Computational protocol: Global analysis of ginsenoside Rg1 protective effects in β-amyloid-treated neuronal cells

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Protocol publication

[…] MS was performed as previously described . Briefly, the tryptic-dried peptides were analyzed using the Agilent HPLC-Chip/TOF MS system with the Agilent 1260 nano-LC system, HPLC Chip-cube MS interface and a 6530 QTOF single quadrupole-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The dried peptide samples were re-resolved in 2% ACN/0.1% FA and concentrated on a large-capacity HPLC Chip (Agilent Technologies). The HPLC chip incorporated an enrichment column (9 mm, 75 mm-inner diameter, 160 nL) and a reverse-phase column (15 cm, 75 mm-inner diameter, packed with Zorbax 300SB-C18 5 mm resins). The peptide separation was performed using a 70-min gradient of 3–45% buffer B (buffer A contained 0.1% FA/dry weight, and buffer B contained 90% ACN/0.1% FA/dry weight) at a flow rate of 300 nL/min. The MS and MS/MS data were acquired in positive ion mode and stored in centroid mode. The chip spray voltage was set at 1950 V and maintained under chip conditions. The drying gas temperature was set at 325°C with a flow rate of 3.5 L/min. A medium isolation (4 m/z) window was used for precursor isolation. A collision energy with a slope of 3.7 V/100 Da and an offset of 2.5 V were used for fragmentation. Additionally, while the MS data were acquired over a mass range of 300–3000 m/z, the MS/MS data were acquired over a mass range of 50–2500 m/z. Reference mass correction was performed using a reference mass of 922.0098. Precursors were set in an exclusion list for 0.5 min after two MS/MS spectra. The MS and MS/MS spectra were extracted using the Mass Hunter Qualitative Analysis B.05.00 software (Agilent Technologies) with default parameters. Database searches using the X! Tandem search engine were performed with a peptide mass tolerance of 20 ppm, an MS/MS tolerance of 0.5 Da, and a strict tryptic specificity (cleavage after lysine and arginine) allowing two missed cleavage sites; carbamidomethylation of Cys was set as a fixed modification, whereas oxidation (M) was considered a variable modification. [...] The MS database search results were analyzed using the Trans-Proteomic Pipeline (Systems Biology, Seattle, WA, USA), all assigned peptides were validated by PeptideProphet and the results filtered with an error rate of 0.05 (FDR of 5%) . Peptide quantification was performed using the XPRESS modules included in the Trans-Proteomic Pipeline. Protein identification and quantification were validated using ProteinProphet and the result cutoff was set to a protein probability of 80%/error rate of 0.05 (FDR 5%) and a ratio of heavy from fixed to 1. SILAC ratios for proteins were quantified using XPRESS software . The elution profiles of the light and heavy peptides were isolated and quantified based on the area of each peptide peak, and the abundance ratio was calculated based on these areas by XPRESS. Quantitative protein ratios were determined based on the average level of quantified peptides. […]

Pipeline specifications

Software tools X! Tandem, PeptideProphet, ProteinProphet
Application MS-based untargeted proteomics
Diseases Alzheimer Disease, Mitochondrial Diseases
Chemicals Amino Acids