Yeast proteomic response to amino acid mix supplementation

Published: 6 December 2022| Version 1 | DOI: 10.17632/sd8zthmrk4.1
Contributors:
,
Pauline Trébulle,
,
Markus Ralser

Description

In this dataset, we investigate the proteomic response to the supplementation of an amino acid mix in a prototrophic wild-type yeast strain.

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The control wild-type strain (BY4741, quadruple knock-in: HIS3, LEU2, URA3 and MET15), herein referred to as the prototrophic wild-type strain, was cultured in 1.4 mL SM media in 96 deep well plates with a starting OD of 0.05 with and without a synthetic supplement mix (Sigma, Cat# Y1896, final concentration of 3.1 g/L). After 10 hours, cell pellets were harvested by centrifugation (4 min, 3,200 g). Samples were prepared as described previously in Messner et al. 2021 and 2 µg of peptides were chromatographically separated on an analytical column (Waters HSS T3, 150 mm x 300 µm, 1.8 µm, maintained at 35C, with a 5µL/min flow rate) installed in a nanoAcquity UPLC (Waters). The chromatographic gradient started at 3% buffer B (acetonitrile with 0.1% formic acid) and 97% buffer A (1% acetonitrile in water with 0.1% formic acid), and ended at 80% buffer B before returning to starting conditions (total runtime 27.5 min). The UPLC was coupled to a SCIEX TripleTOF 6600 with a DuoSpray Turbo V source operated in High Resolution mode. The ion source gas 1 (nebulizer gas), ion source gas 2 (heater gas), and curtain gas were set to 15, 20 and 25. The ion spray voltage was set to 5500 V and the source temperature was set to 75. The acquisition scheme was DIA SWATH with 40 windows and 35 milliseconds accumulation time. The raw files were then processed using DIA-NN 1.8 using a yeast experimental library while further data analysis was carried out using R. Briefly, precursors with Global.Q.Values, Global.PG.Q.Values, Q.Values, PG.Q.Values > 0.01 and non proteotypic were removed. Precursors detected in less than 75% of the replicates in a condition and with a median CV above 0.3 in a condition were excluded before summarization of the protein quantities using maxLFQ.

Institutions

Charite Universitatsmedizin Berlin, Francis Crick Institute

Categories

Proteomics, Amino Acids, Yeast

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