Amol Prakash, Thermo Fisher Scientific, BRIMS, Cambridge, MA, USA
Taha Rezai, Thermo Fisher Scientific, BRIMS, Cambridge, MA, USA
Bryan Krastins, Thermo Fisher Scientific, BRIMS, Cambridge, MA, USA
David Sarracino, Thermo Fisher Scientific, BRIMS, Cambridge, MA, USA
Michael Athanas, Vast Scientific, Wayland, MA, USA
Paul Russo, George Mason University, Fairfax, VA, USA
Mark Ross, George Mason University, Fairfax, VA, USA
Yuan Tian, John Hopkins University, Baltimore, MD, USA Hui Zhang, John Hopkins University, Baltimore, MD, USA
Vathany Kulasingam, Mt. Sinai Hospital, University of Toronto, Toronto, ON, Canada
Igor Batruch, Mt. Sinai Hospital, University of Toronto, Toronto, ON, Canada
Chris Smith, Mt. Sinai Hospital, University of Toronto, Toronto, ON, Canada
Lance Liotta, George Mason University, Fairfax, VA, USA
Emanuel Petricoin, George Mason University, Fairfax, VA, USA
Eleftherios P. Diamandis, Mt. Sinai Hospital, University of Toronto, Toronto, ON, Canada Dan Chan, John Hopkins University, Baltimore, MD, USA
Mary F. Lopez, Thermo Fisher Scientific, BRIMS, Cambridge, MA, USA |
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Targeted SRM assays for protein biomarkers are increasingly being applied in the areas of disease prognostics and therapeutic drug monitoring because they are typically robust and selective, even in complex matrices. However, in order to develop effective assays for diseases or pathologies with multiple intersecting metabolic and/or signaling pathways, multiplexed assays (monitoring panels of peptides) will have to be developed. Unfortunately, designing effective SRM assays still remains a challenge, because the choice of peptides/transitions and the optimization of sensitivity, selectivity and throughput is typically slow, labor intensive and costly. More important, it is currently difficult to transfer assays between labs while retaining a high degree of inter-lab reproducibility.
SRM assays were developed on a TSQ Quantum Ultra triple quadrupole mass spectrometer, Surveyor MS pump, Micro Autosampler and an IonMax Source equipped with a low flow metal needle (ThermoFisher Scientific). Reverse phase separations were carried out on a 1mm X 50mm Hypersil Gold 1.9µm C18 particle. Solvent A was LC-MS grade water with 0.2% (v/v) formic acid, and solvent B was LC-MS grade 30% (v/v) acetonitrile with 0.2% (v/v) formic acid (Optima grade reagents, Fisher Scientific). Pinpoint software (ThermoFisher Scintific) was used for developing SRM assays. The software algorithm predicts candidate peptides and facilitates the choice of multiple fragment ions for SRM assay design, instrument method development, automatic peptide identity confirmation and quantitative data processing.
A variety of SRM assays were monitored across four different laboratories with identical LC/MS-triple-quadrupole platforms. Initially, data were collected from single peptide infusions with analysis of MS and MS/MS under various conditions. Next, a mixture of 64 synthetic peptides was analyzed in order to understand and optimize numerous parameters that affect LC-MS SRM of a moderately complex but defined sample. The 64 peptide mixture was initially optimized in one laboratory and later the method was transferred to the other three locations. In a final series of experiments, the 64 peptide mixture was analyzed by all four laboratories in a background of digested plasma providing level of detection (LOD) and level of quantification (LOQ) information in a complex matrix for all 64 peptides. |
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