The tumor microenvironment and proinflammatory signals significantly alter glycosylation of cell-surface proteins on endothelial cells. By altering the N-glycosylation machinery in the endoplasmic reticulum and Golgi, proinflammatory cytokines promote the modification of endothelial glycoproteins such as vascular endothelial growth factor receptor 2 (VEGFR2) with sialic acid-capped N-glycans. VEGFR2 is a highly N-glycosylated receptor tyrosine kinase involved in pro-angiogenic signaling in physiological and pathological contexts, including cancer. Here, using glycoside hydrolase and kinase assays and immunoprecipitation and MS-based analyses, we demonstrate that N-linked glycans at the Asn-247 site in VEGFR2 hinder VEGF ligand-mediated receptor activation and signaling in endothelial cells. We provide evidence that cell surfaceassociated VEGFR2 displays sialylated N-glycans at Asn-247 and, in contrast, that the nearby sites Asn-145 and Asn-160 contain lower levels of sialylated N-glycans and higher levels of high-mannose N-glycans, respectively. Furthermore, we report that VEGFR2 Asn-247-linked glycans capped with sialic acid oppose ligand-mediated VEGFR2 activation, whereas the uncapped asialo-glycans favor activation of this receptor. We propose that N-glycosylation, specifically the capping of N-glycans at Asn-247 by sialic acid, tunes ligand-dependent activation and signaling of VEGFR2 in endothelial cells.

Human tissue samples commonly preserved as formalin-fixed paraffin-embedded (FFPE) tissues after diagnostic or surgical procedures in the clinic represent an invaluable source of clinical specimens for indepth characterization of signaling networks to assess therapeutic options. Tyrosine phosphorylation (pTyr) plays a fundamental role in cellular processes and is commonly dysregulated in cancer but has not been studied to date in FFPE samples. Additionally, pTyr analysis that may otherwise inform therapeutic interventions for patients has been limited by the requirement for large amounts of frozen tissue. Here we describe a method for highly sensitive, quantitative analysis of pTyr signaling networks, with hundreds of sites quantified from 1-2 10-µm sections of FFPE tissue specimens. A combination of optimized magnetic bead-based sample processing, optimized pTyr enrichment strategies, and TMT multiplexing enabled in depth coverage of pTyr signaling networks from small amounts of input material. Phosphotyrosine profiles of flash frozen and FFPE tissues derived from the same tumors suggested that FFPE tissues preserve pTyr signaling characteristics in patient-derived xenografts and archived clinical specimens. pTyr analysis of FFPE tissue sections from breast cancer tumors as well as lung cancer tumors highlighted patient-specific oncogenic driving kinases, indicating potential targeted therapies for each patient. These data suggest the capability for direct translational insight from pTyr analysis of small amounts of FFPE tumor tissue specimens.

Neurochemical dysregulation underlies many pathologies and can be monitored by measuring the composition of brain interstitial fluid (ISF). Existing in vivo tools for sampling ISF do not enable measuring large rare molecules, such as proteins and neuropeptides, and thus cannot generate a complete picture of the neurochemical connectome. Our micro-invasive platform, composed of a nanofluidic pump coupled to a membrane-free probe, enables sampling multiple neural biomarkers in parallel. This platform outperforms the state of the art in low-flow pumps by offering low volume control (single stroke volumes, <3 nl) and bidirectional fluid flow (<100 nl/min) with negligible dead volume (<30 nl) and has been validated in vitro, ex vivo, and in vivo in rodents. ISF samples (<1.5 μL) can be processed via liquid chromatography–tandem mass spectrometry. These label-free liquid biopsies of the brain could yield a deeper understanding of the onset, mechanism, and progression of diverse neural pathologies.

Formalin fixed paraffin embedded (FFPE) tissues are an invaluable source of clinical specimens. Tyrosine phosphorylation (pTyr) plays a fundamental role in cellular processes and is commonly dysregulated in cancer but has not been studied to date in FFPE samples. We describe a method for quantitative analysis of pTyr signaling networks at an unprecedented sensitivity, with hundreds of sites quantified from 1-2 10-μm sections of FFPE tissue specimens. Phosphotyrosine profiles of flash frozen and FFPE tissues derived from the same tumors suggest that FFPE tissues preserve pTyr signaling characteristics in PDX tumors and archived clinical specimens. Differential activation of oncogenic proteins was observed in triple negative breast cancer tumors as well as lung cancer tumors, highlighting patient specific oncogenic driving kinases and indicating potential targeted therapies for each patient. These data highlight the capability for direct translational insight from pTyr analysis of small amounts of FFPE tumor tissue specimens.

Supplementary Data from Quantitative Analysis of Tyrosine Phosphorylation from FFPE Tissues Reveals Patient-Specific Signaling Networks