Solid-Phase Peptide Capture and Release for Bulk and Single-Molecule Proteomics

Howard, Cecil J.; Floyd, Brendan M.; Bardo, Angela M.; Swaminathan, Jagannath; Marcotte, Edward M.; Anslyn, Eric V.

doi:10.1101/2020.01.13.904540

Cited by 3 publications

(4 citation statements)

References 24 publications

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“…The selective labeling of the C-terminal carboxylic acid not only provides a unique attachment point for immobilization, but its exclusivity also allows for subsequent labeling of internal acidic residues using alternate technologies, such as amide coupling, which can enhance the fluorosequencing method when used in tandem. We implemented the peptide-labeling process for angiotensin (illustrated in Figure 4A) by first conjugating the C-terminal carboxylic acid with a synthesized NB-PEG4-alkyne linker in solution using the above-described and optimized photoredox chemistry, then capturing the peptide on the solid support via its N-terminal amine using a pyridine carboxaldehyde reagent (PCA) (procedure adapted from a previous study 15 ), followed by a two-step labeling process to attach a fluorophore on glutamic acid. The two-step coupling reaction involves an amide coupling using 3azidopropylamine, followed by installing an Atto647N-PEG4-DBCO using standard copper-free click chemistry.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…The labeled peptides were cleaved by reacting the lanterns with 600 μL of trifluoroacetic acid (TFA) cocktail (95% TFA, 2.5% water, and 2.5% triisopropylsilane) at room temperature for 2 h. After blowing dry N 2 to remove the TFA and cold ether precipitation, we solubilized the fluorophore-labeled peptides with 50 μL of the acetonitrile/water mixture. We then deprotected the PCA-modified N-terminus of the peptide by incubating in hydrazine (dimethylaminoethylhydrazine dihydrochloride) at 60 °C for 16 h. 15 Peptide Surface Immobilization. For single-molecule peptide sequencing, a 40 mm German Desag 263 borosilicate glass coverslip (Bioptechs) surface was first cleaned by UV/ozone (Jelight Company) and then functionalized by soaking for 30 min in methanol containing 0.01% azidopropyltriethoxysilane (Gelest, Cat # SIA0777.0) and 4 mM acetic acid.…”

Section: ■ Methodsmentioning

confidence: 99%

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Photoredox-Catalyzed Decarboxylative C-Terminal Differentiation for Bulk- and Single-Molecule Proteomics

et al. 2021

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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 labeling chemistry is designed. In practice, only two functional groups are present in 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 singlemolecule 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 Cterminal decarboxylative labeling with an orthogonal carboxylic acid-labeling technology in tandem to establish a new platform for fluorosequencing.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Section: ■ Methodsmentioning

confidence: 99%

Photoredox-Catalyzed Decarboxylative C-Terminal Differentiation for Bulk- and Single-Molecule Proteomics

et al. 2021

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“…An ability to detect phosphoserine in the analysis was demonstrated, suggesting this strategy could be extended to other post‐translational modifications with the development of suitable labeling methods. Work continues on improving dye and measurement characteristics [63].…”

Section: Image‐based Edman Degradationmentioning

confidence: 99%

Imaging the future: the emerging era of single‐cell spatial proteomics

et al. 2021

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The proteome of a human cell is partitioned within organelles, such as the nucleus, and other subcellular compartments, such as the cytoplasm, forming a myriad of membrane‐bound and membrane‐free ultrastructures. This compartmentalization allows discrete biochemical processes to occur efficiently in isolation, with relevant proteins localized to appropriate niches to fulfill their biological function(s). Proper delivery and dynamic exchange of proteins between compartments underlie the regulation of many cellular processes, such as cell signaling, division, and programmed cell death. To this end, cells deploy dedicated trafficking mechanisms to ensure correct protein localization, as mis‐localization can result in pathology. In addition to trafficking, variation in the expression, modification, and physical associations of proteins within and between cells can result in biological heterogeneity, motivating the need for single‐cell measurements. In this review, we introduce diverse platform technologies developed for subcellular proteomics and high‐throughput systems biology, with the aim of providing mechanistic insights into fundamental cell biological processes underlying healthy and diseased states, and valuable public data resources. In contrast to the rapidly advancing field of single‐cell genomics, the single‐cell spatial proteomics toolbox remains in its infancy, but is poised to make considerable advances in the coming years.

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“…A number of approaches are also described for point selective conjugation and labeling through the N-terminal amine. 20 Another common immobilization strategy is to use the natural binding affinity or recognition of biomolecules. Antibodies are often used in immunoaffinity chromatography, where the immobilized antibodies bind their corresponding epitope target to selectively remove it from a mixture.…”

Section: Introductionmentioning

confidence: 99%

Selective C-Terminal Conjugation of Protease-Derived Native Peptides for Proteomic Measurements

et al. 2022

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Bottom-up proteomic experiments often require selective conjugation or labeling of the N- and/or C-termini of peptides resulting from proteolytic digestion. For example, techniques based on surface fluorescence imaging are emerging as a promising route to high-throughput protein sequencing but require the generation of peptide surface arrays immobilized through single C-terminal point attachment while leaving the N-terminus free. While several robust approaches are available for selective N-terminal conjugation, it has proven to be much more challenging to implement methods for selective labeling or conjugation of the C-termini that can discriminate between the C-terminal carboxyl group and other carboxyl groups on aspartate and glutamate residues. Further, many approaches based on conjugation through amide bond formation require protection of the N-terminus to avoid unwanted cross-linking reactions. To overcome these challenges, herein, we describe a new strategy for single-point selective immobilization of peptides generated by protease digestion via the C-terminus. The method involves immobilization of peptides via lysine amino acids which are found naturally at the C-terminal end of cleaved peptides from digestions of certain serine endoproteinases, like LysC. This lysine and the N-terminus, the sole two primary amines in the peptide fragments, are chemically reacted with a custom phenyl isothiocyanate (EPITC) that contains an alkyne handle. Subsequent exposure of the double-modified peptides to acid selectively cleaves the N-terminal amino acid, while the modified C-terminus lysine remains unchanged. The alkyne-modified peptides with free N-termini can then be immobilized on an azide surface through standard click chemistry. Using this general approach, surface functionalization is demonstrated using a combination of X-ray photoelectron spectroscopy (XPS), ellipsometry, and atomic force microscopy (AFM).

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Solid-Phase Peptide Capture and Release for Bulk and Single-Molecule Proteomics

Cited by 3 publications

References 24 publications

Photoredox-Catalyzed Decarboxylative C-Terminal Differentiation for Bulk- and Single-Molecule Proteomics

Photoredox-Catalyzed Decarboxylative C-Terminal Differentiation for Bulk- and Single-Molecule Proteomics

Imaging the future: the emerging era of single‐cell spatial proteomics

Selective C-Terminal Conjugation of Protease-Derived Native Peptides for Proteomic Measurements

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