U.S. Department of Energy

Pacific Northwest National Laboratory

Evaluation of SRM-, PRM- and DIA-based Targeted Quantification

Introduction 

Mass spectrometry (MS) based proteomics has increasingly been considered as a pivotal technology for biological and biomedical research. Verification studies using targeted proteomics are often required to provide high selectivity, accuracy, and sample throughput while maintaining high sensitivity. Selected reaction monitoring (SRM) has widely been used as a targeted MS technique with high sensitivity and selectivity. With recent advances in MS instrumentation especially the Orbitrap based hybrid mass spectrometers, two novel approaches, namely parallel reaction monitoring (PRM) and data independent acquisition (DIA), were also introduced for targeted proteomics analysis. Herein, we systematically assessed the analytical characteristics of SRM-, PRM-, and DIA-based targeted quantification and demonstrated the complementarity of these techniques for fit-for-purpose applications.

Methods 

Twenty peptides were selected for the initial evaluation of the 3 techniques. The heavy stable isotope-labeled peptides were synthesized and spiked into the samples as the internal standard. SRM was performed on the Thermo TSQ Vantage triple quadrupole instrument while PRM and DIA were carried out on the Thermo Q Exactive HF instrument with the same LC system and setup. Each transition and its corresponding collision energy were optimized for SRM while HCD of target precursor ions was acquired with an isolation window of 2 and 5 m/z for PRM and DIA, respectively. The performance of the 3 techniques was further evaluated using 133 and 110 peptides associated with EGFR pathway and prostate cancer, respectively. Data were analyzed using Skyline. 

Preliminary data 

The reverse response curve was created for the 20 peptides in HMEC cell digest to characterize the linear dynamic range and lower limit of quantification (LLOQ) of the 3 techniques. Overall, all 20 target peptides were quantified by SRM while 18 and 15 peptides were quantified by PRM and DIA, respectively; 6 peptides showed better LLOQ values by SRM over PRM and DIA while 3 peptides showed better LLOQ values by PRM over SRM and DIA. SRM also exhibited slightly better reproducibility. An averaged CV values were 8.3, 10.0, and 12.2 by SRM, PRM, and DIA, respectively. One of the challenges in SRM is potential interferences in complex samples due to the low mass resolution and accuracy achievable on the triple quadrupole instrument. PRM and DIA data acquired on high-resolution Q Exactive instrument are more flexible for minimizing interferences by requiring higher mass accuracy and post-acquisition transition refinement and peak integration, contributing to better selectivity and quantification accuracy for 8 of the target peptides. To further test the potential impact of multiplexing levels on the analytical performance, SRM and PRM were also performed for 110 peptides associated with prostate cancer. Two injection times (60 and 150 ms) were used for PRM. SRM and PRM quantified 44 and 48 peptides, respectively. Both ion injection time settings in PRM showed the same level of peptide detection and high correlation with SRM (R2>0.98); however the use of longer injection time resulted in better MS response and reproducibility. We are currently applying the 3 techniques to quantify 133 EGFR pathway peptides. Front-end enrichment techniques will also be tested for their impact on the sensitivity and selectivity of the 3 targeted quantification methods. 

Novel aspect 

Evaluation of popular targeted MS quantification techniques and the potential impact of multiplexing and front-end enrichment/separation on their analytical performances

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