Exploration of cell state heterogeneity using single-cell proteomics through sensitivity-tailored data-independent acquisition.

Single-cell resolution analysis of complex biological tissues is fundamental to capture cell-state heterogeneity and distinct cellular signaling patterns that remain obscured with population-based techniques. The limited amount of material encapsulated in a single cell however, raises significant technical challenges to molecular profiling. Due to extensive optimization efforts, single-cell proteomics by Mass Spectrometry (scp-MS) has emerged as a powerful tool to facilitate proteome profiling from ultra-low amounts of input, although further development is needed to realize its full potential. To this end, we carry out comprehensive analysis of orbitrap-based data-independent acquisition (DIA) for limited material proteomics. Notably, we find a fundamental difference between optimal DIA methods for high- and low-load samples. We further improve our low-input DIA method by relying on high-resolution MS1 quantification, thus enhancing sensitivity by more efficiently utilizing available mass analyzer time. With our ultra-low input tailored DIA method, we are able to accommodate long injection times and high resolution, while keeping the scan cycle time low enough to ensure robust quantification. Finally, we demonstrate the capability of our approach by profiling mouse embryonic stem cell culture conditions, showcasing heterogeneity in global proteomes and highlighting distinct differences in key metabolic enzyme expression in distinct cell subclusters.

In vivo Anti-inflammatory Activity of Lipidated Peptidomimetics Pam-(Lys-ßNspe)6-NH2 and Lau-(Lys-ßNspe)6-NH2 Against PMA-Induced Acute Inflammation.

Host Defense Peptides (HDPs) are key components of innate immunity that exert antimicrobial, antibiofilm, and immunomodulatory activities in all higher organisms. Synthetic peptidomimetic analogs were designed to retain the desirable pharmacological properties of HDPs while having improved stability toward enzymatic degradation, providing enhanced potential for therapeutic applications. Lipidated peptide/ß-peptoid hybrids [e.g., Pam-(Lys-ßNspe)6-NH2 (PM1) and Lau-(Lys-ßNspe)6-NH2 (PM2)] are proteolytically stable HDP mimetics displaying anti-inflammatory activity and formyl peptide receptor 2 antagonism in human and mouse immune cells in vitro. Here PM1 and PM2 were investigated for their in vivo anti-inflammatory activity in a phorbol 12-myristate 13-acetate (PMA)-induced acute mouse ear inflammation model. Topical administration of PM1 or PM2 led to attenuated PMA-induced ear edema, reduced local production of the pro-inflammatory chemokines MCP-1 and CXCL-1 as well as the cytokine IL-6. In addition, diminished neutrophil infiltration into PMA-inflamed ear tissue and suppressed local release of reactive oxygen and nitrogen species were observed upon treatment. The obtained results show that these two peptidomimetics exhibit anti-inflammatory effects comparable to that of the non-steroidal anti-inflammatory drug indomethacin, and hence possess a potential for treatment of inflammatory skin conditions.

Lipidated α-peptide/ß-peptoid hybrids with potent anti-inflammatory activity.

In this study, we investigated, optimized, and characterized a novel subclass of host defense peptide (HDP) mimics based on α-peptide/ß-peptoid hybrid oligomers with an alternating cationic/hydrophobic design with respect to their ability to modulate the pro-inflammatory response by human primary leukocytes upon exposure to bacterial components. Structure-activity studies revealed that certain lipidated α-peptide/ß-peptoid hybrid oligomers possess anti-inflammatory activities in the submicromolar range with low cytotoxicity, and that the anti-inflammatory activity of the HDP mimics is dependent on the length and position of the lipid element(s). The resulting lead compound, Pam-(Lys-ßNSpe)6-NH2, blocks LPS-induced cytokine secretion with a potency comparable to that of polymyxin B. The mode of action of this HDP mimic appears not to involve direct LPS interaction since it, in contrast to polymyxin B, displayed only minor activity in the Limulus amebocyte lysate assay. Flow cytometry data showed specific interaction of a fluorophore-labeled lipidated α-peptide/ß-peptoid hybrid with monocytes and granulocytes indicating a cellular target expressed by these leukocyte subsets.