Billy Molloy (Presenter)
Waters
Authorship: Billy J Molloy 1, Donald JL Jones 2,3, Johannes PC Vissers 1, Donald P Cooper 1, James I Langridge 1
(1) Waters Wilmslow, United Kingdom (2) Department of Cancer Studies / Cardiovascular Research Centre, Glenfield Hospital, Leicester, United Kingdom
| Short Abstract Translational and biomarker verification studies are challenged in that they not only require the analysis of large sample cohorts with high-throughput, but also demand high sensitivity, high resolution and selectivity over a large dynamic range. Targeted LC-MS/MS based assays afford analyte quantification with the reproducibility and throughput required in order to rapidly assess biomarker performance. Multiple Reaction Monitoring (MRM), using tandem quadrupole mass spectrometry, is an enabling technology that provides speed and selectivity, whilst miniaturized LC systems offer additional improved sensitivity. Here, the application of micro-fluidics coupled to a novel tandem quadrupole MS/MS system, using a multi-point internal calibration method for the quantitation of peptides and proteins is presented and considered for speed, sensitivity, accuracy/ bias and selectivity. |
Long Abstract
Introduction
Translational and biomarker verification studies are challenged in that they not only require the analysis of large sample cohorts with high-throughput, but also demand high sensitivity, high resolution and selectivity over a large dynamic range. Targeted LC-MS/MS based assays afford analyte quantification with the reproducibility and throughput required in order to rapidly assess biomarker performance. Multiple Reaction Monitoring (MRM), using tandem quadrupole mass spectrometry, is an enabling technology that provides speed and selectivity, whilst miniaturized LC systems offer additional improved sensitivity. Here, the application of micro-fluidics coupled to a novel tandem quadrupole MS/MS system, using a multi-point internal calibration method for the quantitation of peptides and proteins is presented and considered for speed, sensitivity, accuracy/ bias and selectivity.
Methods
LC peptide separations were performed with a 0.15 × 100 mm ionKey/MS micro-fluidics device packed with BEH C18 1.7 μm stationary phase. The reversed phase gradient was 2-30% B over 45 min at a flow rate of 1 µL/min with the device temperature maintained at 35 ºC. Samples were injected/loaded directly on-column or using a 0.3 × 50 mm precolumn configuration packed with 5 µm Symmetry C18. In this instance, the samples were loaded at a flow rate of 15 µL/min for 1 min. MRM experiments were performed using a prototype tandem quadrupole mass spectrometer. Dwell and interscan delay times were automatically calculated by the operating software based on a minimum number of data points across a chromatographic peak.
Preliminary Data
Initial benchmarking of the MS system, fitted with a novel ion guide that enables improved and selective ion transmission, alongside with detector advances, was conducted by contrasting the sensitivity and precision performance for six stable isotope labelled peptides spike into biological matrix (tryptic digested, undepleted human plasma) against four other MS systems, including two tandem quadrupole and two high-resolution time-of-flight instruments, all operated in MRM mode of acquisition. On average, S/N was found to be two-fold better compared to the next most sensitive instrument in MRM mode of acquisition. %CV performance values were found to be similar to the two other tandem quadrupole instruments and approximately two fold better than the time-of-flight instruments.
A multi-point internal calibration method was used to achieve within sample calibration. This technique was assessed with regard to quantitative precision. In addition, since external standard response calibration runs are not required, throughput gains were determined. Performance metrics of the method will be demonstrated and the results contrasted with those obtained with conventional MRM quantitation approaches for the direct analysis, without applying any type of fractionation or enrichment, of three human plasma peptides, representing putative blood-based markers of cardiovascular disease that span over five orders of dynamic concentration range.
Novel aspect
High sensitivity detection of peptides in biological matrices using a novel tandem quadrupole MS system and quantitation method.
References & Acknowledgements:
1. MRM-based multiplexed quantitation of 67 putative cardiovascular disease biomarkers in human plasma. Domanski et al. Proteomics. 2012 Apr;12(8):1222-43.
2. Advances in Quadrupole and Time-of-Flight Mass Spectrometry for Peptide MRM based Translational Research Analysis. Mbasu et al. Proteomics. 2016 (in press)
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