Liu, C. Z. | Search bioRxiv

bioRxiv2020

Interstitial spaces are continuous across tissue and organ boundaries in humans

Bodies have "reticular networks" comprising collagens, elastin, glycosaminoglycans, and other extracellular matrix components, that are continuous within and around all organs. Fibrous tissue coverings of nerves and blood vessels create structural continuity beyond organ boundaries. We recently described fluid flow through such human fibrous tissues. It remains unclear whether these interstitial spaces are continuous through the body or are discontinuous, confined within individual organs. We investigated IS continuity using two approaches. Non-biological particles (tattoo pigment, colloidal silver) were tracked within colon and skin interstitial spaces and into adjacent fascia. We also exploited hyaluronic acid, a macromolecular component of interstitial spaces. Both techniques demonstrate continuity of interstitial spaces within and across organ boundaries, including within perineurium and vascular adventitia traversing organs and the spaces between them. We suggest a body-wide network of fluid-filled interstitial spaces with significant implications for molecular signaling, cell trafficking, and the spread of malignant and infectious disease.

pathology↗

bioRxiv2020

Improved discrimination of asymmetric and symmetric arginine dimethylation by optimization of the normalized collision energy in LC-MS proteomics

Protein arginine methylation regulates diverse biological processes including signaling, metabolism, splicing, and transcription. Despite its important biological roles, arginine methylation remains an understudied post-translational modification. Partly, this is because the two forms of arginine dimethylation, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), are isobaric and therefore indistinguishable by traditional mass spectrometry techniques. Thus, there exists a need for methods that can differentiate these two modifications. Recently, it has been shown that the ADMA and SDMA can be distinguished by the characteristic neutral loss (NL) of dimethylamine and methylamine, respectively. However, the utility of this method is limited because the vast majority of dimethylarginine peptides do not generate measurable NL ions. Here, we report that increasing the normalized collision energy (NCE) in a higher-energy collisional dissociation (HCD) cell increases the generation of the characteristic NL that distinguish ADMA and SDMA. By analyzing both synthetic and endogenous methyl-peptides, we identify an optimal NCE value that maximizes NL generation and simultaneously improves methyl-peptide identification. Using two orthogonal methyl peptide enrichment strategies, high pH strong cation exchange (SCX) and immunoaffinity purification (IAP), we demonstrate that the optimal NCE increases improves NL-based ADMA and SDMA annotation and methyl peptide identifications by 125% and 17%, respectively, compared to the standard NCE. This simple parameter change will greatly facilitate the identification and annotation of ADMA and SDMA in mass spectrometry-based methyl-proteomics to improve our understanding of how these modifications differentially regulate protein function.

biochemistry↗