutes. After reduction, 2.7 uL of 550 mM iodoacetamide was added and the solutions were incubated in the dark at room temperature for 15 minutes. Reduced and alkylated protein solutions were digested using trypsin at a ratio of 1:200 (w/w trypsin:protein) and incubating at 37°C. After overnight incubation at 37°C, the samples were acidified by the addition of trifluoroacetic acid (TFA, Thermo-Fischer) to 1%. Samples were then frozen and stored at -20°C until analysis.

Dataset Information

Label free quantification of LC-MS analysis of Dental pulp cells

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