Af luorescent sensor for catecholamines,N S510, is presented. The sensor is based on aq uinolone fluorophore incorporating ab oronic acid recognition element that gives it high affinity for catecholamines and at urn-on response to norepinephrine.T he sensor results in punctate staining of norepinephrine-enriched chromaffin cells visualizedu sing confocal microscopyi ndicating that it stains the norepinephrine in secretory vesicles.A mperometry in conjunction with total internal reflection fluorescence (TIRF) microscopy demonstrates that the sensor can be used to observe destaining of individual chromaffin granules upon exocytosis.N S510 is the highest affinity fluorescent norepinephrine sensor currently available and can be used for measuring catecholamines in live-cell assays.
Methods for the selective labeling of biogenic functional groups on peptides are being developed and used in the workflow of both current and emerging proteomics technologies, such as single-molecule fluorosequencing. To achieve successful labeling with any one method requires that the peptide fragments contain the functional group for which the labeling chemistry is designed. In practice, only two functional groups are present on every peptide fragment regardless of the protein cleavage site, namely, an N-terminal amine and a C-terminal carboxylic acid. Developing a global-labeling technology, therefore, requires one to specifically target the N- and/or C-terminus of peptides. In this work, we showcase the first successful application of photocatalyzed C-terminal decarboxylative-alkylation for peptide mass-spectrometry and single molecule protein sequencing, that can be broadly applied to any proteome. We demonstrate that peptides in complex mixtures generated from enzymatic digests from bovine serum albumin, as well as protein mixtures from yeast and human cell extracts, can be site-specifically labeled at their C-terminal residue with a Michael acceptor. Using two distinct analytical approaches, we characterize C-terminal labeling efficiencies of greater than 50% across complete proteomes and document the proclivity of various C-terminal amino acid residues for decarboxylative-labeling, showing histidine and tryptophan to be the most disfavored. Finally, we combine C-terminal decarboxylative labeling with an orthogonal carboxylic acid labeling technology in tandem, to establish a new platform for fluorosequencing.
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