Mass spectrometric analysis data of Pyruvate Kinase from E. histolytica

Published: 7 October 2019| Version 1 | DOI: 10.17632/d9cvddpp53.1
Contributor:
Samudrala Gourinath

Description

To Identify the purified recombinant protein, we have performed Mass spectrometric analysis to prove it as Pyruvate Kinase from E. histolytica Sample Preparation for Mass spectrometric studies The protein spot was manually excised from the SDS-PAGE gel, and it was de-stained with 50 mM sodium thiosulphate and 15 mM potassium ferricyanide, and reduced with 5 mM TCEP and was finally alkylated with 50 mM iodoacetamide in the dark at room temperature for 30 min. Further, the gel particles were digested with (0.02μg/μl) trypsin in 50 mM ammonium bicarbonate and incubated at 37°C for 16–18 h (overnight). Digests were cleaned using a C18 silica cartridge to remove the salt and dried using a speed-vacuum centrifuge. The dried pellet was resuspended in buffer A (5% acetonitrile, 0.1% formic acid). Mass Spectrometric analysis of peptide mixtures The experiment was performed using EASY-nLC 1000 system (Thermo Fisher Scientific) coupled to Thermo Fisher-Q Exactive equipped with nano-electrospray ion source. Peptide mixture (1.0µg) was resolved using 25 cm PicoFrit column (360µm outer diameter, 75µm inner diameter, 10µm tip) filled with 1.9 µm of C18-resin (Dr Maeisch, Germany). The peptides were loaded with buffer A and eluted with a 0–40% gradient of buffer B (95% acetonitrile, 0.1% formic acid) at a flow rate of 300 nl/min for 100 min. MS data were acquired using a data-dependent top 10 method dynamically choosing the most abundant precursor ions from the survey scan. Data Processing The RAW files generated were analyzed with Proteome Discoverer (v2.2) against the UniProt Entamoeba histolytica reference proteome database. For Sequest search, the precursor and fragment mass tolerances were set at 10 ppm and 0.5 Da, respectively. The protease used to generate peptides, i.e. enzyme specificity was set for trypsin/P (cleavage at the C terminus of “K/R: unless followed by “P”) along with maximum missed cleavages value of two. Carbamidomethyl on cysteine as fixed modification and oxidation of methionine and N-terminal acetylation were considered as variable modifications for database search. Both peptide spectrum match and protein false discovery rate were set to 0.01 FDR.

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Institutions

Jawaharlal Nehru University

Categories

Mass Spectrometry

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