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PXD023308

PXD023308 is an original dataset announced via ProteomeXchange.

Dataset Summary
TitleMudPIT analyses of the proteins co-purified with RAP01-HA, RAP14-HA, and negative controls from late asexual Plasmodium falciparum parasites
DescriptionHA-affinity purification - Purified late asexual parasites from parental and HA-tagged PF3D7_0105200 (RAP01-HA) and PF3D7_1470600 (RAP14-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 RAP14-HA along with 4 negative controls experiments.
HostingRepositoryMassIVE
AnnounceDate2022-01-26
AnnouncementXMLSubmission_2022-01-26_11:17:56.696.xml
DigitalObjectIdentifier
ReviewLevelNon peer-reviewed dataset
DatasetOriginOriginal dataset
RepositorySupportSupported dataset by repository
PrimarySubmitterLaurence Florens
SpeciesList scientific name: Plasmodium falciparum 3D7; NCBI TaxID: 36329; scientific name: Homo sapiens; common name: human; NCBI TaxID: 9606;
ModificationListS-carboxamidomethyl-L-cysteine; L-methionine sulfoxide
InstrumentLTQ
Dataset History
RevisionDatetimeStatusChangeLog Entry
02020-12-24 08:49:10ID requested
12022-01-26 11:17:57announced
Publication List
no publication
Keyword List
submitter keyword: RAP (RNA binding domain abundant in Apicomplexans), Inducible TetR-aptamer system, HA affinity purification
Contact List
Laurence Florens
contact affiliationThe Stowers Institute for Medical Research
contact emaillaf@stowers.org
lab head
Laurence Florens
contact affiliationStowers Institute for Medical Research
contact emaillaf@stowers.org
dataset submitter
Full Dataset Link List
MassIVE dataset URI
Dataset FTP location
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