Proteins are vital in all biological systems as they constitute the main structural and functional components of cells. Recent advances in mass spectrometry have brought the promise of complete proteomics by helping draft the human proteome. Yet, this commonly used protein sequencing technique has fundamental limitations in sensitivity. Here we propose a method for single-molecule (SM) protein sequencing. A major challenge lies in the fact that proteins are composed of 20 different amino acids, which demands 20 molecular reporters. We computationally demonstrate that it suffices to measure only two types of amino acids to identify proteins and suggest an experimental scheme using SM fluorescence. When achieved, this highly sensitive approach will result in a paradigm shift in proteomics, with major impact in the biological and medical sciences.

Proteomic analyses provide essential information on molecular pathways of cellular systems and the state of a living organism. Mass spectrometry is currently the first choice for proteomic analysis. However, the requirement for a large amount of sample renders a small-scale proteomics study challenging. Here, we demonstrate a proof of concept of single-molecule FRET-based protein fingerprinting. We harnessed the AAA+ protease ClpXP to scan peptides. By using donor fluorophore-labeled ClpP, we sequentially read out FRET signals from acceptor-labeled amino acids of peptides. The repurposed ClpXP exhibits unidirectional processing with high processivity and has the potential to detect low-abundance proteins. Our technique is a promising approach for sequencing protein substrates using a small amount of sample.

Human aging is associated with loss of function and regenerative capacity. Human bone marrow derived mesenchymal stromal cells (hMSCs) are involved in tissue regeneration, evidenced by their capacity to differentiate into several lineages and therefore are considered the golden standard for cell-based regeneration therapy. Tissue maintenance and regeneration is dependent on stem cells and declines with age and aging is thought to influence therapeutic efficacy, therefore, more insight in the process of aging of hMSCs is of high interest. We, therefore, hypothesized that hMSCs might reflect signs of aging. In order to find markers for donor age, early passage hMSCs were isolated from bone marrow of 61 donors, with ages varying from 17–84, and clinical parameters, in vitro characteristics and microarray analysis were assessed. Although clinical parameters and in vitro performance did not yield reliable markers for aging since large donor variations were present, genome-wide microarray analysis resulted in a considerable list of genes correlating with human age. By comparing the transcriptional profile of aging in human with the one from rat, we discovered follistatin as a common marker for aging in both species. The gene signature presented here could be a useful tool for drug testing to rejuvenate hMSCs or for the selection of more potent, hMSCs for cell-based therapy.

peer-reviewed)

tracked with changes in diffusion time of the larger mAb-antigen complex. With this approach, we measured the binding affinities of Alexa-488 labeled streptavidin and anti-streptavidin immunoglobulin G2 (IgG2) in buffer and neat serum, and found that binding is~3-fold tighter in serum versus buffer. Through control experiments in viscosity-matched sucrose solution, we found that differences in viscosity may account for some but not all of this effect. Serum contains high concentrations of co-solutes of various sizes including albumin and g-globulins. We tested the effects of macromolecular crowding on binding using physiological concentrations of bovine serum albumin as well as polymeric crowders, and found that crowding also partially accounts for the tighter binding of the streptavidin-IgG2 pair in serum. Conversely, serum does not affect the binding affinity of anti-streptavidin IgG1 to streptavidin. Accurate characterization of therapeutic proteins in relevant conditions is integral to assessing their safety and efficacy. The implications of these results with respect to drug development are discussed.