Targeted MS approaches for biomarker verification

Antibody-based assays have been the most common methods of choice for targeted studies, but their low protein throughput makes them more suitable for final validation phases focused on a restricted list of biomarkers. Highly multiplexed targeted MS-based techniques have gained in popularity for verification studies, in order to evaluate a large number of potential biomarkers coming from discovery studies and prioritize the most promising candidates to enter into further validation phases ²⁰². In addition, the improvement in sample throughput of the MS technologies facilitates their use in larger sample sets in initial validation phases (Figure 13C).

In targeted MS approaches, a list of peptides representing the proteins to be measured must be selected before the actual MS acquisition and measured by repeated MS/MS events through acquisition windows. The data points extracted from the MS/MS events allow to recreate the chromatic elution profiles of the peptides that can be integrated for further quantification. As for DDA, targeted acquisition relies on the filtering of the precursors through a narrow mass window of 1 m/z before MS/MS fragmentation, significantly improving the selectivity compared to DIA experiments. However, targeted experiments are limited in the number of peptides that can be monitored per analysis. In order to maximize the number of measurable peptides, the liquid chromatography retention time of each peptide should be determined in a preliminary analysis to be able to monitor the fragment ions of specific peptides in the corresponding time windows. This is called scheduled acquisition ²⁸⁸. Whereas DDA acquisition requires the detection of a precursor in a survey scan to trigger a MS/MS event, targeted acquisition systematically performs MS/MS events during the acquisition windows which are centered on the elution times of each targeted

Proteomic approaches for biomarker identification

53

Introduction

peptide. Therefore, targeted acquisition does not generate missing data and replicates can be quantitatively compared as they perfectly overlap.

! Selected Reaction Monitoring (SRM)

The SRM acquisition performed on a triple quadrupole mass spectrometer, which was elected "Method of the year 2012" by Nature Methods, became the reference technique for MS-based quantitative methods in proteomics ²⁸⁹.

Figure 14. Comparison of the SRM and PRM acquisitions. A. SRM acquisition performed on a triple quadrupole mass spectrometer. B. PRM acquisition performed on a quadrupole-orbitrap mass spectrometer.

The SRM acquisition is performed by the selection of a precursor ion by the first quadrupole (Q1), followed by the fragmentation of the selected ion in the second quadrupole (q2) operated as a collision cell. Finally, one fragment ion is selected by the third quadrupole (Q3) and reaches the detector to generate a signal ²⁹⁰ (Figure 14A). The first and the third quadrupoles are usually operated to filter ions with a mass width of 0.7 to 1 m/z. A pair of a precursor associated with a fragment ion is called an SRM transition and usually three to five transitions are recorded per peptide to improve the confidence of the measurements.

Regarding data processing, the quantification is usually based on the area under the peak of the elution profiles of the targeted fragment ions for improved accuracy and precision.

LC-SRM is a multiplexed acquisition method that allows for the accurate quantification of a hundred of peptides in a single LC-MS analysis. It achieves a good selectivity due to the two levels of mass filtering on the precursors and the fragment ions, and high reproducibility across many samples and different laboratories 289,291,292. The possibility of using internal standards such as stable isotope labeled peptides or proteins brings several advantages for quantitative analysis including: i) control of the signal variation (see section 5.1.2.1., page

Proteomic approaches for biomarker identification

54

Introduction

45); ii) a strong confidence in the peptide identification due to the co-elution of the endogenous peptide and internal standard and the similarity of the fragmentation pattern;

and iii) the possibility to perform absolute quantification if the concentration of the internal standard is known ^249,293.

SRM assays usually cover a linear dynamic range of 5 orders of magnitude ²⁸⁸, and provide a good sensitivity. However, as for most LC-MS based proteomic methods, a depletion or enrichment step can be required to quantify very low abundance proteins ^230,294. The method development in this approach is time consuming and static, as the SRM transition list must be defined prior acquisition. Since only a limited number of transitions are monitored for each peptide, the presence of interferences may jeopardize the data analysis and may require the reanalysis of the samples, decreasing the throughput of the technique ²⁵⁹. Therefore, it is important to validate the specificity and sensitivity of the SRM transitions in a representative number of samples before the analysis of the full sample set ²⁹³. The information required for the method development prior to the SRM analysis is shown in Figure 15.

! Parallel Reaction Monitoring (PRM)

Targeted MS-based approaches are also feasible with high-resolution accurate mass spectrometers, and the PRM acquisition performed in these instruments recently arose as a promising alternative to SRM ^259,295. The configuration of the LC-PRM shares some similarities with the LC-SRM performed on a triple quadrupole, but the third quadrupole is replaced by a high-resolution mass analyzer (TOF or orbitrap). In the case of a quadrupole-orbitrap configuration, a specific precursor ion is selected by the quadrupole and transferred via the C-trap to the collision cell where it is fragmented. The fragment ions are then transferred back to the C-trap and injected into the orbitrap to be analyzed at high resolution (Figure 14B) ²⁹⁶.

This acquisition method permits the detection of all fragment ions of the targeted precursor ion in one MS/MS event whereas the SRM method requires one MS/MS event per fragment ion ^275,277. The data processing is performed by extraction of the ion chromatograms (XIC) of the fragment ions of interest and, as for SRM and DIA data, quantification can be performed by integration of the areas of the elution profiles.

The PRM acquisition has several advantages over the SRM approach ²⁵⁹. The high resolution dramatically reduces the risk of interferences from the background and the accurate mass improves the confidence on the fragment ions identities. In addition, because it is based on full MS/MS spectra, all potential fragment ions are recorded, instead of only the 3-5 usually targeted in SRM. A reference spectra can be used to select the fragment

Proteomic approaches for biomarker identification required prior to the MS analysis (Figure 15). Moreover, the presence of interfering ions can be solved by using alternative fragment ions without the need of a re-acquisition, as it is the case with SRM. The PRM mode is also fully compatible with the use of isotope labeled peptides, with all the associated benefits previously described ^257,297.

Although the advantages of application of these high-resolution accurate mass spectrometers in clinical studies have been evaluated ^241,298, only few studies have already employed this technique for biomarker searches in clinical ²⁹⁹ or cell lines samples ^300–302, and none of them for targeting a high number of candidate biomarkers.

Figure 15. Steps required for the development of an SRM and a PRM method. Adapted from Gallien et al. 2011 ²⁹².