Extending Single Molecule Imaging to Proteome Analysis by Quantitation of Fluorescent Labeling Homogeneity in Complex Protein Samples

Fluorescence-based electrophoresis has been widely used for proteome analysis in which every protein species in cells is labeled with a fluorescent dye, separated by electric migration, and quantified using fluorescence detection. The ultimate limit of sensitivity for this approach could be reached by single-molecule fluorescence imaging and counting individual proteins, requiring exhaustive fluorescent labeling of proteins across molecular populations and species. However, it remains unclear how homogeneous the fluorescence labeling of individual protein molecules of each species is across the proteome. To address this question, we developed a method to measure the labeling homogeneity based on a single-molecule fluorescence counting assay. Our results reveal that the proportion of proteins labeled with at least one dye, called labeling occupancy (LO), was 35% for fluorescently labeled BSA using existing protocols. We then found that the LO could be improved to 82% under high pH and surfactant-rich conditions. Furthermore, when a proteome sample from a human cell lysate was analyzed, the total LO was 71%, whereby the values varied between 50 and 90% for low and high molecular weight proteome fractions, respectively. The results support the possibility of sensitive detection of proteins using single-molecule counting with fluorescent labeling at the proteome scale

Charge-Controlled Nanoprecipitation as a Modular Approach to Ultrasmall Polymer Nanocarriers: Making Bright and Stable Nanoparticles

Ultrasmall polymer nanoparticles are rapidly gaining importance as nanocarriers for drugs and contrast agents. Here, a straightforward modular approach to efficiently loaded and stable sub-20-nm polymer particles is developed. In order to obtain ultrasmall polymer nanoparticles, we investigated the influence of one to two charged groups per polymer chain on the size of particles obtained by nanoprecipitation. Negatively charged carboxylate and sulfonate or positively charged trimethylammonium groups were introduced into the polymers poly(d,l-lactide-<i>co</i>-glycolide) (PLGA), polycaprolactone (PCL), and poly(methyl methacrylate) (PMMA). According to dynamic light scattering, atomic force and electron microscopy, the presence of one to two charged groups per polymer chain can strongly reduce the size of polymer nanoparticles made by nanoprecipitation. The particle size can be further decreased to less than 15 nm by decreasing the concentration of polymer in the solvent used for nanoprecipitation. We then show that even very small nanocarriers of 15 nm size preserve the capacity to encapsulate large amounts of ionic dyes with bulky counterions at efficiencies >90%, which generates polymer nanoparticles 10-fold brighter than quantum dots of the same size. Postmodification of their surface with the PEG containing amphiphiles Tween 80 and pluronic F-127 led to particles that were stable under physiological conditions and in the presence of 10% fetal bovine serum. This modular route could become a general method for the preparation of ultrasmall polymer nanoparticles as nanocarriers of contrast agents and drugs

Oxyluciferin Derivatives: A Toolbox of Environment-Sensitive Fluorescence Probes for Molecular and Cellular Applications

In this work, we used firefly oxyluciferin (<b>OxyLH</b>_<b>2</b>) and its polarity-dependent fluorescence mechanism as a sensitive tool to monitor biomolecular interactions. The chromophores, <b>OxyLH</b>_<b>2</b>, and its two analogues, <b>4-MeOxyLH</b> and <b>4,6′-DMeOxyL</b>, were modified trough carboxylic functionalization and then coupled to the N-terminus part of Tat and NCp7 peptides of human immunodeficiency virus type-1 (HIV-1). The photophysical properties of the labeled peptides were studied in live cells as well as in complex with different oligonucleotides in solution. By monitoring the emission properties of these derivatives we were able, for the first time, to study <i>in vitro</i> biomolecular interactions using oxyluciferin as a sensor. As an additional application, cyclopropyl-oxyluciferin (<b>5,5-Cpr-OxyLH</b>) was site-specifically conjugated to the thiol group (Cys-232) of the human protein α-1 antytripsin to investigate its interaction with porcine pancreatic elastase. Our data demonstrate that <b>OxyLH</b>_<b>2</b> and its derivatives can be used as fluorescence reporters for monitoring biomolecular interactions