The identification and quantification of proteins lags behind DNA sequencing methods in scale, sensitivity and dynamic range. Here we show that sparse amino acid sequence information can be obtained for individual protein molecules for thousands to millions of molecules in parallel. We demonstrate selective fluorescent labeling of cysteine and lysine residues in peptide samples, immobilization of labeled peptides on a glass surface, and imaging by total internal reflection microscopy to monitor reductions in each molecule’s fluorescence following consecutive rounds of Edman degradation. The obtained sparse fluorescent sequence of each molecule was then assigned to its parent protein in a reference database. We demonstrate the method on synthetic and naturally-derived peptide molecules in zeptomole-scale quantities. We also fluorescently label phosphoserines and demonstrate single-molecule, positional readout of the phosphorylated sites. We measured >93% efficiencies for dye labeling, survival, and cleavage; further improvements should empower studies of increasingly complex proteomic mixtures, with the high sensitivity and digital quantification offered by single molecule sequencing.

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A small molecule library of pyrido[2,3-d]pyrimidine-2,4-dione derivatives 6-16 was synthesized from 6-amino-1,3-disubstituted uracils 18, characterized, and screened for inhibitory activity against eukaryotic elongation factor-2 kinase (eEF-2K). To understand the binding pocket of eEF-2K, structural modifications of the pyrido[2,3-d]pyrimidine were made at three regions (R(1), R(2), and R(3)). A homology model of eEF-2K was created, and compound 6 (A-484954, Abbott laboratories) was docked in the catalytic domain of eEF-2K. Compounds 6 (IC50=420nM) and 9 (IC50=930nM) are found to be better molecules in this preliminary series of pyrido[2,3-d]pyrimidine analogs. eEF-2K activity in MDA-MB-231 breast cancer cells is significantly reduced by compound 6, to a lesser extent by compound 9, and is unaffected by compound 12. Similar inhibitory results are observed when eEF-2K activity is stimulated by 2-deoxy-d-glucose (2-DOG) treatment, suggesting that compounds 6 and 9 are able to inhibit AMPK-mediated activation of eEF-2K to a notable extent. The results of this work will shed light on the further design and optimization of novel pyrido[2,3-d]pyrimidine analogs as eEF-2K inhibitors.

Polymer topology dictates dynamic and mechanical properties of materials. For most polymers, topology is a static characteristic. In this article, we present a strategy to chemically trigger dynamic topology changes in polymers in response to a specific chemical stimulus. Starting with a dimerized PEG and hydrophobic linear materials, a lightly cross-linked polymer, and a cross-linked hydrogel, transformations into an amphiphilic linear polymer, lightly cross-linked and linear random copolymers, a cross-linked polymer, and three different hydrogel matrices were achieved via two controllable cross-linking reactions: reversible conjugate additions and thiol−disulfide exchange. Significantly, all the polymers, before or after topological changes, can be triggered to degrade into thiol-or amine-terminated small molecules. The controllable transformations of polymeric morphologies and their degradation herald a new generation of smart materials.

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