Multiple Reaction Monitoring assays targeting modified peptides from Plasmodium histones at 0, 18 and 36 hours post-invasion

ABSTRACT: Acid-extracted histones from the 0-hpi_2, 18-hpi_1 and 36-hpi_2 samples previously analyzed for PTMs by MudPIT were digested with endoproteinase Arg-C (RC) or endoproteinase Lys-C followed by Glu-C (KCEC). Digested histone peptides were separated on a 15cm nano-reverse phase (RP) column using a 4-hr RP-HPLC separation. The data was acquired on a LTQ-Velos-Orbitrap in low resolution mode. Based on the available fragmentation data of the modified peptides, we selected between three to seven MS/MS fragment ions for transitions. The criteria for selecting the fragment ions were that the fragments included the amino acid residue(s) modified in the peptide. The Multiple Reaction Monitoring (MRM) method was built by including the parent mass, parent mass selection window, product mass and mass selection windows for these three to seven product ions. Average and monoisotopic m/z values were used for the parent ion mass and the product ions, respectively. The number of simultaneously assayable peptides was limited to 12. SRM and the MS/MS spectra for the same parent ion were collected consecutively within the same run. Each of the 24 precursor ions targeted for SRM was assayed at least twice independently in each of the 3 samples analyzed (Supplementary Files 0-, 18-, 36-hpi_MRM-Assays_Overview.xlsx). The 24 peptides targeted for SRM were validated manually to confirm the presence of the relevant transitions in the SRM spectra followed by their corresponding MS/MS spectrum. When a peptide was not observed in one of the 3 stages analyzed, the unfiltered PSMs (below cut-off) were also manually assessed to ensure that the modified peptide expression was indeed stage-specific and not missed due to the conservative selection criteria. Annotated MS/MS spectra for all modified peptides are provided in Supplementary Files 0-, 18-, 36-hpi_ MRMs_MS2-Annotated-Spectra.pdf.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (ncbitaxon:9606) Plasmodium Falciparum 3d7 (ncbitaxon:36329)

SUBMITTER: Laurence Florens 

PROVIDER: MSV000079792 | MassIVE | Thu Jun 02 12:26:00 BST 2016

SECONDARY ACCESSION(S): PXD004268

REPOSITORIES: MassIVE