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

Abstract

The field of proteomics has expanded recently with more sensitive techniques for the bulk measurement of peptides as well as single-molecule techniques. One limiting factor for some of these methods is the need for multiple chemical derivatizations and highly pure proteins free of contaminants. We demonstrate a solid-phase capture strategy suitable for the proteolysis, purification, and subsequent chemical modification of peptides. We use this resin on an HEK293T cell lysate and perform one-pot proteolysis, capture, and derivatization to generate a cellular proteome that identified over 40,000 bead-bound peptides. We also show that this capture can be reversed in a traceless manner, such that it is amenable for singlemolecule proteomics techniques. With this technique, we perform a fluorescent labeling and C-terminal derivatization on a peptide and subject it to fluorosequencing, demonstrating that washing the resin is sufficient to remove excess dyes and other reagents prior to singlemolecule protein sequencing.Scheme 1. Formation of the N-terminal imidazolidinone cap by pyridinyl carboxaldehyde [2] .

Photoredox-catalyzed decarboxylative C-terminal differentiation for bulk and single molecule proteomics

“…The selective labeling of the C -terminal carboxylic acid not only provides a unique attachment point for immobilization, but its exclusivity 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 solid support via’ its N -terminal amine using a pyridine carboxaldehyde reagent (PCA) (procedure adapted from 15 ), followed by a two-step labeling process to attach a fluorophore on the glutamic acid. The two-step coupling reaction involves an amide coupling using 3-azidopropylamine followed by installing an Atto647N-PEG4-DBCO using standard copper-free click chemistry.…”

“…We then de-protected the PCA modified N-terminus of the peptide by incubating in a hydrazine (dimethylaminoethylhydrazine dihydrochloride) at 60 °C for 16h. 15…”

“…A number of techniques have been developed for the selective and efficient labeling of the N -terminal amino group of peptides and proteins (as opposed to the amine of lysine residues) 14,15 . Corresponding methods for attaching a synthetic reporter to the C -terminus of peptides and proteins that also keep alternate side chain acidic residues (i.e., aspartate and glutamate) pristine are few and seldom well optimized 16–18 .…”

The emerging landscape of single-molecule protein sequencing technologies

“…Furthermore, the reliance on chemical labeling leads to partial sequencing of the peptide, with the unidentified remainder inferred by comparison to a reference proteome. In addition, inefficient labeling can lead to errors that must be modeled into the reference proteome comparison, spurring the development of new protocols to increase yields 10 . Exciting new proposals could add the dimension of protonation-based sequencing.…”

“…A more general methodology has also emerged where the N-terminal amine condenses with 2-pyridinecarboxaldehyde, forming an imine structure that further reacts via cyclization with the nearby amide nitrogen of the second amino acid to form a stable imidazolidinone product 134 . This reaction has recently been shown to be useful for single-molecule peptide sequencing as a method for the immobilization of peptides onto a solid-phase resin, multiple chemical derivatization steps without purification and subsequent traceless release before fluorosequencing 10 .…”