Here we have undertaken a pilot study analysing the proteome of MD2 pineapple using Liquid Chromatography coupled with Mass Spectrometry (LC-MS/MS) and identified 3,818 peptides from 1,781 candidate proteins in the annotated V3 genome sequenced. In addition, a further 603 peptide identifications were found that mapped exclusively to an independent MD2 transcriptome-derived database and were not found in the standard V3 annotated proteome. Peptide identifications derived from the MD2 transcripts were also cross-referenced to a more recent and complete MD2 genome annotation, resulting in 402 non overlapping peptides with either of the genomes. Subsequently, cross-referencing these peptide identifications via their parent transcripts, 30 high-quality gene candidates novel to the V3 pineapple genomes were identified. These analyses add to the knowledge of experimentally validated pineapple genes and demonstrate the utility of transcript-derived proteomics to discover both novel genes and genetic structure in plant genome, and can help improve its annotation.