Mass spectrometry-based proteomics enables the global identification and quantification of proteins and their posttranslational modifications in complex biological samples. However, proteomic analysis requires a complete and accurate reference set of proteins and is therefore largely restricted to model organisms with sequenced genomes. Here, we demonstrate the feasibility of deep genome-free proteomics by using a reference proteome derived from heterogeneous mRNA data. We identify more than 11,000 proteins with 99% confidence from the unfertilized Xenopus laevis egg and estimate protein abundance with approximately 2-fold precision. Our reference database outperforms the provisional gene models based on genomic DNA sequencing and references generated by other methods. Surprisingly, we find that many proteins in the egg lack mRNA support and that many of these proteins are found in blood or liver, suggesting that they are taken up from the blood plasma, together with yolk, during oocyte growth and maturation, potentially contributing to early embryogenesis. To facilitate proteomics in nonmodel organisms, we make our platform available as an online resource that converts heterogeneous mRNA data into a protein reference set. Thus, we demonstrate the feasibility and power of genome-free proteomics while shedding new light on embryogenesis in vertebrates. •Genome-free proteomics identifies more than 11,000 proteins in the Xenopus laevis egg•Each protein’s expression level is predicted with approximately 2-fold precision•Many blood plasma proteins are taken up from oocyte during growth in the ovary•Web tool generates proteomic reference sets from mRNA data for any organism Wühr et al. demonstrate the feasibility of deep proteomics, without the use of a sequenced genome, using the example of the egg of the African clawed frog Xenopus laevis. The authors identify more than 11,000 proteins and can predict each protein’s expression level with approximately 2-fold precision.