Diet-induced dyslipidemia induces metabolic and migratory adaptations in regulatory T cells

A hallmark of advanced atherosclerosis is inadequate immunosuppression by regulatory T(Treg) cells inside atherosclerotic lesions. Dyslipidemia has been suggested to alter Treg cellmigration by affecting the expression of specific membrane proteins, thereby decreasing Treg cellmigration towards atherosclerotic lesions. Besides membrane proteins, cellular metabolism has beenshown to be a crucial factor in Treg cell migration. We aimed to determine whether dyslipidemiacontributes to altered migration of Treg cells, in part, by affecting cellular metabolism.Dyslipidemia was induced by feeding Ldlr-/- mice a Western-type diet for 16-20weeks and intrinsic changes in Treg cells affecting their migration and metabolism were examined.Dyslipidemia was associated with altered mTORC2 signaling in Treg cells, decreased expression ofmembrane proteins involved in migration, including CD62L, CCR7 and S1Pr1, and decreased Tregcell migration towards lymph nodes. Furthermore, we discovered that diet-induced dyslipidemiainhibited mTORC1 signaling, induced PPARδ activation and increased fatty acid (FA) oxidation inTreg cells. Moreover, mass-spectrometry analysis of serum from Ldlr-/- mice with normolipidemia ordyslipidemia showed increases in multiple PPARδ ligands during dyslipidemia. Treatment with asynthetic PPARδ agonist increased the migratory capacity of Treg cells in vitro and in vivo in an FAoxidation dependent manner. Furthermore, diet-induced dyslipidemia actually enhanced Treg cellmigration into the inflamed peritoneum and into atherosclerotic lesions in vitro.Altogether, our findings implicate that dyslipidemia does not contribute toatherosclerosis by impairing Treg cell migration as dyslipidemia associated with an effector-likemigratory phenotype in Treg cells.Analytical BioScience

Bridging the gap between molecular and elemental mass spectrometry: Higher energy collisional dissociation (HCD) revealing elemental information

Molecular mass spectrometry has been applied to simultaneously obtain molecular and elemental information from metal-containing species. Energy tuning of the higher-energy collision dissociation (HCD) fragmentation cell allows the controlled production of typical peptide fragments or elemental reporter ions informing about the metallic content of the analyzed species. Different instrumental configurations and fragmentation techniques have been tested, and the efficiency extracting the elemental information has been compared. HCD fragmentation operating at very high energy led to the best results. Platinum, lanthanides, and iodine reporter ions from peptides interacting with cisplatin, peptides labeled with lanthanides-MeCAT-IA, and iodinated peptides, respectively, were obtained. The possibility to produce abundant molecular and elemental ions in the same analysis simplifies the correlation between both signals and open pathways in metallomics studies enabling the specific tracking of metal-containing species. The proposed approach has been successfully applied to in solution standards and complex samples. Moreover, interesting preliminary MALDI-imaging experiments have been performed showing similar metal distribution compared to laser ablation (LA)-ICPMS

Bacterial surface layer proteins as a novel capillary coating material for capillary electrophoretic separations.

A novel concept for stable coating in capillary electrophoresis, based on recrystallization of surface layer proteins on hydrophobized fused silica capillaries, was demonstrated. Surface layer protein A (SlpA) from Lactobacillus acidophilus bacteria was extracted, purified and used for coating pre-silanized glass substrates presenting different surface wettabilities (either hydrophobic or hydrophilic). Contact angle determination on SlpA-coated hydrophobic silica slides showed that the surfaces turned to hydrophilic after coating (53 ± 5°), due to a protein monolayer formation by protein-surface hydrophobic interactions. Visualization by atomic force microscopy demonstrated the presence of a SlpA layer on methylated silica slides displaying a surface roughness of 0.44 ± 0.02 nm. Additionally, a protein layer was visualized by fluorescence microscopy in methylated silica capillaries coated with SlpA and fluorescein isothiocyanate-labeled. The SlpA-coating showed an outstanding stability, even after treatment with 20 mM NaOH (pH 12.3). The electroosmotic flow in coated capillaries showed a partial suppression at pH 7.50 (3.8 ± 0.5 10(-9) m(2) V(-1) s(-1)) when compared with unmodified fused silica (5.9 ± 0.1 10(-8) m(2) V(-1) s(-1)). To demonstrate the potential of this novel coating, the SlpA-coated capillaries were applied for the first time for electrophoretic separation, and proved to be very suitable for the isotachophoretic separation of lipoproteins in human serum. The separations showed a high degree of repeatability (absolute migration times with 1.1-1.8% coefficient-of-variation (CV) within a day) and 2-3% CV inter-capillary reproducibility. The capillaries were stable for more than 100 runs at pH 9.40, and showed to be an exceptional alternative for challenging electrophoretic separations at long-term use.</p

Bacterial surface layer proteins as a novel capillary coating material for capillary electrophoretic separations.

