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Communication Dans Un Congrès Année : 2011

Comparison of different liquid chromatography systems for the detection of low-level biomarkers in biological fluids by Selected Reaction Monitoring

Résumé

Detection of low-abundant proteins in biological samples is usually hampered by limited sensitivity and dynamic range of mass spectrometers. In the Selected Reaction Monitoring (SRM) mode, the dynamic range problem is alleviated, as target parent and fragment ions are respectively selected in a narrow mass range window by the two quadrupole analyzers, filtering out other highly abundant peptide ions that otherwise would lead to signal suppression in a full MS scan. Nonetheless, SRM analysis of weakly concentrated molecule results in low ion currents in the mass spectrometer, and intrinsic sensitivity of the instrument may then become the limiting factor of the analysis. Injecting higher amounts of analyte in the mass spectrometer could thus in this case help to improve the detection of species present at very low level in a complex matrix. Here, we tested different sample processing methods and LC-MS instrumental settings to optimize the detection of biomarker candidates in two biological fluids. Increasing amounts of sample were loaded either on a 75μm I.D. capillary column coupled to a nanospray source, or on a 2mm I.D. column coupled to an ESI source. In each case, several proteins were monitored by SRM on a 5500QTrapTM mass spectrometer (ABSciex), in order to determine the optimal loading capacity of the chromatographic system. In the case of the nanoLC setting, samples were loaded on C18 precolumns with different loading capacity, and gradients of different lengths were tested in order to increase the amount of material loaded on the system without affecting the linearity of the MS response. For each experimental setting, the limit of detection by SRM of candidate biomarkers, spiked at different concentrations in plasma or cerebrospinal fluid, was determined. Our results indicate that the loss of sensitivity of conventional HPLC compared to nanoLC is balanced by the higher amount of sample that can be injected in the instrument, making this instrumental configuration an interesting alternative for the detection of low-abundant species, as long as the absolute quantity of starting material does not represent a limiting factor.
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Dates et versions

hal-00960690 , version 1 (18-03-2014)

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  • HAL Id : hal-00960690 , version 1

Citer

Florence Roux-Dalvai, Violette Gautier, Alexandre Stella, Jérôme Lemoine, Anne Gonzales de Peredo, et al.. Comparison of different liquid chromatography systems for the detection of low-level biomarkers in biological fluids by Selected Reaction Monitoring. Congrès français de Spectrométrie de Masse et d'Analyse Protéomique (SMAP 2011), Sep 2011, Avignon, France. ⟨hal-00960690⟩
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