The emerging landscape of single-molecule protein sequencing technologies

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.This Perspective describes new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell proteomics.</p

Single-molecule fret for protein characterization

The invention provides an analysis method (100) for characterization of a tagged protein (10) using FRET donor-acceptor pair chromophores (20), wherein the FRET donor- acceptor pair chromophores (20) comprise a first chromophore (21) and a second chromophore (22), wherein the FRET donor-acceptor pair chromophores (20) have a donor excitation radiation range, a donor emission radiation range and an acceptor emission radiation range, wherein one of the FRET donor-acceptor pair chromophores (23) is excitable by donor excitation radiation (51) in the donor excitation radiation range, wherein the other of the FRET donor-acceptor pair chromophores (24) is configured to provide acceptor emission radiation (54) in the acceptor emission radiation range upon excitation with donor excitation radiation (51) in the donor excitation radiation range of the one of the FRET donor-acceptor pair chromophores (23) when the first chromophore (21) and the second chromophore (22) are configured within a predetermined distance, wherein the tagged protein (10) comprises a first amino acid (11) tagged with a first tag (31) and a second amino acid (12) tagged with a second tag (32), wherein the first tag (31) comprises the first chromophore (21) or is associated to the first chromophore (21), wherein the second tag (32) comprises an oligonucleotide, and wherein the analysis method (100) comprises: a barcode exposure stage (110) comprising: (i) exposing the tagged protein (10) to a barcode (42), wherein the barcode (42) is configured to hybridize with the second tag (32), and wherein the barcode (42) comprises the second chromophore (22), (ii) providing radiation having a wavelength selected from the donor excitation radiation range to the tagged protein (10), and (iii) measuring emission in the donor emission radiation range and the acceptor emission radiation range to provide an emission signal.</p

Light sensitive phenacyl crosslinked dextran hydrogels for controlled delivery

Stimuli-responsive soft materials enable controlled release of loaded drug molecules and biomolecules. Controlled release of potent chemotherapeutic or immunotherapeutic agent is crucial to reduce unwanted side effects. In an effort to develop controlled release strategies that can be triggered using Cerenkov luminescence, we have developed polymer hydrogels that can release doxorubicin, bovine serum albumin and immunoglobulin G using light (254 nm – 375 nm) as a trigger. We describe the synthesis and photochemical characterisation of two light sensitive phenacyl bis-azide crosslinkers that are used to prepare transparent self-supporting hydrogel patches. One crosslinker was designed to optimize the overlap with the Cerenkov luminescence emission window, bearing an -extended phenacyl core, resulting in a high quantum yield (14%) of photocleavage when irradiated with 375 nm light. We used the extended phenacyl crosslinker for the preparation of protein-loaded dextran hydrogel patches, which showed efficient and selective dosed release of bovine serum albumin or immunoglobulin G after irradiation with 375 nm light. Based on the high quantum yield, efficient release and large overlap with the Cerenkov window, we envision application of these photosensitive soft materials in radiation targeted drug release

Highly sensitive CE-ESI-MS analysis of N-glycans from complex biological samples

In-depth characterization of complex glycomes is complicated by the immense structural diversity of glycans. Here, the authors present a mass spectrometry-based strategy for untargeted, sensitive glycan profiling and identify 167 N-glycan compositions in total human plasma

High-Speed Super-Resolution Imaging Using Protein-Assisted DNA-PAINT

Super-resolution imaging allows for the visualization of cellular structures on a nanoscale level. DNA-PAINT (DNA point accumulation in nanoscale topology) is a super-resolution method that depends on the binding and unbinding of DNA imager strands. The current DNA-PAINT technique suffers from slow acquisition due to the low binding rate of the imager strands. Here we report on a method where imager strands are loaded into a protein, Argonaute (Ago), which allows for faster binding. Ago preorders the DNA imager strand into a helical conformation, allowing for 10 times faster target binding. Using a 2D DNA origami structure, we demonstrate that Ago-assisted DNA-PAINT (Ago-PAINT) can speed up the current DNA-PAINT technique by an order of magnitude, while maintaining the high spatial resolution. We envision this tool to be useful for super-resolution imaging and other techniques that rely on nucleic acid interactions.</p

Conditional Copper-Catalyzed Azide Alkyne Cycloaddition by Catalyst Encapsulation

Supramolecular encapsulation is known to alter chemical properties of guest molecules. Here we apply this strategy of molecular encapsulation to temporally control the catalytic activity of a stable Cu(I)-carbene catalyst. Encapsulation of the Cu(I)-carbene catalyst by supramolecular host cucurbit[7]uril (CB[7]) resulted in the complete inactivation of a copper catalyzed alkyne-azide cycloaddition (CuAAC) reaction. The addition of a chemical signal achieved the near instantaneous activation of the catalyst, by releasing the catalyst from the inhibited CB[7] catalyst complex. To broaden the scope of our on demand CuAAC reaction, we demonstrated the protein labelling of Vinculin using the Cu(I)-carbene catalyst, to inhibit its activity by encapsulation with CB[7], and to initiate labelling at any moment by adding a specific signal molecule. <br /