Quantification of Protein Glycosylation Using Nanopores

Abstract

Although nanopores can be used for single-molecule sequencing of nucleic acids using low-cost portable devices, the characterization of proteins and their modifications has yet to be established. Here, we show that hydrophilic or glycosylated peptides translocate too quickly across FraC nanopores to be recognized. However, high ionic strengths (i.e., 3 M LiCl) and low pH (i.e., pH 3) together with using a nanopore with a phenylalanine at its constriction allows the recognition of hydrophilic peptides, and to distinguish between mono- and diglycosylated peptides. Using these conditions, we devise a nanopore method to detect, characterize, and quantify post-translational modifications in generic proteins, which is one of the pressing challenges in proteomic analysis.

Selective Detection and Characterization of Small Cysteine-Containing Peptides with Cluster-Modified Nanopore Sensing

“…These blockages can be analyzed to ascertain properties of the molecule in question. Recent efforts by nanopore researchers have focused on a variety of peptide and protein applications that include sequencing, − detection of various peptide modifications, , and peptide sensing and analysis . Most efforts that apply nanopore detection to peptide sensing have utilized high-throughput detection to enable construction of size distributions, while fewer studies have used applications that utilize modifications to the nanopore environment to facilitate long-time extended interrogation of single molecules. , Some of these pore modification studies have the advantage of enabling clear identification of the molecule of interest without the need for constructing current blockade or transit time distributions. − …”

Unidirectional Single-File Transport of Full-Length Proteins Through a Nanopore

“…Finally, although we use GdmCl for threading/stretching a protein in the pore, we note that combining this mode with enzyme-driven protein motion is possible, for example, by using an asymmetric buffer in which: 1) protein unfolding is mediated in a denaturant chamber, 2) electroosmotic forces are used to keep the protein taut at the pore, and 3) enzyme-mediated motion on another chamber of the pore is used to move the protein through the pore in a discrete manner. Other improvements such as changing the electrolyte type 59 , pore variant [60][61][62] , and voltage waveform, can be made to further enhance the ability of our method to obtain more unique and unequivocal fingerprint signatures from full-length proteins.…”

Mass Spectrometry-Based Glycoproteomic Workflows for Cancer Biomarker Discovery

“…It is clear that the current discoveries surrounding glycosylation in cancer generated from mass spectrometry-based techniques are the tip of the iceberg for understanding these important PTMs. 30 , 35 We expect that continued advances in specific glycan-specific enrichments, including the development of nanobodies 149 and the use of nanopores, 150 combined with standardized protocols and user-friendly software strategies will allow more laboratories to adopt discovery glycoproteomics. Deep visual proteomics combines the capabilities of workflow A and C in Figure 2 to allow for the spatial resolution of both glycans and deep global proteomics.…”