N-Glycan profile of the cell membrane as a probe for lipopolysaccharide-induced microglial neuroinflammation uncovers the effects of common fatty acid supplementation.

Altered N-glycosylation of proteins on the cell membrane is associated with several neurodegenerative diseases. Microglia are an ideal model for studying glycosylation and neuroinflammation, but whether aberrant N-glycosylation in microglia can be restored by diet remains unknown. Herein, we profiled the N-glycome, proteome, and glycoproteome of the human microglia following lipopolysaccharide (LPS) induction to probe the impact of dietary and gut microbe-derived fatty acids-oleic acid, lauric acid, palmitic acid, valeric acid, butyric acid, isobutyric acid, and propionic acid-on neuroinflammation using liquid chromatography-tandem mass spectrometry. LPS changed N-glycosylation in the microglial glycocalyx altering high mannose and sialofucosylated N-glycans, suggesting the dysregulation of mannosidases, fucosyltransferases, and sialyltransferases. The results were consistent as we observed the restoration effect of the fatty acids, especially oleic acid, on the LPS-treated microglia, specifically on the high mannose and sialofucosylated glycoforms of translocon-associated proteins, SSRA and SSRB along with the cell surface proteins, CD63 and CD166. In addition, proteomic analysis and in silico modeling substantiated the potential of fatty acids in reverting the effects of LPS on microglial N-glycosylation. Our results showed that N-glycosylation is likely affected by diet by restoring alterations following LPS challenge, which may then influence the disease state.

Profiling Intact Glycosphingolipids with Automated Structural Annotation and Quantitation from Human Samples with Nanoflow Liquid Chromatography Mass Spectrometry.

Sphingolipids are an essential subset of bioactive lipids found in most eukaryotic cells that contribute to membrane biophysical properties and are involved in cellular differentiation, recognition, and mediating interactions. The described nanoHPLC-ESI-Q/ToF methodology utilizes known biosynthetic pathways, accurate mass detection, optimized collision-induced disassociation, and a robust nanoflow chromatographic separation for the analysis of intact sphingolipids found in human tissue, cells, and serum. The methodology was developed and validated with an emphasis on addressing the common issues experienced in profiling these amphipathic lipids, which are part of the glycocalyx and lipidome. The high sensitivity obtained using nanorange flow rates with robust chromatographic reproducibility over a wide range of concentrations and injection volumes results in confident identifications for profiling these low-abundant biomolecules.

Quantitative glycoproteomics of high-density lipoproteins.

In this work, we developed a targeted glycoproteomic method to monitor the site-specific glycoprofiles and quantities of the most abundant HDL-associated proteins using Orbitrap LC-MS for (glyco)peptide target discovery and QqQ LC-MS for quantitative analysis. We conducted a pilot study using the workflow to determine whether HDL protein glycoprofiles are altered in healthy human participants in response to dietary glycan supplementation.

Improving identification of low abundance and hydrophobic proteins using fluoroalcohol mediated supramolecular biphasic systems with quaternary ammonium salts.

In this study, a newly discovered Supramolecular Biphasic System (S-BPS) was used in bottom-up proteomics of the Saccharomyces cerevisiae strain of yeast. We took advantage of S-BPS in bottom-up proteomics of this strain of yeast as the protein sample, while the results were compared to routinely used solubilizing reagents, such as urea, and sodium dodecyl sulfate (SDS). With the S-BPS, we identified 3043 proteins as compared to 2653 proteins that were identified in the control system. Interestingly, of the additional 390 proteins characterized by the S-BPS, 300 proteins were low abundance (less than 4000 molecules/cell). Remarkably, the identification of proteins at very low abundance (less than 2000 molecule/cell) was improved by 106%. This suggests that the S-BPS is particularly advantageous for detecting low abundance proteins. Gene Ontology (GO) analysis was conducted to find fractionation pattern of proteins in our two-phase system, and in nearly every gene ontology category, the S-BPS provided greater coverage than the control experiment, i.e., coverage for integral membrane proteins and mitochondrial ribosome proteins are improved by 18% and 58%, respectively. The improvements in proteins coverage for low abundance and membrane proteins can be attributed to the strong solubilizing power of the amphiphile-rich phase of this S-BPS and its capability for concomitant extraction, fractionation, and enrichment of the complex proteomics samples. Each phase has selectivity towards specific yeast protein groups, this selectivity is generally based on pI and hydrophobicity of proteins. Therefore, more hydrophobic proteins and acidic proteins exhibit greater affinities for the amphiphile-rich phase due to the hydrophobic effect and electrostatic interactions.

Fluoroalcohol - Induced coacervates for selective enrichment and extraction of hydrophobic proteins.

As previously reported, fluoroalcohols can induce coacervation in aqueous solutions of amphiphilic compounds with subsequent formation of two-phase systems, where one phase is enriched in amphiphile and fluoroalcohol and the other is primarily an aqueous - rich phase. This study focuses on the use of simple coacervates made of a single component amphiphile induced by a fluoroalcohol for extraction and enrichment of proteins. 1,1,1,3,3,3-Hexafluoroisopropanol (HFIP) and 2,2,2-trifluoroethanol (TFE) were used to induce coacervation in the aqueous solutions of a cationic amphiphile, cetyltrimethylammonium bromide (CTAB) or tetra-n-butylammonium bromide (TBAB). Cationic amphiphiles (CTAB, TBAB) formed two-phase coacervate systems in a basic pH and/or sufficient ionic strength depending on the strength of coacervator (HFIP or TFE). The phase diagrams for TBAB paired with HFIP or TFE coacervates were created. By increasing the concentration of coacervator (HFIP or TFE) at a constant surfactant concentration, transition from a single liquid phase to a two or multiple phase mixture, and then eventually to a single liquid phase was observed. TBAB/HFIP mixture without additives showed a unique three-phase system before transitioning to a two-phase system upon increasing HFIP concentration. However, salt addition eliminated this three-phase region and expanded the region of two-phase formation. Select two-phase systems composed of TBAB and a perfluoroalcohol (HFIP or TFE) were utilized to extract model proteins of ranging hydrophobicity. All coacervate phases extracted bacteriorhodopsin, a membrane protein, and gramicidin, a very hydrophobic polypeptide ion channel. The most hydrophilic protein in the mixture, ribonuclease A, remained in aqueous phases. The coacervates formed from TBAB/TFE/200 mM NaCl mixture and TBAB/HFIP mixture exhibited the most selectivity in extracting proteins of high hydrophobicity. The partition coefficient (P) for each protein was calculated using the ratio of the protein concentration in the coacervate to that in the aqueous-rich phases. TBAB (50 mM)/HFIP (8%, v/v) coacervate showed remarkable selectivity and a high partition coefficient (>100) for both bacteriorhodopsin and gramicidin. Thus, this system may potentially be beneficial for facile fractionation of hydrophobic and membrane proteins in proteomics applications.