KTYDSYLGDDYVR Linearity A Comparative Study between Selected Reaction Monitoring (SRM) on 4000 Q-TRAP™ and 5600 Triple ToF™ Mass Spectrometers and pseudo-SRM on an Orbitrap Velos™ Instrument David N. Potier*, Andrew J. Williamson and Anthony D. Whetton Stem Cell and Leukaemia Proteomics Laboratory (SCALPL), University of Manchester, UK Introduction SRM has long been used to quantify molecules of interest1 such as small molecules2 or peptides3. Instrumentation advances have allowed SRM to be performed on different machines A pseudo-SRM can also be performed on the Orbitrap Velos™. The claim that these instruments are able to perform SRM-type analysis requires testing. This, combined with increased sensitivity over previous generations of instrument, make their SRM performance relative to a 4000 Q-TRAP™ an interesting comparison. In this study, we perform linearity testing across seven orders of magnitude using ten different peptides in the background of an E. Coli tryptic digest. From this, we assess the limit of detection for each machine. Sample Preparation Method A six protein mix digest (Dionex) was made up at concentrations ranging from 1 amol to 1 pmol in the background of an E. Coli tryptic digest (500 ng, Waters). Ten different peptides were monitored in these samples. All samples were run in triplicate. 4000 Q-TRAP™/5600 Triple ToF™ SRM Method Four SRM transitions were set up to identify each target peptide as shown in Figure 1. Quantitation was performed on the transition giving the most intense response. Samples were analysed on the 4000 Q-TRAP™ and the 5600 Triple ToF™ mass spectrometers. FIGURE 1: A schematic of an SRM, with precursor and product ion selection taking place in Q1 and Q3 respectively, and fragmentation occurring in the collision cell in Q2. Orbitrap Velos™ pseudo-SRM Method The same four pseudo-SRM transitions were set up to identify each target peptide as shown in Figure 2. Quantitation was performed on the transition giving the most intense response. Samples were analysed in the LTQ of an Orbitrap Velos™ mass spectrometer. FIGURE 2: A schematic of a pseudo-SRM, with precursor ion selection taking place in the linear ion trap, fragmentation occurring in an HCD collision cell, & product ion selection taking place in the orbitrap. Detector Q0 Q1 Quadrupole /ToF Collision Cell Results KTYDSYLGDDYVR Linearity GYLAVAVVK Linearity Variance Conclusion Both the 4000 Q-TRAP and Orbitrap Velos™ provide a linear response across up to six orders of magnitude on average, with the similar variances. The machine with the lowest limit of detection was the Orbitrap Velos™, with LOD’s as low as 1 amol in a complex background. 5600 Triple ToF™ data is presently being acquired to compare to the above. References 1. W. H. Phillips, Jr., K. Ota and N. A. Wade, J Anal Toxicol, 1989, 13, 268-273. 2. Y.-Q. Xia, J. D. Miller, R. Bakhtiar, R. B. Franklin and D. Q. Liu, Rapid Commun. Mass Spectrom., 2003, 17, 1137-1145. 3. L. Anderson and C. L. Hunter, Mol Cell Proteomics, 2006, 5, 573-588. Acknowledgements We thank BBSRC, LLR and Philips for funding this work. We also thank Dr. David Knight for the use of his Q-TRAP™ and Dr. Stacey Warwood for her assistance in this project.