MassIVE MSV000086636

Complete Public PXD023308

MudPIT analyses of the proteins co-purified with RAP01-HA, RAP21-HA, and negative controls from late asexual Plasmodium falciparum parasites

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Description

[NOTE: PF3D7_1470600 previously named RAP14 is now named RAP21]. HA-affinity purification - Purified late asexual parasites from parental and HA-tagged PF3D7_0105200 (RAP01-HA) and PF3D7_1470600 (RAP21-HA) lines were suspended in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 % Triton X-100, 1 mM AEBSF and EDTA-free protease inhibitor cocktail (Roche). After lysis by passing through a 26G needle 25 times, the soluble extracts were treated with 100 units of DNase I (NEB) for 10 min at room temperature and centrifuged at 14,000g for 15 min at 4C. The lysates were precleared with Dynabeads Protein A (Invitrogen) for 1h at 4C. The Rb anti-HA antibody (1:100, Abcam ab9110) was added in each sample and incubated overnight at 4C. Dynabeads Protein A were used to precipitate antibody-protein complexes and were washed with buffer A (1 % Triton X-100, 1 mM EDTA in PBS), buffer B (wash buffer A, 0.5 M NaCl) and buffer C (1 mM EDTA in PBS). Proteins were eluted using 0.1 M glycine, pH 2.8, and neutralized using 2 M Tris-HCl, pH 8.0. Then proteins were precipitated in 20% TCA followed by cold acetone washes. Multidimensional Protein Identification Technology - TCA-precipitated proteins were urea-denatured, reduced, alkylated, and digested with endoproteinase Lys-C (Roche) followed by modified trypsin (Promega). Peptide mixtures were loaded onto 100-um fused silica microcapillary columns packed with 5-um C18 reverse phase (Aqua, Phenomenex), strong cation exchange resin (Luna, Phenomenex), and 5-um C18 Aqua. Loaded microcapillary columns were placed in-line with a Quaternary Agilent 1100 series HPLC pump and a LTQ linear ion trap mass spectrometer equipped with a nano-LC electrospray ionization source (ThermoScientific, San Jose, CA). Fully automated 11-step MudPIT runs were carried out on the electrosprayed peptides. Tandem mass (MS/MS) spectra were interpreted using ProluCID against a database consisting of 5527 non-redundant (NR) Plasmodium falciparum 3D7 proteins (PlasmoDB-42 release), 36661 NR human proteins (NCBI, 2018-03-30 release), 419 usual contaminants (human keratins, IgGs, and proteolytic enzymes). To estimate false discovery rates (FDRs), the amino acid sequence of each NR protein entry was randomized, which resulted in a total search space of 85246 NR sequences. All cysteines were considered as fully carboxamidomethylated (+57 Da statically added), while methionine oxidation was searched as a differential modification. DTASelect and swallow, an in-house developed software (v.0.0.1, https://github.com/tzw-wen/kite), were used to filter ProLuCID search results at given FDRs at the spectrum, peptide, and protein levels. Here, all controlled FDRs were less than 1.2%. Three immunoprecipitations were performed each for RAP01-HA and RAP21-HA along with 4 negative controls experiments. [doi:10.25345/C5R795] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: RAP (RNA binding domain abundant in Apicomplexans) ; Inducible TetR-aptamer system ; HA affinity purification

Contact

Principal Investigators:
(in alphabetical order) 		Laurence Florens, The Stowers Institute for Medical Research, USA
Submitting User: laflorens

Publications

Hollin T, Abel S, Falla A, Pasaje CFA, Bhatia A, Hur M, Kirkwood JS, Saraf A, Prudhomme J, De Souza A, Florens L, Niles JC, Le Roch KG.
Functional genomics of RAP proteins and their role in mitoribosome regulation in Plasmodium falciparum.
Nat Commun. 2022 Mar 11;13(1):1275. Epub 2022 Mar 11.

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