Automated Trapping Column Exchanger for High-Throughput Nanoflow Liquid Chromatography

As compared to conventional high-performance liquid chromatography (HPLC) techniques, nanoflow HPLC exhibits improved sensitivity and limits of detection. However, nanoflow HPLC suffers from low throughput due to instrument failure (e.g., fitting fatigue and trapping column failure), limiting the utility of the technique for clinical and industrial applications. To increase the robustness of nanoflow HPLC, we have developed and tested a trapping column exchanging robot for autonomous interchange of trapping columns. This robot makes reproducible, automated connections between the active trapping column and the rest of the HPLC system. The intertrapping column retention time is shown to be sufficiently reproducible for scheduled selected reaction monitoring assays to be performed on different trapping columns without rescheduling the selection windows

Clinical characteristics of the study population.

<p>Clinical characteristics of the study population.</p

Protein quantified in the targeted proteomics analysis.

<p>Protein quantified in the targeted proteomics analysis.</p

Odds ratios for type 2 diabetes according to HDL endothelial protective functions and selected dyslipidemia and common risk factors.

<p>Multivariate logistic regression models included the listed dyslipidemia and other cardiovascular risk factors and odds ratios for continuous variables are presented per 1-SD increase for <b>(A)</b> HDL ability to stimulate eNOS activation, and <b>(B)</b> for the suppression of NFκB activation.</p

Vaisar et al, Type 2 Diabetes is Associated with Loss of HDL Endothelium Protective Functions

Targeted proteomics analysis of HDL from people with and without T2D. Attached files contain Skyline documents with relevant SRM-LCMS data. Data is directly accessible at PanoramaWeb Public, https://panoramaweb.org/labkey/VaisarHDL_T2D.url.<br>Original publication - Vaisar et al, PlosOne 2018.<br

Protective effects of HDL on endothelial cells are impaired in diabetes, correlate with S1P and negatively associate with <i>in vivo</i> measure of endothelial dysfunction.

<p><b>(A)</b> The ability of HDL to suppress NFκB activation was measured as phosphorylation of p65 in HMEC after 16 h incubation with HDL (50 μg/mL) followed by 4 h stimulation with TNFα (n = 41 per group). <b>(B)</b> The ability of HDL to stimulate eNOS Ser1179 phosphorylation was measured in BAEC after 30 min incubation with HDL (50 μg/mL) (n = 38 non-diabetic, n = 41 diabetic subjects). (Data is expressed relative to cells not treated with HDL). <b>(C)</b> Sphigosine-1-phosphate concentration measured by LC-MS is reduced in patients with diabetes and <b>(D)</b> correlates positively with HDL ability to stimulate eNOS phosphorylation (n = 40 non-diabetic, n = 39 diabetic subjects; *excluded outlier and <i>P</i>-value after exclusion). <b>(E)</b> The ability of HDL to stimulate eNOS is inversely correlated negatively with level of P-selectin in plasma, an <i>in vivo</i> measure of endothelial dysfunction (Pearson correlation coefficient; n = 81 after an outlier exclusion).</p

Table_1_Circulating sphingolipids and subclinical brain pathology: the cardiovascular health study.docx

BackgroundSphingolipids are implicated in neurodegeneration and neuroinflammation. We assessed the potential role of circulating ceramides and sphingomyelins in subclinical brain pathology by investigating their association with brain magnetic resonance imaging (MRI) measures and circulating biomarkers of brain injury, neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) in the Cardiovascular Health Study (CHS), a large and intensively phenotyped cohort of older adults.MethodsBrain MRI was offered twice to CHS participants with a mean of 5 years between scans, and results were available from both time points in 2,116 participants (mean age 76 years; 40% male; and 25% APOE ε4 allele carriers). We measured 8 ceramide and sphingomyelin species in plasma samples and examined the associations with several MRI, including worsening grades of white matter hyperintensities and ventricular size, number of brain infarcts, and measures of brain atrophy in a subset with quantitative measures. We also investigated the sphingolipid associations with serum NfL and GFAP.ResultsIn the fully adjusted model, higher plasma levels of ceramides and sphingomyelins with a long (16-carbon) saturated fatty acid were associated with higher blood levels of NfL [β = 0.05, false-discovery rate corrected P (PFDR) = 0.004 and β = 0.06, PFDR = FDR = 0.03]. We did not observe any associations with GFAP blood levels, white matter grade, ventricular grade, mean bilateral hippocampal volume, or total brain volume.ConclusionOverall, our comprehensive investigation supports the evidence that ceramides and sphingomyelins are associated with increased aging brain pathology and that the direction of association depends on the fatty acid attached to the sphingosine backbone.</p

