Article Title: Carboxyl-Ester Lipase Maturity-Onset Diabetes of the Young Is Associated With Development of Pancreatic Cysts and Upregulated MAPK Signaling in Secretin-Stimulated Duodenal Fluid
Figure Lengend Snippet: The identification of a patient-specific protein signature in pancreatic juice. Protein quantitation was performed using isobaric labeling with TMT and LC-MS/MS analysis on an LTQ-Orbitrap Velos mass spectrometer. A : An unsupervised clustering analysis identified clearly different expression patterns in CEL -mutation carriers compared with controls with analyses performed by an investigator (F.M.) blinded to the samples. B : Log 2 plot of secretin-stimulated duodenal protein abundances and ratios of levels in the three CEL -mutation carriers compared with the three controls. Note the lack of skewed distribution of pancreatic proteins in the log 2 plot, indicating that a majority of the proteins had been detected despite degradation. Using “no-enzyme”-specific searches, we identified 757 proteins in the secretin-stimulated duodenal juice, of which 115 proteins were pancreatic specific based on a previous report ( 17 ). C : Immunoblot validation examples of proteins identified with stippled lines indicate gel crops. In addition to the three diabetic CEL -mutation carriers (D1, D2, D3) and three nonfamily controls (N1, N2, N3), we also added secretin-stimulated duodenal juice samples from two prediabetic CEL -mutation carriers (P1, P2) and from the CEL -mutation carrier with the KRAS -mutated pancreatic ductal adenocarcinoma (C1). D : Visualization of the MAPK-targeted proteins identified by Biobase ExPlain 3.0 tool and alphanumeric codes referring to PubMed identification in Supplementary Table 3 . E : Heat map demonstrating differential clustering of kinase activities in the secretin-stimulated duodenal juice and pancreatic tissue of CEL -mutation carriers and controls. F : Vertical bar graphs showing significant differences in kinase activities for PKC, CH1, and CH2 in secretin-stimulated duodenal juice. The multiplexed kinase activity assay allows the absolute quantitation of kinase activities by measuring the amount of phosphorylation of 60 peptide substrates with known motifs for many kinases. Vertical bar graphs showing differences in kinase activities for PKC, CH1, CH2, and MAPK in secretin-stimulated duodenal juice. PDAC, pancreatic ductal adenocarcinoma; mut, mutation; log 2 ratio, log 2 of the ratio of MS abundance for the sum of the three controls divided by the sum of the three diabetic mutation carriers; log 2 total, log 2 of the total MS abundance for a protein as the sum of the abundances of three controls and the three diabetic mutation carriers.
Article Snippet: The peptides from proteolytic degradation assays were analyzed by liquid chromatography (LC)-MS on a high-resolution Exactive Orbitrap mass spectrometer (Thermo Scientific, Rockford, IL).
Techniques: Protein Quantitation, Labeling, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Expressing, Mutagenesis, Kinase Assay, Quantitation Assay