Studies of Surface Preparation for the Fluorosequencing of Peptides

Abstract

Silica passivating agents have shown great success in minimizing nonspecific protein binding to glass surfaces for imaging and microscopy applications. Amine-derivatized surfaces are commonly used in conjugation with amide coupling agents to immobilize peptides/proteins through C-terminal or side-chain carboxylic acids. In the case of the single-molecule fluorosequencing of peptides, attachment occurs via the C-terminus and nonspecific surface binding has previously been a source of error in peptide identification. Here, we employ fluorosequencing as a high-throughput, single-molecule sensitivity assay to identify and quantify the extent of nonspecific binding of peptides to amine-derivatized surfaces. We show that there is little improvement when using common passivating agents in combination with the surface derivatizing agent 3-amino­propyl-tri­ethoxy­silane (APTES) to couple the peptides to the modified surface. Furthermore, many xanthene fluorophores have carboxylic acids in the appended phenyl ring at positions ortho and meta or ortho and para, and the literature shows that conjugation through the ortho position is not favored. Because xanthene-derived fluorophores are commonly used for single-molecule applications, we devised a novel assay to probe the conjugation of peptides via their fluorophores relative to their C-termini on silane-derivatized surfaces. We find significant attachment to the ortho position, which is a warning to those attempting to immobilize fluorophore-labeled peptides to silica surfaces via amide coupling agents. However, eliminating all amines on the surface by switching to 3-azido­propyl-tri­ethoxy­silane (AzTES) for coupling via copper-catalyzed azide–alkyne cycloaddition (CuAAC) and omitting additional passivation agents allowed us to achieve a high level of C-terminally bound peptides relative to nonspecifically or ortho-phenyl-bound, fluorophore-labeled peptides. This strategy substantially improves the specificity of peptide immobilization for single-molecule fluorosequencing experiments.

Estimating error rates for single molecule protein sequencing experiments

“…Background sigma (the dispersion of the zero-count distribution) is computed automatically through a different mechanism. During signal processing with sigproc_v2 , the image processing routine in the fluorosequencing data analysis package plaster SigProc ( https://github.com/erisyon/plaster_v1 , as in [ 22 , 23 ]) signal is extracted from the central pixels of a peak, and a local background value is subtracted from the signal. The local background is computed by averaging the values of pixels just outside those used to compute the signal.…”

Amino acid sequence assignment from single molecule peptide sequencing data using a two-stage classifier

“…These radiometry raw reads simulate the fluorescent intensity data we would expect to collect from processing raw single molecule microscope images, a process currently performed for experimental data using the algorithm SigProc (Part of Erisyon's tool Plaster, https:// github.com/erisyon/plaster_v1), as in [10,11].…”

Robust and scalable single-molecule protein sequencing with fluorosequencing

“…For synthetic peptides, this was achieved using Fmoc-propargylglycine as the C-terminal amino acid building block. After making this modification we observed a 23-fold improvement in correctly sequenceable peptides (Hinson et al 2021). We therefore moved exclusively to immobilizing all peptides in this work by azide-alkyne click chemistry.…”