Computational protocol: Proteome and Acetyl-Proteome Profiling of Camellia sinensis cv. ‘Anji Baicha’ during Periodic Albinism Reveals Alterations in Photosynthetic and Secondary Metabolite Biosynthetic Pathways

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Protocol publication

[…] To detect acetylation, the peptide mixture after affinity purification was loaded onto a reversed-phase pre-column (Acclaim PepMap 100; Thermo Fisher Scientific). Peptides were separated using a reversed-phase analytical column (Acclaim PepMap RSLC; Thermo Fisher Scientific). Briefly, the peptide mixture was separated on a linear gradient of 8–25% buffer containing 98% acetonitrile and 0.1% formic acid for 20 min followed by 25–40% buffer for 12 min; the buffer concentration was increased up to 80% in 4 min and held at 80% for the last 4 min. The flow rate was 400 nl/min. Results were analyzed with a Q Exactive Plus hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific). Peptides were subjected to nanospray ionization source followed by MS/MS in conjunction with ultra-performance liquid chromatography. Intact peptides were acquired at a resolution of 70,000. Peptides were selected for MS/MS using a normalized collision energy setting of 30, and ion fragments were detected at a resolution of 17,500. A data-dependent “top 20” method was used to identify the most abundant precursor ions (mass range 350–1,800 m/z) above a threshold ion count of 5E3 in the MS survey scan with 15.0-s dynamic exclusion. The electrospray voltage used was 2.0 kV.MS/MS data were processed using MaxQuant software with an integrated Andromeda search engine (v. The tandem MS data were searched against the C. sinensis genome dataset (Xia et al., ) concatenated with a reverse decoy database. Trypsin/P was specified as the cleavage enzyme allowing up to four missing cleavages, four modifications per peptide, and five charges. The mass error was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. Carbamidomethylation on cysteine was specified as a fixed modification. Oxidation of methionine, acetylation of lysine, and N-terminal acetylation were set as variable modifications. False discovery rate thresholds for peptide, protein, and modification site were specified at 1%. The minimum peptide length was set to 7. All other parameters were set to the default values specified by MaxQuant, and >0.75 was used as the site localization probability. Relative quantification was performed as shown in the Supplementary Materials and Methods, which also describes LC-MS/MS measurements and analysis of data for complete peptide mixtures. Bioinformatics analyses, such as functional annotation and enrichment, motif, and functional interaction network analyses were performed as previously described (Xu et al., ). […]

Pipeline specifications

Software tools MaxQuant, Andromeda
Application MS-based untargeted proteomics
Diseases Albinism