A significant consequence of protein phosphorylation is to alter protein-protein interactions, leading to dynamic regulation of the components of protein complexes that direct many core biological processes. Recent proteomic studies have populated databases with extensive compilations of cellular phosphoproteins and phosphorylation sites and a similarly deep coverage of the subunit compositions and interactions in multiprotein complexes. However, considerably less data are available on the dynamics of phosphorylation, composition of multiprotein complexes or that define their interdependence. We describe a method to identify candidate phosphoprotein complexes by combining phosphoprotein affinity chromatography, separation by size, denaturing gel electrophoresis, protein identification by tandem mass spectrometry, and informatics analysis. Toward developing phosphoproteome profiling, we have isolated native phosphoproteins using a phosphoprotein affinity matrix, Pro-Q Diamond resin (Molecular Probes-Invitrogen). This resin quantitatively retains phosphoproteins and associated proteins from cell extracts. Pro-Q Diamond purification of a yeast whole cell extract followed by 1-D PAGE separation, proteolysis and ESI LC-MS/MS, a method we term PA-GeLC-MS/MS, yielded 108 proteins, a majority of which were known phosphoproteins. To identify proteins that were purified as parts of phosphoprotein complexes, the Pro-Q eluate was separated into two fractions by size, <100 kDa and >100 kDa, before analysis by PAGE and ESI LC-MS/MS and the component proteins queried against databases to identify protein-protein interactions. The <100 kDa fraction was enriched in phosphoproteins indicating the presence of monomeric phosphoproteins. The >100 kDa fraction contained 171 proteins of 20-80 kDa, nearly all of which participate in known protein-protein interactions. Of these 171, few are known phosphoproteins, consistent with their purification by participation in protein complexes. By comparing the results of our phosphoprotein profiling with the informational databases on phosphoproteomics, protein-protein interactions and protein complexes, we have developed an approach to examining the correlation between protein interactions and protein phosphorylation.

Culture-based methods to measure Escherichia coli ( E. coli ) are used by beach administrators to inform whether bacteria levels represent an elevated risk to swimmers. Since results take up to 12 h, statistical models are used to forecast bacteria levels in lieu of test results; however they underestimate days with elevated fecal indicator bacteria levels. Quantitative polymerase chain reaction (qPCR) tests return results within 3 h but are 2–5 times more expensive than culture-based methods. This paper presents a prediction model which uses limited deployments of qPCR tested sites with inter-beach correlation to predict when bacteria will exceed acceptable thresholds. The model can be used to inform management decisions on when to warn residents or close beaches due to exposure to the bacteria. Using data from Chicago collected between 2006 and 2016, the model proposed in this paper increased sensitivity from 3.4 percent to 11.2 percent–a 230 percent increase. We find that the correlation between beaches are substantial enough to provide higher levels of precision and sensitivity to predictive models. Thus, limited deployments of qPCR testing can be used to deliver better predictions for beach administrators at lower cost and less complexity.

9Culture-based methods to measure Escherichia coli (E. coli) are used by beach administrators to inform whether bacteria levels represent an elevated risk to swimmers. Since results take up to 12 hours, statistical models are used to forecast bacteria levels in lieu of test results; however they underestimate days with elevated fecal indicator bacteria levels. Quantitative polymerase chain reaction (qPCR) tests return results within 3 hours but are 2 to 5 times more expensive than culture-based methods. This paper presents a prediction model which uses limited deployments of qPCR tested sites with inter-beach correlation to predict when bacteria will exceed acceptable thresholds. The model can be used to inform management decisions on when to warn residents or close beaches due to exposure to the bacteria. Using data from Chicago collected between 2006 and 2016, the model proposed in this paper increased sensitivity from 3.4 percent to 11.2 percent-a 230 percent increase. We find that the correlation between beaches are substantial enough to provide higher levels of precision and sensitivity to predictive models. Thus, limited deployments of qPCR testing can be used to deliver better predictions for beach administrators at lower cost and less complexity.