Fig. 1.
Workflow of Spectral Library Generation. A, Workflow: 846 genes from the human ORFeome bank were introduced via Gateway cloning into a wheat germ expression vector (vector map in supplemental Fig. S1A) containing a 5′-FLEXII-Tag (sequence in supplemental Fig. S1B) resulting in a clone library with 801 of the selected 846 ORFs (95%). FLEXII-tagged proteins were expressed in cell-free wheat germ expression system and enriched via Ni-NTA beads. Eighty-four percent of the expressed clones yielded visible bands on SDS-PAGE (details in supplemental Fig. S1C). The purified proteins were in solution trypsinized and the peptide mixtures were analyzed in pools of eight by LC-MS/MS on a Q-Exactive mass spectrometer. Database searches were performed by the ProteinPilot software and a global FDR cutoff of 1% was chosen for peptide/protein identification resulting in 735 identified proteins, that is an identification rate of 90%; this included proteins not visible by SDS-PAGE. Absolute quantification was carried out using synthetic peptide-based isotope dilution mass spectrometry when necessary. MS data are publicly available at Peptide Atlas (www.peptideatlas.org). B, Sequence coverage distribution for the 735 identified proteins. C, Distribution of the observed number of peptides per protein for the 735 identified proteins.