A novel concept for stable coating in capillary electrophoresis, based on recrystallization of surface layer proteins on hydrophobized fused silica capillaries, was demonstrated. Surface layer protein A (SlpA) from Lactobacillus acidophilus bacteria was extracted, purified and used for coating pre-silanized glass substrates presenting different surface wettabilities (either hydrophobic or hydrophilic). Contact angle determination on SlpA-coated hydrophobic silica slides showed that the surfaces turned to hydrophilic after coating (53 ± 5°), due to a protein monolayer formation by protein-surface hydrophobic interactions. Visualization by atomic force microscopy demonstrated the presence of a SlpA layer on methylated silica slides displaying a surface roughness of 0.44 ± 0.02 nm. Additionally, a protein layer was visualized by fluorescence microscopy in methylated silica capillaries coated with SlpA and fluorescein isothiocyanate-labeled. The SlpA-coating showed an outstanding stability, even after treatment with 20 mM NaOH (pH 12.3). The electroosmotic flow in coated capillaries showed a partial suppression at pH 7.50 (3.8 ± 0.5 10(-9) m(2) V(-1) s(-1)) when compared with unmodified fused silica (5.9 ± 0.1 10(-8) m(2) V(-1) s(-1)). To demonstrate the potential of this novel coating, the SlpA-coated capillaries were applied for the first time for electrophoretic separation, and proved to be very suitable for the isotachophoretic separation of lipoproteins in human serum. The separations showed a high degree of repeatability (absolute migration times with 1.1-1.8% coefficient-of-variation (CV) within a day) and 2-3% CV inter-capillary reproducibility. The capillaries were stable for more than 100 runs at pH 9.40, and showed to be an exceptional alternative for challenging electrophoretic separations at long-term use

Thiol-free reducing agents in electrophoretic separations and FASP proteolytic digestions for the analysis of metal-binding proteins

The analysis of the complexes between metal-based chemotherapeutic drugs and proteins in biological samples, such as cisplatin or oxaliplatin, can be a challenge due to metal strong reactivity towards S-donor molecules such as dithiothreitol (DTT) or β-mercaptoethanol (BME), usually employed as reducing agents in electrophoretic separations and proteolytic digestions for LC–MS/MS analysis. • This protocol describes the use of the thiol-free reducing trialkylphosphines, such as tributylphosphine (TBP) and tris(2-carboxyethyl)phosphine (TCEP) as suitable reagents for the preservation of the metal-protein complexes during OFFGEL-IEF and SDS-PAGE separations, respectively. • Moreover, the filter-aided sample preparation (FASP) method is presented as an advantageous option to perform tryptic in-solution digestions of metal–protein complexes in combination with OFFGEL-IEF separations. • The FASP procedure allows including previous reduction and alkylation steps in addition to proteolysis, ensuring the preservation of the metal–protein complexes. The limited time that proteins remain in contact with the reducing agent, either TBP or even DTT, during FASP could be a key factor for its extraordinary performance on the digestion of metal–protein complexes

Differences in binding kinetics, bond strength and adduct formation between Pt-based drugs and S- or N-donor groups: A comparative study using mass spectrometry techniques

Pt-S and Pt-N interactions resulting from the coordination of cisplatin, oxaliplatin and carboplatin to two synthetic peptides that differ from each other in one amino acid (Met or His) have been thoroughly studied in this work. The degree of Pt-binding was determined by inductively coupled plasma mass spectrometry after the separation of the Pt-complexes from the unreacted drugs by size exclusion chromatography. Cisplatin and oxaliplatin showed high affinity for the peptides from the first hours of incubation, although the peptides required longer incubation times to obtain the same platination degrees with cisplatin than with oxaliplatin. Once the reactions reached their maximum binding degrees, the complexes with oxaliplatin began to dissociate, revealing binding reversibility, while a pseudo steady-state was observed for cisplatin until the last day of incubation. Conversely, the equilibrium was not reached for carboplatin and the His-peptide after 30 days, showing a binding degree of 16%, versus 78% for the Met-peptide. The S-donor group also presented an important influence on the reactivity and the adduct formation. The reaction rate for the Met-peptide was faster than the hydrolysis of oxaliplatin and carboplatin, and all the drugs, except oxaliplatin, were able to coordinate up to 3 different donor groups, which were identified by nanospray mass spectrometry. Since structural characterization of metal-complexes often represents an analytical challenge during electrophoretic separations, the strength of Pt-Met and Pt-His bonds was also evaluated under these conditions. The nature of the electrophoretic agents and the incubation times used were the parameters that most affected the stability. Higher Pt losses were found for the Met-peptide (35–90%) than for the His-peptide (16–48%), indicating that Pt-Met bonds were kinetically preferred while Pt-His interactions were thermodynamically favored