Additional file 2 of Circulating ceramides and sphingomyelins and the risk of incident cardiovascular disease among people with diabetes: the strong heart study

Additional file 2: Table S1. Risk of incident CVD per two-fold higher sphingolipid level in SHFS. Table S2. Odds of incident CVD per two-fold higher sphingolipid level in SHS. Table S3. Sensitivity analysis—associations of sphingolipids with incident CVD risk after adjustment for HDL and Triglycerides, Fibrinogen, and Chronic Kidney Disease

Paraoxonase‑3 Is Depleted from the High-Density Lipoproteins of Autoimmune Disease Patients with Subclinical Atherosclerosis

Patients with autoimmune diseases have a significantly increased risk of developing cardiovascular disease. In disease, high-density lipoprotein (HDL) particles lose their anti-inflammatory and antioxidant properties and become dysfunctional. The purpose of this study was to test the hypothesis that alterations in the HDL proteomic profile are associated with subclinical atherosclerosis and HDL dysfunction in patients with autoimmune diseases such as systemic lupus erythematosus (SLE) and type 1 diabetes. Targeted proteomics was used to quantify the relative abundance of 18 proteins in HDL from SLE patients with and without atherosclerotic plaque detectable by carotid ultrasound. Changes in the proteomic profile were compared against the in vitro ability of HDL to protect against lipid oxidation. The same proteins were quantified in HDL from patients with type 1 diabetes with or without coronary artery calcification as determined by computed tomography. In each population, paraoxonase-3 (PON3), a potent antioxidant protein, was depleted from the HDL of patients with subclinical atherosclerosis. PON3 expression in HDL was positively correlated with HDL antioxidant function. These results suggest that PON3 may be an important protein in preventing atherosclerosis and highlight the importance of antioxidant proteins in the prevention of atherosclerosis in vivo

Optimized Protocol for Quantitative Multiple Reaction Monitoring-Based Proteomic Analysis of Formalin-Fixed, Paraffin-Embedded Tissues

Despite a clinical, economic, and regulatory imperative to develop companion diagnostics, precious few new biomarkers have been successfully translated into clinical use, due in part to inadequate protein assay technologies to support large-scale testing of hundreds of candidate biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues. Although the feasibility of using targeted, multiple reaction monitoring mass spectrometry (MRM-MS) for quantitative analyses of FFPE tissues has been demonstrated, protocols have not been systematically optimized for robust quantification across a large number of analytes, nor has the performance of peptide immuno-MRM been evaluated. To address this gap, we used a test battery approach coupled to MRM-MS with the addition of stable isotope-labeled standard peptides (targeting 512 analytes) to quantitatively evaluate the performance of three extraction protocols in combination with three trypsin digestion protocols (i.e., nine processes). A process based on RapiGest buffer extraction and urea-based digestion was identified to enable similar quantitation results from FFPE and frozen tissues. Using the optimized protocols for MRM-based analysis of FFPE tissues, median precision was 11.4% (across 249 analytes). There was excellent correlation between measurements made on matched FFPE and frozen tissues, both for direct MRM analysis (<i>R</i>² = 0.94) and immuno-MRM (<i>R</i>² = 0.89). The optimized process enables highly reproducible, multiplex, standardizable, quantitative MRM in archival tissue specimens