Disulfide Reduction, Alkylation, and Tryptic Digestion

Proteins were disulphide-reduced by 25 mM DTT at 37 °C for 40 min. Cysteines were alkylated by 50 mM iodoacetamide. Proteins were digested with sequencing-grade trypsin (Promega) at 37 °C overnight and the supernatant was desalted using C18 solid-phase cartridges and lyophilized. The dried peptides were reconstituted in 0.1% FA and loaded onto an Acclaim PepMap 100 C18 LC column (Thermo Fisher) utilizing a Thermo Easy nLC 1000 LC system (Thermo Fisher) connected to Q Exactive HF mass spectrometer (Thermo Fisher) and analyzed^{66 (link)}.

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Wu Y., Tang L., Huang H., Yu Q., Hu B., Wang G., Ge F., Yin T., Li S, & Yu X. (2023). Phosphoglycerate dehydrogenase activates PKM2 to phosphorylate histone H3T11 and attenuate cellular senescence. Nature Communications, 14, 1323.

Publication 2023

sequencing-grade trypsin Acclaim PepMap 100 C18 LC column Thermo Easy nLC 1000 LC system Q Exactive HF mass spectrometer

Cysteines Disulphide Iodoacetamide Peptides Promega Proteins Trypsin

Corresponding Organization : Renmin Hospital of Wuhan University

Other organizations : Hubei University, Wuhan Sports University, Wuhan No.1 Hospital, Institute of Hydrobiology, Chinese Academy of Sciences

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Variable analysis

independent variables

Disulphide reduction by 25 mM DTT at 37 °C for 40 min
Cysteine alkylation by 50 mM iodoacetamide

dependent variables

Proteomic analysis using Q Exactive HF mass spectrometer

control variables

Trypsin digestion at 37 °C overnight
Desalting using C18 solid-phase cartridges
Lyophilization of dried peptides
Reconstitution in 0.1% FA

controls

No positive or negative controls were explicitly mentioned.

Annotations

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