Developing Soybean Seed Lysine-N-epsilon-acetylome

Intact proteins, as well as Glu-C, and Lys-C/trypsin-derived peptides extracted from developing soybean seeds were immunoenriched using antibodies against acetyllysine residues. Enriched proteins were separated by SDS-PAGE prior to tryptic digestions and enriched peptides were analyzed directly, in both cases using a Thermo LTQ-Orbitrap XL. In separate LC-MS/MS runs fragmentation was achieved using CID, ETD, or a combination of the two mediated by a data-dependent decision tree. Individual RAW files were converted to mzXML format for mining with MS-GFDB (v2012.06.07) using default settings of the msconvert executable within the Trans-Proteomic Pipeline v4.6. Searches with MS-GFDB were performed with default methods and parameters, except as noted: number of allowed isotope errors was set at zero, and fully enzymatic peptides were specified, i.e. no non-enzymatic termini. Data mining was additionally performed with a local Mascot server, v2.3.1, and ZCore v1.17, both accessed through Proteome Discoverer v1.3 (Thermo Scientific), as well as Sequest HT v1.3, a reimplementation of SEQUEST within Proteome Discoverer v1.4. Tandem mass spectra were searched in Proteome Discoverer with allowed missed cleavages set to four. Prior to Mascot and Sequest searches, ETD spectra were further filtered to remove peaks resulting from precursors, charge-reduced precursors, and neutral losses thereof with mass windows of 4 Da, 2 Da, and 2 Da, respectively.