The purpose of this application is to acquire a hybrid triple quadrupole-ion trap mass spectrometer, specifically the QTRAP 5500 from Applied Biosystems, with unique capabilities to support actively ongoing studies that aim to uncover critical physiological pathways and causes of pediatric diseases. This instrument will be used exclusively for quantitative mass spectrometric applications by 9 NIH funded investigators at the Joseph Stokes, Jr. Research Institute of the Children's Hospital of Philadelphia. Ongoing projects initiated by these investigators have employed genomic discovery platforms, biochemical, molecular, different mass spectrometric and orthogonal experiments to discover proteins of interest, posttranslational modifications, proteolytic processing and networks of proteins that execute biological functions. Therefore all these projects are well advanced past the stages of discovery and specific and practical protein and peptide targets have been identified. The immediate challenge for these projects is to move beyond qualitative descriptions to explore these discoveries in depth by quantifying changes in proteins and peptides. This goal cannot be accomplished with typical antibody based western blot, ELISA or even with our current linear ion trap mass spectrometers since these approaches are often neither feasible nor practical due to lack of sensitivity, specificity, and/or throughput. Targeted peptide quantification is becoming the clear choice as a highly selective and sensitive methodological approach for quantitative evaluation of dynamic changes of proteins, protein networks, posttranslational modifications, validation and utilization of biological markers. Towards this goal the PI and co-Investigator in collaboration with the users of the proposed instrument are actively developing methodologies that employ stable isotope labeled peptides and multiple reaction monitoring (MRM) workflows for the quantification of proteins in human samples as well as in animal and cellular model systems. Multiple reaction monitoring is emerging as the standard technique for quantitative liquid chromatography tandem mass spectrometry (LC/MS/MS) experiments;however generation and application of these MRM workflows with our current ion trap platforms is limited. Therefore the installation and use of the proposed mass spectrometer is vital for enabling the development and implementation of methodologies for targeted peptide quantification to generate sizeable new knowledge and insights. Overall, the proposed projects although biologically diverse are united by the need to develop, validate and implement quantitative hypothesis-driven mass spectrometric approaches. Thus, while each research project stands alone in scientific value and integrity the added value of this combined proposal is substantial.
The instrument will have a major and immediate impact on the research programs of 9 NIH-funded investigators that are engaged in both basic and translational research aimed to uncover fundamental physiological processes and mechanisms of disease at the Children's Hospital of Philadelphia. The requested instrumentation will facilitate the quantification of potentially novel biological markers for the prognosis and diagnosis of pediatric diseases.
| Doulias, Paschalis-Thomas; Raju, Karthik; Greene, Jennifer L et al. (2013) Mass spectrometry-based identification of S-nitrosocysteine in vivo using organic mercury assisted enrichment. Methods 62:165-70 |
