The need for mass-spectrometric determination of protein identity severely limits the throughput of many whole-cell proteomic analyses. We have developed an approach to physically connect in vivo-expressed proteins to their encoding messenger RNAs (mRNAs). This genotype–phenotype linkage enables us to carry out a variety of whole-proteome assays and determine the identity of each protein using nucleic acid sequencing. We show that the resulting in vivo mRNA display technology enables determination of protein subcellular localization and protein–protein interactions, with high sensitivity and specificity, using simple purification followed by high-throughput sequencing. In vivo mRNA display should enable more efficient interrogation of proteome behavior in a variety of applications that are currently limited by the cost and throughput of mass-spectrometric analysis.

Large-scale proteomic methods are essential for the functional characterization of proteins in their native cellular context. However, proteomics…

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