The wild-type S. Typhimurium and mutant strains were grown in LB medium to log or stationary phase. R-proteins were collected by sucrose gradient centrifugation and digested with trypsin at 37°C for 18 h, then desalted on C18 column (Waters Wat051910). The acetylated peptides were enriched by anti-Kac antibody beads (PTMScan acetyl lysine motif (Kac) Kit, Cell Signal Technology) and then taken for LC–MS/MS analysis (Thermo Fisher Scientific). Mass spectrometric data were analyzed using the Maxquant software (version no. 1.3.0.5) for database search. To quantify the acetylation levels of r-proteins, the ratios of average area (representing peptide intensity) of acetylated peptides to the average area of total lysine-containing peptides were calculated.
Lc ms ms analysis
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LC–MS/MS (Liquid Chromatography-Tandem Mass Spectrometry) is an analytical technique that combines the separation capabilities of liquid chromatography with the high sensitivity and specificity of mass spectrometry. This technique is used to identify and quantify a wide range of molecules, including pharmaceuticals, environmental pollutants, and biological compounds, in complex samples.
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Lab products found in correlation
3 protocols using lc ms ms analysis
1
Quantifying Ribosomal Protein Acetylation
2
Celastrol Target Identification in Astrocytes
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Pull-down/LC-MS/MS was performed to identify the targets of celastrol in astrocytes according to a previous description [23 (link)]. Astrocytes were treated with celastrol probe (cel-p) (4 μM) with or without a competitor (celastrol, 8×), for 2 h. After collecting the protein lysate of cells, click chemistry reaction was conducted with biotin azide (50 μM), sodium ascorbate (NaVc) (1 mM), tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) (100 μM), and CuSO4 (1 mM), which were purchased from Sigma-Aldrich (Sigma-Aldrich Co., Ltd.). Then, pre-cooled acetone was added to precipitate the proteins, which were re-dissolved in 0.1% SDS. Washed agarose beads were then added to pull down the proteins. Subsequently, the proteins were digested and isolated from the beads, followed by spin drying. The samples were labeled with distinct TMT10 isobaric peptide tags, desalted, and submitted for LC-MS/MS analysis (Thermo Fisher Scientific Inc., Boston, MA, USA).
3
Proteomic Analysis of SpltNPV-C3 OBs
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The proteins of the SpltNPV-C3 OBs were separated via SDS‒PAGE using an 8–15% gradient gel. The protein bands were collected into a 1.5 mL centrifuge tube for LC‒MS/MS analysis (Thermo Fisher Scientific, MA, USA). LC‒MS/MS analysis and protein identification were performed by Shanghai Omicsolution Co. The raw files of the MS spectra were searched against the putative protein database SpltNPV-C3 (NC_011616).
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