Robust and scalable single-molecule protein sequencing with fluorosequencing.

The need to accurately survey proteins and their modifications with ever higher sensitivities, particularly in clinical settings with limited samples, is spurring development of new single molecule proteomics technologies. Fluorosequencing is one such highly parallelized single molecule peptide sequencing platform, based on determining the sequence positions of select amino acid types within peptides to enable their identification and quantification from a reference database. Here, we describe substantial improvements to fluorosequencing, including identifying fluorophores compatible with the sequencing chemistry, mitigating dye-dye interactions through the use of extended polyproline linkers, and developing an end-to-end workflow for sample preparation and sequencing. We demonstrate by fluorosequencing peptides in mixtures and identifying a target neoantigen from a database of decoy MHC peptides, highlighting the potential of the technology for high sensitivity clinical applications.

Estimating error rates for single molecule protein sequencing experiments.

The practical application of new single molecule protein sequencing (SMPS) technologies requires accurate estimates of their associated sequencing error rates. Here, we describe the development and application of two distinct parameter estimation methods for analyzing SMPS reads produced by fluorosequencing. A Hidden Markov Model (HMM) based approach, extends whatprot, where we previously used HMMs for SMPS peptide-read matching. This extension offers a principled approach for estimating key parameters for fluorosequencing experiments, including missed amino acid cleavages, dye loss, and peptide detachment. Specifically, we adapted the Baum-Welch algorithm, a standard technique to estimate transition probabilities for an HMM using expectation maximization, but modified here to estimate a small number of parameter values directly rather than estimating every transition probability independently, which should help prevent overfitting. We demonstrate a high degree of accuracy on simulated data, but on experimental datasets, we observed that the model needed to be augmented with an additional error type, N-terminal blocking. This, in combination with data pre-processing, results in reasonable parameterizations of experimental datasets that agree with controlled experimental perturbations. A second independent implementation using a hybrid of DIRECT and Powell's method to reduce the root mean squared error (RMSE) between simulations and the real dataset was also developed. We compare these methods on both simulated and real data, finding that our Baum-Welch based approach outperforms DIRECT and Powell's method by most, but not all, criteria. Although some discrepancies between the results exist, we also find that both approaches provide similar error rate estimates from experimental single molecule fluorosequencing datasets.

Evaluating the Effect of Dye-Dye Interactions of Xanthene-Based Fluorophores in the Fluorosequencing of Peptides.

A peptide sequencing scheme utilizing fluorescence microscopy and Edman degradation to determine the amino acid position in fluorophore-labeled peptides was recently reported, referred to as fluorosequencing. It was observed that multiple fluorophores covalently linked to a peptide scaffold resulted in a decrease in the anticipated fluorescence output and worsened the single-molecule fluorescence analysis. In this study, we report an improvement in the photophysical properties of fluorophore-labeled peptides by incorporating long and flexible (PEG)10 linkers at the peptide attachment points. Long linkers to the fluorophores were installed using copper-catalyzed azide-alkyne cycloaddition conditions. The photophysical properties of these peptides were analyzed in solution and immobilized on a microscope slide at the single-molecule level under peptide fluorosequencing conditions. Solution-phase fluorescence analysis showed improvements in both quantum yield and fluorescence lifetime with the long linkers. While on the solid support, photometry measurements showed significant increases in fluorescence brightness and 20 to 60% improvements in the ability to determine the amino acid position with fluorosequencing. This spatial distancing strategy demonstrates improvements in the peptide sequencing platform and provides a general approach for improving the photophysical properties in fluorophore-labeled macromolecules.

Studies of Surface Preparation for the Fluorosequencing of Peptides.

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.

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

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-release 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 survey peptide capture biases from over 40 000 unique peptides from a cellular proteome. We also show that this capture can be reversed in a traceless manner, such that it is amenable for single-molecule 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 single-molecule protein sequencing.

Highly parallel single-molecule identification of proteins in zeptomole-scale mixtures.

The identification and quantification of proteins lags behind DNA-sequencing methods in scale, sensitivity, and dynamic range. Here, we show that sparse amino acid-sequence information can be obtained for individual protein molecules for thousands to millions of molecules in parallel. We demonstrate selective fluorescence labeling of cysteine and lysine residues in peptide samples, immobilization of labeled peptides on a glass surface, and imaging by total internal reflection microscopy to monitor decreases in each molecule's fluorescence after consecutive rounds of Edman degradation. The obtained sparse fluorescent sequence of each molecule was then assigned to its parent protein in a reference database. We tested the method on synthetic and naturally derived peptide molecules in zeptomole-scale quantities. We also fluorescently labeled phosphoserines and achieved single-molecule positional readout of the phosphorylated sites. We measured >93% efficiencies for dye labeling, survival, and cleavage; further improvements should enable studies of increasingly complex proteomic mixtures, with the high sensitivity and digital quantification offered by single-molecule sequencing.