Constructing a Dual-Species Compound Library

The human and maize spectral libraries (project specific, obtained via fractionated sample analysis using data-dependent acquisition LC-MS/MS) used to generate the two-species compound library have been described previously^{12 (link)}. The maize library was filtered to exclude peptides matched to either the NCBI human redundant database (April 25^th, 2018) or the UniProt^{27 (link)} human canonical proteome (3AUP000005640). The human library was filtered to include only peptides matched to the latter. In both cases, filtering was performed with leucine and isoleucine treated as the same amino acid. The libraries were merged, resulting in a library containing only precursor ions matched to either human or maize proteomes, but not both. To enable the use of the library by all of the software tools under consideration, the library was converted to the OpenMS-compatible format with the use of DIA-NN. Following the protocol of Navarro and co-workers^{21 (link)}, only precursor ions associated with at least six fragment ions were retained in the library, and all fragments but the top six (ordered by their reference intensities) were discarded. This was done to ensure that there is no bias in terms of the distribution of the number of annotated fragments between human and maize precursors. In addition, although DIA-NN can take advantage of large numbers of fragment ions described in the spectral library, many software tools tend to perform poorly if the number of fragment ions is not restricted, e.g. Spectronaut and Skyline only use the top six fragments by default. Reference retention times (Biognosys iRT scale) below -60.0 were adjusted to -60.0, to enable efficient linear retention time prediction by Skyline and OpenSWATH, as the respective precursors were observed to elute concomitantly. A low number of precursor ions had to be removed from the spectral library, so that the library could be imported error-free into Skyline (Supplementary Note 9). The resulting compound spectral library contained 202310 human precursor ions and 9781 maize precursor ions.