Proteome discoverer 2.2 platform

Manufactured by Thermo Fisher Scientific

The Proteome Discoverer 2.2 platform is a comprehensive software solution designed for the analysis and identification of proteins in complex biological samples. It provides a unified workflow for mass spectrometry-based proteomics data processing, peptide and protein identification, and quantification.

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4 protocols using proteome discoverer 2.2 platform

Quantitative Proteomics of E. coli

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2 mg E. coli cell pellets were lysed in 8 M urea using pulsed sonication. The supernatant was extracted, and then sequentially reduced, alkylated, and digested overnight with trypsin. The protein digests were labelled with 10-plex Tandem Mass Tag (TMT) reagents (Thermo Fisher Scientific, San Jose, CA) [18 (link)]. Labelled protein digests were then pooled, and desalted. High-pH reverse phase liquid chromatography (bRPLC) [19 (link)] was carried out to separate the peptide mixture into 24 fractions. Each fraction was analyzed on an Orbitrap Lumos (Thermo Fisher Scientific) based nanoLCMS system.
Peptide and protein IDs were assigned by searching the resulting LCMS raw data against the Uniprot E. coli database supplemented with the Bax-Intein-CBD sequence using the Sequest HT algorithm. Quantitation values were extracted using the Proteome Discoverer 2.2 platform (Thermo Fisher Scientific). 10 channels’ sample amounts were normalized using the total report ion intensities of their corresponding channels. Individual proteins were quantified based on the normalized report ion intensities of their unique peptides.
We used 2-fold cutoff and t-test p values less than 0.05 to select for changed proteins.

He Y., Chen Y., Morris D.L., Lee D.Y, & Tjandra N. (2019). Bax expression is optimal at low oxygen tension and constant agitation. Protein expression and purification, 165, 105501.

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Quantitative Proteomics of E. coli

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He Y., Chen Y., Morris D.L., Lee D.Y, & Tjandra N. (2019). Bax expression is optimal at low oxygen tension and constant agitation. Protein expression and purification, 165, 105501.

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Mass Spectrometry-Based Proteomics Analysis

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Whole cell protein was prepared by using a phase transfer surfactant method¹⁹ and then trypsinized and desalted. Peptide samples were dissolved in 0.1% formic acid and loaded into a nano‐flow UHPLC (Easy‐nLC 1000 system; Thermo Fisher Scientific) online‐coupled to an Orbitrap mass spectrometer equipped with a nanospray ion source (Q‐Exactive Plus; Thermo Fisher Scientific) to obtain MS/MS spectra as described previously.²⁰ For MS/MS data analysis, we used the Sequest HT (Thermo Fisher Scientific) and Mascot ver 2.5 (Matrix Science) algorithms embedded in the Proteome Discoverer 2.2 platform (Thermo Fisher Scientific), and the peak lists were searched against the UniProt human databases. The tolerance of precursor ions and that of fragment ions were set to 10 ppm and 0.02 Da, respectively.

Murakami T., Takasawa A., Takasawa K., Akimoto T., Aoyama T., Magara K., Saito Y., Ota M., Kyuno D., Yamamoto S., Hasegawa T., Saito T, & Osanai M. (2020). Aberrant expression of junctional adhesion molecule‐A contributes to the malignancy of cervical adenocarcinoma by interaction with poliovirus receptor/CD155. Cancer Science, 112(2), 906-917.

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Profiling Adenoviral Protein Interactions

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U2OS cells infected with adenovirus expressing TMEM55B-WT-GFP or TMEM55B-P66A-GFP cells treated with or without 300 μM NaAsO₂ for 2 h. Cell lysates were subjected to immunoprecipitation by using GFP beads. Immunoprecipitated proteins were subjected to SDS–PAGE, and gel bands from corresponding molecular weights were excised for enzymatic digestion. Briefly, the samples were first reduced with TCEP (tris(2-carboxyethyl)phosphine, Sigma-Aldrich) and alkylated with CAA (chloroacetamide, Sigma-Aldrich), and then digested with chymotrypsin (Promega). The resulting peptide mixtures were analyzed with an Orbitrap Fusion Lumos that is equipped with a Dionex Ultimate 3000 nanoLC system (ThermoFisher Scientific). Peptide IDs and phosphorylation sites were assigned with Mascot V2.5 (Matrix Science). The confidence of phosphorylation site localization is assessed with ptmRS node in Proteome Discoverer 2.2 platform (ThermoFisher Scientific). All peptides were filtered out at 1% false discovery rate (FDR) and their relative abundances were compared based on the areas under curve (AUC) of their corresponding chromatographic peaks.

Jeong E., Willett R., Rissone A., La Spina M, & Puertollano R. (2024). TMEM55B links autophagy flux, lysosomal repair, and TFE3 activation in response to oxidative stress. Nature Communications, 15, 93.

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