Proteases and ubiquitin proteins discovered in pooled plasma from <i>T. b. rhodesiense</i>-infected patients with parasitologically confirmed late stage human African trypanosomiasis.

<p>Proteases and ubiquitin proteins discovered in pooled plasma from <i>T. b. rhodesiense</i>-infected patients with parasitologically confirmed late stage human African trypanosomiasis.</p

Summary of patient information.

<p>Plasma and CSF were collected from <i>T. b. rhodesiense</i>-infected patients confirmed to have late stage human African trypanosomiasis at Lwala Hospital in central Uganda.</p

List of human plasma proteins with mass spectrometric molar intensities similar to those from the most abundant trypanosome proteins found in plasma from sleeping sickness patients.

*<p>Protein concentrations were retrieved from reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071463#pone.0071463-Hortin1" target="_blank">[19]</a>. Human proteins reported to be depleted by the LC20 IgY14 and Supermix LC10 columns <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071463#pone.0071463-Patel1" target="_blank">[26]</a> have been omitted from this table.</p

Variant Surface Glycoproteins (VSGs) discovered in pooled plasma from <i>T. b. rhodesiense</i>-infected patients confirmed to have late-stage human African trypanosomiasis.

<p>Variant Surface Glycoproteins (VSGs) discovered in pooled plasma from <i>T. b. rhodesiense</i>-infected patients confirmed to have late-stage human African trypanosomiasis.</p

Chaperones and protein isomerases identified in pooled plasma from <i>T. b. rhodesiense</i>-infected patients with parasitologically confirmed late stage human African trypanosomiasis.

<p>Chaperones and protein isomerases identified in pooled plasma from <i>T. b. rhodesiense</i>-infected patients with parasitologically confirmed late stage human African trypanosomiasis.</p

ELISA detection of trypanosome antigens in plasma from <i>T. b. rhodesiense</i> -infected patients with late-stage human African trypanosomisis.

<p>All samples were tested in duplicate and the average response is shown. Error bars represent 1 standard deviation.</p

Automated Microchromatography Enables Multiplexing of Immunoaffinity Enrichment of Peptides to Greater than 150 for Targeted MS-Based Assays

Immunoaffinity enrichment of peptides coupled with analysis by stable isotope dilution multiple reaction mass spectrometry has been shown to have analytical performance and detection limits suitable for many biomarker verification studies and biological applications. Prior studies have shown that antipeptide antibodies can be multiplexed up to 50 in a single assay without significant loss of performance. Achieving higher multiplex levels is relevant to all studies involving precious biological material as this minimizes the amount of sample that must be consumed to measure a given set of analytes and reduces the assay cost per analyte. Here we developed automated methods employing the Agilent AssayMAP Bravo microchromatography platform and used these methods to characterize the performance of immunoaffinity enrichment of peptides up to multiplex levels of 172. Median capture efficiency for the target peptides remained high (88%) even at levels of 150-plex and declined to 70% at 172-plex compared to antibody performance observed at standard lower multiplex levels (<i>n</i> = 25). Subsequently, we developed and analytically characterized a multiplexed immuno-multiple reaction monitoring-mass spectrometry (immuno-MRM-MS) assay (<i>n</i> = 110) and applied it to measure candidate protein biomarkers of cardiovascular disease in plasma of patients undergoing planned myocardial infarction. The median lower limit of detection of all peptides was 71.5 amol/μL (nM), and the coefficient of variation (CV) was less than 15% at the lower limit of quantification. The results demonstrate that high multiplexed immuno-MRM-MS assays are readily achievable using the optimized sample processing and peptide capture methods described here

Longer-term lentiviral knockdown of <i>Brsk1</i> and <i>Camkk2</i> stimulates PDX1 and insulin protein expression in αTC1 cells.

<p>Cells were infected with lentivirus carrying expression cassettes that encode shRNAs against (A) <i>Brsk1</i>, (B) <i>Camkk2</i>, (C) <i>Stk11</i>, and were selected with puromycin for 10 days. <i>Pdx1</i> mRNA expression (white bars) was measured jointly with the expression of each of the shRNA-targeted genes (black bars). Data represent fold change (in log₂ scale), compared to the average of control empty vectors. Data represent mean and standard deviation of four independent experiments. The significance was determined by t-test. *<i>p</i><0.05 and **<i>p</i><0.01. (D–S) Nuclei (blue), insulin (green), and Pdx1 (red) immunofluorescence, and merged images in 10-day shRNA-expressing αTC1 cells: (D–G) control virus, (H–K) sh1_Camkk2, (L–O) sh1_Brsk1, (P–S) sh1_Stk11. Scale bar = 100 µm.</p

Gene-expression analysis of alpha and beta cell lines reveals higher metabolic activity in the alpha cell line.

<p>Gene sets with increased expression in (A) alpha or (B) beta cell lines were identified by performing gene-set enrichment analysis (GSEA) on gene-expression profiling data, resulting in an enrichment score profile for each gene set (green line). Individual members of each gene set (vertical black bars) are enriched in either alpha cells (blue) or beta cells (red). To validate the predicted differences in cellular respiration between alpha and beta cells, we determined (C) extracellular acidification rate (ECAR) and (D) oxygen consumption rate (OCR) of alpha cells (red), BRD7389-treated alpha cells (black), βTC3 cells (blue), and INS-1E cells (brown). Glucose (Glu), oligomycin (Oligo), 2-deoxyglucose (DOG), CCCP, and rotenone/antimycin A (Rot/Ant) were added at the indicated times. Data represent the average and standard deviation of 18 biological replicates.</p

Summary of proteomic studies.

<p>*including 467 phosphorylated kinase peptides.</p><p>Total proteome results based on 24 SCX fractions over two SILAC experiments. pSTY peptide results are based on 12 SCX fractions over two SILAC experiments.</p