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 fluorescent 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 reductions in each molecule’s fluorescence following 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 demonstrate the method on synthetic and naturally-derived peptide molecules in zeptomole-scale quantities. We also fluorescently label phosphoserines and demonstrate single-molecule, positional readout of the phosphorylated sites. We measured >93% efficiencies for dye labeling, survival, and cleavage; further improvements should empower studies of increasingly complex proteomic mixtures, with the high sensitivity and digital quantification offered by single molecule sequencing.

Single molecule spectroscopic methods are used to obtain detailed information on the polarity and rigidity of molecular-scale environments found in thin poly(vinyl alcohol) (PVA) and poly(methyl methacrylate) (PMMA) films. Nile Red is employed as a highly sensitive spectroscopic probe of environmental properties in these experiments. Fluorescence spectra are recorded for numerous single molecules and their peak positions and widths determined by fitting the spectra to Gaussian functions. The spectral data are analyzed using a new model for the dependence of the Nile Red charge-transfer transition on the properties of the surrounding medium. This model is based on previous work by Marcus (Marcus, R. A. J. Phys. Chem. 1990, 94, 4963). Additional information required for the analysis is obtained from extensive bulk solution-phase absorption and fluorescence studies. A broad inhomogeneous distribution of environments is found for PVA. The results are shown to depend significantly on PVA film water content, with the results for hydrated films indicating the presence of less rigid environments. In contrast to the PVA results, two distinct classes of environments are found in the PMMA films. On the basis of an analysis of the data using the aforementioned model, it is concluded that the two environments differ in rigidity but have nearly identical polarity.

The nanoscale properties of organically modified sol−gel-derived silicate thin films are studied in detail by single-molecule spectroscopic methods. For these studies, the solvent-sensitive probe Nile Red is doped into the films at nanomolar concentrations. Spectroscopic data are obtained for films prepared from sols containing different mole fractions of isobutyltrimethoxysilane and tetraethoxysilane. The data are analyzed using a model based on Marcus theory, providing important new information on static local film properties such as polarity and the extent of specific dopant−matrix interactions. Data on dynamic phenomena related to local matrix rigidity is also obtained. In general, throughout the range of samples studied, the most polar environments are also found to be the most rigid. With regard to their static properties, broad heterogeneous distributions are found in films of predominantly inorganic composition. In several instances, bimodal distributions are also observed, which result from specific chemical interactions and likely involve hydrogen bonding of the dye to the silicate matrix and/or to entrapped solvent. As the organic content of the film is increased, the film environments become less polar, less rigid, and more homogeneous. In addition, the effects of specific chemical interactions become dramatically less apparent. With respect to dynamic film properties, two distinct distributions are observed in films of intermediate organic/inorganic composition, reflecting the presence of environments differing in their rigidity. Studies of time-dependent single-molecule fluorescence fluctuations provide support for the conclusions derived from the spectroscopic data.

Single-molecule fluorescence spectroscopy is used to compare the nanoscale properties of organically modified sol−gel-derived silicate thin films prepared from different silicate precursors. Sols containing different mole fractions of isobutyltrimethoxysilane (BTMOS) and/or 3-(triethoxysilyl)propionitrile (CNS) and tetraethoxysilane (TEOS) are used for film preparation. The solvent-sensitive dye Nile red is doped into these films at nanomolar concentrations and is used to probe their nanoscale environments. The single-molecule fluorescence spectra obtained are analyzed using a form of Marcus theory for charge-transfer transitions. Important information on local film polarity and rigidity is obtained. The results show that films derived from CNS-containing sols are more polar than those prepared using BTMOS, as expected. Data obtained from a series of films as a function of film organic content (i.e., CNS or BTMOS, the remainder being TEOS) show that the local film environments become distinctly less polar and less rigid as the film organic content increases. However, the CNS and BTMOS sample series exhibit markedly different behaviors as a function of film organic content. CNS-containing materials exhibit gradual changes in their nanoscale polarity and rigidity, whereas BTMOS-containing materials exhibit a “discrete” change in these properties for films of greater than approximately 50% organic content. The latter result is attributed to phase separation and/or the formation of micelle-like domains in BTMOS-derived films. Comparisons between cohydrolyzed and separately hydrolyzed sols prepared from similar binary and ternary silane mixtures also show evidence for phase separation. Importantly, the single-molecule data indicate that the average and most common film environments are distinctly different in virtually all films studied.

Single molecule confocal microspectroscopic methods are used to characterize individual molecular-scale environments in silicate thin films for the first time. Rhodamine dyes doped into the materials at nanomolar levels are used as probes of the physicochemical environment in which each molecule is entrapped. The results are compared to those obtained from dye-doped organic polymer films. Static fluorescence spectra and time-dependent fluorescence signals recorded for a large number of single molecules show the silicate materials to be highly inhomogeneous in comparison to the polymer films. Histograms of the fluorescence maxima for encapsulated Rhodamine B show a full width at half-maximum of 13.3 nm for the silicate host framework and 6.7 nm for the polymer film. The integrated fluorescence signal from single molecules, recorded with millisecond time resolution, under continuous illumination conditions, is also sensitive to the local environment. The time-dependent signal traces show dramatic intensity fluctuations for some molecules and none for others. The fluctuations occur most frequently for the silicate-entrapped dye. In the present work, the signal fluctuations are proposed to result from time-dependent variations in the molecular environment, which in turn cause changes in the excitation and emission characteristics of the molecules. The photophysical phenomena behind these fluctuations include quantum yield variations, intersystem crossing to a long-lived dark state, and, to a lesser extent, spectral diffusion. All such effects are highly dependent upon the physicochemical properties of the molecular-scale environment, as shown by comparison to results obtained for the polymer samples. The results are used as further evidence for the heterogeneous nature of entrapment in silicate host structures. The dynamic nature of many of the molecular-scale environments in these materials is demonstrated as well.

Single-molecule spectroscopy is used to characterize the microenvironments found in silicate thin films dried under different conditions. Local film properties are assigned on the basis of the fluorescence emission characteristics of individual dopant (rhodamine B) molecules. The samples studied include those characterized immediately after being spin cast onto a glass substrate (fresh samples) and after drying at ≈80°C in a vacuum oven for at least 12 h (dried samples). The single-molecule fluorescence spectra shift to the red for films dried under more rigorous conditions, reflecting increased average film polarity. The distribution of fluorescence emission maxima also broadens slightly with drying, reflecting an increase in film heterogeneity. Bimodal distributions in the widths of the emission maxima are observed. These distributions exhibit a narrowing of the single-molecule emission with drying, pointing to greater microenvironmental rigidity. Studies of the time-dependent emission characteristics of the single molecules show the total number of photons emitted (prior to bleaching) by the molecules in the dried films is four (3.6 ( 0.6) times greater than in the fresh films. A 4-fold (4.3 ( 0.7) increase in the average survival time of the molecules is also observed, proving that increased dye emission from the dried films results primarily from an increase in dye stability, rather than an increase in fluorescence quantum yield. It is also shown that the single-molecule emission fluctuates more rapidly in the dried films, possibly due to an increase in the rate of triplet formation and/or an increase in the triplet lifetime. Increased dopant stability is attributed to reduced oxygen and dye mobility within the more dense, highly cross-linked silicate network of the dried films. FTIR studies of the thin films provide additional support for these conclusions.

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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] .