Dataset Information

The Leishmania mexicana promastigote flagellar proteome

ABSTRACT: Leishmania are protists (class: Kinetoplastida) with a single multifunctional flagellum used for motility, attachment to the sand fly vector and sensory functions. To discover the protein composition of the L. mexicana promastigote flagellum, we fractionated L. mexicana promastigotes by mechanical shearing and differential centrifugation into four fractions: detergent soluble (Fs) and insoluble (Fi) fractions of isolated flagella and detergent soluble (Cs) and insoluble fractions (Ci) of deflagellated cell body remnants. A label-free quantitation method (SINQ; Trudgian et al., 2011, Proteomics 10.1002) was used to identify proteins enriched in each of the four fractions. This PRIDE upload contains .RAW, .MGF and .XML files, as well as the SINQ quantification output file “SINQ_raw_data” and the genome sequence annotation used for protein identification “MFiebig_20140619” (Fiebig et al., 2015, PLoS Pathogens II:e1005186). XML files are named SUB7467; MSS10859. .RAW and .MGF files are structured as follows (for each fraction there are eight files, corresponding to individual gel pieces): “Ci: C131121_030; C131121_031; C131121_032; C131121_033; C131121_034; C131121_035; C131121_036; C131121_037”, “Cs: C131121_040; C131121_041; C131121_042; C131121_043; C131121_044; C131121_045; C131121_046; C131121_047”, “Fi: C131121_010; C131121_011; C131121_012; C131121_013; C131121_014; C131121_015; C131121_016; C131121_017”, “Fs: C131121_020; C131121_021; C131121_022; C131121_023; C131121_024; C131121_025; C131121_026; C131121_027”.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Leishmania Mexicana Mhom/gt/2001/u1103

TISSUE(S): Promastigote

DISEASE(S): Cutaneous Leishmaniasis,Leishmaniasis

SUBMITTER: Tom Beneke

LAB HEAD: Eva Gluenz

PROVIDER: PXD011057 | Pride | 2018-12-14

REPOSITORIES: Pride

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Publications

Genetic dissection of a Leishmania flagellar proteome demonstrates requirement for directional motility in sand fly infections.

Beneke Tom T Demay François F Hookway Edward E Ashman Nicole N Jeffery Heather H Smith James J Valli Jessica J Becvar Tomas T Myskova Jitka J Lestinova Tereza T Shafiq Shahaan S Sadlova Jovana J Volf Petr P Wheeler Richard John RJ Gluenz Eva E

PLoS pathogens 20190626 6

The protozoan parasite Leishmania possesses a single flagellum, which is remodelled during the parasite's life cycle from a long motile flagellum in promastigote forms in the sand fly to a short immotile flagellum in amastigotes residing in mammalian phagocytes. This study examined the protein composition and in vivo function of the promastigote flagellum. Protein mass spectrometry and label free protein enrichment testing of isolated flagella and deflagellated cell bodies defined a flagellar pr ...[more]

PMID: 31242261

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Project description:Multidimensional peptide fractionation is widely used in proteomics to reduce the complexity of peptide mixtures prior to mass spectrometric analysis. Here, we describe the sequential use of strong cation exchange and reversed phase liquid chromatography in both basic and acidic pH buffers for separating tryptic peptides from complex mixtures of proteins. Strong cation exchange exclusively separates peptide by their charge state into neutral, singly and multi-charged species. To further reduce complexity, each peptide group was separated by reversed phase liquid chromatography at basic pH and the resultant fractions were analyzed by LC-MS/MS. This workflow was applied to a soluble protein lysate from mouse embryonic fibroblast cells were more than 5000 proteins from 30470 peptides were identified. The high selectivity displayed during the SCX step (95% to 100%) and the overlaps between proteins identified from the SCX-separated peptide groups, result in additional assets of the procedure that significantly increase the confidence of protein identifications. Raw data files were processed with Mascot Distiller (2.2), the mgf files were searched with Mascot Daemon (2.4), all data was stored in ms_lims Fixed modifications: cysteine alkylation Variable modifications: methionine oxidation, protein N-terminus acetylation, pyroglutamate formation of N-terminal glutamine, cyclic carbamate formation of N-terminal threonine or serine Enzyme: trypsin/P with 2 missed cleavages allowed Precursor mass tolerance: 10 ppm Peptide fragment mass tolerance: 0.5 Da Database: Swiss Prot mouse (version 56.4) The xml files of RH0 - RH1 - RH2 CID were created with pride converter 2.5.5 because this is still able to link to ms_lims. The old converter can not deal with ETD spectra, so the xml file of RH2 ETD was created with the new pride converter 2.0.3. But there is no link possible with ms_lims here, the dat files were used for xml file creation. Because of this, there's a small difference between the number of proteins and peptides in the xml file and the ones reported in the manuscript. Therefor, an extra excel file is submitted that contains all of the ETD identifications that were used in the manuscript.

			Action	DRS
	C131121_010.mgf	Mgf
	C131121_010.raw	Raw
	C131121_011.mgf	Mgf
	C131121_011.raw	Raw
	C131121_012.mgf	Mgf