Biology subjects

Hersemann, L.

Publications and source records attributed to Hersemann, L..

4 recordsLinked to original sources

bioRxiv2021

FastCAT accelerates absolute quantification of proteins by using multiple short non-purified chimeric standards

Absolute (molar) quantification of clinically relevant proteins determines their reference values in liquid and solid biopsies. FastCAT (for Fast-track QconCAT) method employs multiple short (<50 kDa) stable-isotope labeled chimeric proteins (CPs) composed of concatenated quantotypic (Q-) peptides representing the quantified proteins. Each CP also comprises scrambled sequences of reference (R-) peptides that relate its abundance to a single protein standard (BSA). FastCAT not only alleviates the need in purifying CP or using SDS-PAGE, but also improves the accuracy, precision and dynamic range of the absolute quantification by grouping Q-peptides according to the expected abundance of target proteins. We benchmarked FastCAT against the reference method of MS Western and tested it in the direct molar quantification of neurological markers in the human cerebrospinal fluid at the low ng/mL level.

biochemistry↗

bioRxiv2021

A combined transcriptional and dynamic roadmap of single human pancreatic endocrine progenitors reveals proliferative capacity and differentiation continuum

Basic helix-loop-helix genes, particularly proneural genes, are well-described triggers of cell differentiation, yet limited information exists on their dynamics, notably in human development. Here, we focus on Neurogenin 3 (NEUROG3), which is crucial for pancreatic endocrine lineage initiation. Using a double reporter to monitor endogenous NEUROG3 transcription and protein expression in single cells in 2D and 3D models of human pancreas development, we show peaks of expression for the RNA and protein at 22 and 11 hours respectively, approximately two-fold slower than in mice, and remarkable heterogeneity in peak expression levels all triggering differentiation. We also reveal that some human endocrine progenitors proliferate once, mainly at the onset of differentiation, rather than forming a subpopulation with sustained proliferation. Using reporter index-sorted single-cell RNA-seq data, we statistically map transcriptome to dynamic behaviors of cells in live imaging and uncover transcriptional states associated with variations in motility as NEUROG3 levels change, a method applicable to other contexts.

developmental biology↗

bioRxiv2021

Median based absolute quantification of proteins using Fully Unlabelled Generic Internal Standard

By reporting molar abundances of proteins, absolute quantification determines their stoichiometry in complexes, pathways or networks. Typically, absolute quantification relies either on protein-specific isotopically labelled peptide standards or on a semi-empirical calibration against the average abundance of peptides chosen from arbitrary selected proteins. In contrast, a generic protein standard FUGIS (for Fully Unlabelled Generic Internal Standard) requires no isotopic labelling, chemical synthesis or external calibration and is applicable to quantifying proteins of any organismal origin. The median intensity of peptide peaks produced by the digestion of FUGIS is used as a single point calibrant to determine the molar abundance of any co-digested protein. Powered by FUGIS, median based absolute quantification (MBAQ) outperformed other available methods of untargeted proteome-wide absolute quantification.

biochemistry↗

bioRxiv2021

The novel Rab5 effector FERRY links early endosomes with the translation machinery

Localized translation is vital to polarized cells and requires precise and robust distribution of different mRNAs and ribosomes across the cell. However, the underlying molecular mechanisms are poorly understood and important players are lacking. Here we show that the novel Rab5 effector Five-subunit Endosomal Rab5 and RNA/ribosome intermediarY, FERRY complex recruits mRNAs and ribosomes to early endosomes, through direct mRNA interaction. FERRY displays preferential binding to certain groups of transcripts, including mRNAs encoding mitochondrial proteins. Deletion of FERRY subunits reduces the endosomal localization of transcripts in cells and has a significant impact on mRNA and protein levels. Clinical studies show that genetic disruption of FERRY causes severe brain damage. We found that, in neurons, FERRY co-localizes with mRNA on early endosomes and mRNA loaded FERRY-positive endosomes are in close proximity of mitochondria. FERRY thus transforms endosomes into mRNA carriers and plays a key role in regulating mRNA distribution and transport.

cell biology↗