Reversible protein phosphorylation plays a pivotal role in the regulation of cellular signaling pathways. Current approaches in phosphoproteomics focus on analysis of the global phosphoproteome in a single cellular state or of receptor stimulation time course experiments, often with a restricted number of time points. Although these studies have provided some insights into newly discovered phosphorylation sites that may be involved in pathways, they alone do not provide enough information to make precise predictions of the placement of individual phosphorylation events within a signaling pathway. Protein disruption and site-directed mutagenesis are essential to clearly define the precise biological roles of the hundreds of newly discovered phosphorylation sites uncovered in modern proteomics experiments. We have combined genetic analysis with quantitative proteomic methods and recently developed visual analysis tools to dissect the tyrosine phosphoproteome of isogenic Zap-70 tyrosine kinase null and reconstituted Jurkat T cells. In our approach, label-free quantitation using normalization to copurified phosphopeptide standards is applied to assemble high density temporal data within a single cell type, either Zap-70 null or reconstituted cells, providing a list of candidate phosphorylation sites that change in abundance after T cell stimulation. Stable isotopic labeling of amino acids in cell culture (SILAC) ratios are then used to compare Zap-70 null and reconstituted cells across a time course of receptor stimulation, providing direct information about the placement of newly observed phosphorylation sites relative to Zap-70. These methods are adaptable to any cell culture signaling system in which isogenic wild type and mutant cells have been or can be derived using any available phosphopeptide enrichment strategy. Molecular & Cellular Proteomics 8:2418 -2431, 2009.The reversible phosphorylation of serine, threonine, and tyrosine residues directly controls many cellular processes, leading to the activation of a coordinated network of additional phosphorylation events across multiple proteins over time. Clearly, there are benefits to individually identifying and characterizing specific components of a particular pathway, such as a phosphorylation site on a given protein, the kinase responsible for the modification, or the proteins interacting subsequently. However, a thorough understanding of these signaling pathways at the molecular level ultimately requires a global, simultaneous evaluation of these phosphorylation events as they occur over time.Currently, the most common method for assessing widescale changes in the proteome is two-dimensional gel electrophoresis (1), but this methodology is relatively low throughput and not optimal for the analysis of low abundance and hydrophobic signaling proteins (2). Recent publications describe alternate approaches for assessing changes in phosphorylation patterns based primarily on LC/MS methodologies (3-8). A variety of promising purification approaches have been dev...

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Background. Recent studies have compared CTA to stress testing and MPI using older Na-I SPECT cameras and traditional rest-stress protocols, but are limited by often using optimized CTA protocols but suboptimal MPI methodology. We compared CTA to stress testing with modern SPECT MPI using high-efficiency CZT cameras and stress-first protocols in an ED population.Methods. In a retrospective, non-randomized study, all patients who underwent CTA or stress testing (ETT or Tc-99m sestamibi SPECT MPI) as part of their ED assessment in 2010-2011 driven by ED attending preference and equipment availability were evaluated for their disposition from the ED (admission vs discharge, length of time to disposition), subsequent visits to the ED and diagnostic testing (within 3 months), and radiation exposure. CTA was performed using a 64-slice scanner (GE Lightspeed VCT) and MPI was performed using a CZT SPECT camera (GE Discovery 530c). Data were obtained from prospectively acquired electronic medical records and effective doses were calculated from published conversion factors. A propensity-matched analysis was also used to compare outcomes in the two groups.Results. A total of 1,458 patients underwent testing in the ED with 192 CTAs and 1,266 stress tests (327 ETTs and 939 MPIs). The CTA patients were a lower-risk cohort based on age, risk factors, and known heart disease. A statistically similar proportion of patients was discharged directly from the ED in the stress testing group (82% vs 73%, P 5 .27), but their time to disposition was longer (11.0 ± 5 vs 20.5 ± 7 hours, P < .0001). There was no significant difference in cardiac return visits to the ED (5.7% CTA vs 4.3% stress testing, P 5 .50), but more patients had follow-up studies in the CTA cohort compared to stress testing (14% vs 7%, P 5 .001). The mean effective dose of 12.6 ± 8.6 mSv for the CTA group was higher (P < .0001) than 5.0 ± 4.1 mSv for the stress testing group (ETT and MPI). A propensity score-matched cohort showed similar results to the entire cohort.Conclusions. Stress testing with ETT, high-efficiency SPECT MPI, and stress-only protocols had a significantly lower patient radiation dose and less follow-up diagnostic testing than CTA with similar cardiac return visits. CTA had a shorter time to disposition, but there was a trend toward more revascularization than with stress testing. (J Nucl Cardiol 2014;21:305-18.)