Bruno Domon1, Lina Ancheva1, Sebastien Gallien1, Antoine Lesur1, Guy Berchem2
1Luxembourg Clinical Proteomics Center, Strassen, Luxembourg, 2Laboratory of Experimental Hemato-Oncology, Strassen, Luxembourg
Targeted proteomics analyses of biomarker are routinely performed on triple quadrupole mass spectrometers, which present limited selectivity, due to low resolution of the mass filters. The targeted analyses of clinical samples carried out on a quadrupole-orbitrap mass spectrometer using parallel reaction monitoring (PRM) showed a significant gain in sensitivity and selectivity. A new acquisition method leveraging the presence of internal standards showed a dramatic improvement of the reliability and the precision of the analyses. The method has a broad applicability to the quantitative analysis of clinical samples, such as lung cancer plasma samples to discriminate the disease stages and subtypes; and to tissue samples to map driver mutations (EGFR and KRAS).
Targeted proteomics analyses of biomarkers are routinely performed on triple quadrupole mass spectrometers in selected reaction monitoring (SRM) mode. However, the low resolution quadrupole mass filters present limited selectivity, especially for complex samples such as bodily fluids. Hybrid mass spectrometers with high resolution and accurate mass capabilities overcome this limitation, and have opened new avenues in quantitative proteomics. The targeted analyses of clinical samples carried out on a quadrupole-orbitrap mass spectrometer using parallel reaction monitoring (PRM) showed a significant gain in sensitivity and selectivity. The technique decouples the data acquisition from the data analysis and offers flexible experimental design as well as iterative data analysis.
The development of a new acquisition method leveraging the presence of internal standards has dramatically improved the reliability and the precision of the analyses.
In order to fully leverage the potential of the PRM technique and to apply it to clinical assays, we have designed a new data acquisition scheme called internal standard triggered – parallel reaction monitoring (IS-PRM). This method relies on the addition of internal standards (i.e., isotopically labeled peptides) to adjust on-the-fly the acquisition parameters (resolution and fill time) to drive in real-time the measurement of endogenous peptides (corresponding to the proteins of interest). This generates precise and high confidence results as the acquisition time is managed to maximize the time effectively devoted to each analyte while keeping an appropriate cycle time. In the IS-PRM approach, the acquisition alternates between two modes: a fast low resolution “watch mode” and a “quantification mode” using optimized parameters ensuring high quality quantitative data. A redefined PRM workflow presenting specific characteristics was developed. First, the acquisition method contains limited information about the analytes (elution time and optimum precursor m/z); it also includes instrument parameters, which are adjusted according to the distribution of the peptide elution times and the intended trade-off between the scale of the experiment and the selectivity and sensitivity of measurements. Second, the data processing method defines an extended list of fragments to be used to assess peptide identity and to perform quantification. The same procedure was also applied to IS-PRM to set the parameters in real-time to measure the endogenous peptides.
The solution allows a fully automated execution of PRM experiments.
A module allows the creation of spectral libraries from the analysis of synthetic reference peptides (including meta-information such as their normalized elution time). It is also used to generate the acquisition methods by compiling the information for the targeted peptides specified in a query. It has specific options for the type of experiment (screening versus quantification), and the acquisition mode. The acquisition method is directly uploaded to the instrument controlling software. The data processing method, also based on the spectral library, indicates the fragments (primary and alternative, with their relative intensity) to be used for peptide identity confirmation and fragment qualification in actual analyses. A computational tool supports this iterative process and ultimately provides peptide qualified fragment ions whose traces are extracted. The relevant PRM traces output can be directly uploaded to Skyline to perform the actual quantification while reducing the volume of data. A proof-of-principle was performed in IS-PRM mode to quantify 600 endogenous peptides representing 338 proteins in bodily fluids. The developed method has a broad applicability and enables routine quantitative PRM analyses. It was applied to the analysis of clinical samples, more specifically to lung cancer markers in plasma samples and, it allowed a clear discrimination of the disease stages and subtypes. The approach was also applied to map driver mutations (EGFR and KRAS) in lung cancer tissues samples, in order to assist the clinicians to select adequate treatments.