A streamlined platform for phosphoproteome mapping of human tissues

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ABSTRACT Recent technological advances in genomics, transcriptomics, and proteomics allow for rapid generation of comprehensive 3D-human tissue maps for biomolecules DNAs, RNAs, and proteins at the single-cell resolution in the HuBMAP consortium. However, single-cell technologies for characterizing functional modifications are lagging far behind but equally important as these existing omics technologies. Protein phosphorylation is one of the most important modifications and often used as an indicator of signaling pathway activation (cell functional state). The lack of high-spatial-resolution phosphoproteomic characterization of human tissues in the HuBMAP consortium represents a significant knowledge gap for achieving a more complete understanding of how tissue heterogeneity impacts human health. The objective of this TTD application is to address this gap by developing a convenient streamlined platform for enabling automated high-resolution 3D-phosphoproteome mapping of human tissues. The project feasibility is strongly supported by our recent progress in many aspects of technology development: 1) Carrier-assisted sample preparation (CASP) for both global and targeted proteomics analysis of 1-100 cells; 2) A boosting to amplify signal with isobaric labeling (BASIL) strategy for high-throughput single- cell proteomics; 3) BASIL/Tip-IMAC (immobilized metal affinity chromatography) for rapid phosphoproteomic analysis of small numbers of cells; 4) Advanced liquid chromatography (LC) separation and disruptive mass spectrometry (MS) technologies (e.g., multi-emitter array technology and sub-ambient pressure ionization with nanoelectrospray (SPIN) source) for improving MS detection sensitivity. In the UG3 phase, Aim 1 will focus on the development of a streamlined platform through 1) improving phospho-recovery by developing an improved CASP/online IMAC platform for automated processing and phospho-enrichment, and 2) leveraging multiple disruptive technologies developed at our group with integration of a high-efficiency multi-emitter SPIN (mSPIN) source and BASIL-based sample multiplexing for significantly improving MS sensitivity by ~50-fold and sample throughput by >20-fold. The streamlined platform will allow for precise quantification of ~1,000 phosphosites in single cells and ~7000 phosphosites in 10 cells with >1000 samples per day. Aim 2 will demonstrate the streamlined platform for enabling 2D-phosphoproteome mapping of mouse uterine tissues when combined with laser capture microdissection (LCM) and standard tube-based voxel collection. In the UH3 phase (Aim 3) we will further optimize the streamlined platform for automated robust phosphoproteomic analysis of LCM-dissected human tissue sections. We then will validate the streamlined platform for high-resolution 3D-phosphoproteome mapping of human breast and uterine tissues and other human tissues from the HuBMAP consortium. An easy- to-use visualization tool will be developed to generate 3D maps that can be quickly and easily accessible by the research community. With its antibody-free feature, the streamlined platform can be equally applicable to any types of tissues. We envision that the streamlined platform will become an indispensable tool for high-resolution 3D-phosphopeoteome mapping of human tissues in the HuBMAP consortium and extend the HuBMAP toolbox for 3D-mapping of functional modifications. In turn, it will make substantial contributions to improve our understanding of tissue biology and accelerate the movement toward precision medicine.