Definition of Targeted Quantitative Proteomics
Based on liquid chromatography-mass spectrometry (LC-MS/MS), shotgun proteomics exhibits robust protein identification capabilities; however, its sensitivity in protein quantification still requires improvement. Conversely, emerging targeted proteomics techniques offer enhanced sensitivity and reproducibility for protein quantification. If shotgun proteomics is akin to large-scale blind screening of samples, targeted proteomics is the focused and high-quality quantitative analysis of a predefined set of proteins of interest.
Targeted proteomics quantification, also referred to as targeted proteinomics quantification, addresses the limitations of untargeted quantitative proteomics. It boasts high throughput, accuracy, and reproducibility advantages. Leveraging earlier data from differential proteomics/transcriptomics/genomics analyses, this approach validates biomarkers in large biological sample sizes. Targeted proteomics selectively collects and analyzes quantitative mass spectrometry data for peptides of interest, achieving higher sensitivity and precision across hundreds or thousands of samples. Following the addition of stable isotope standards to samples, targeted proteomics allows for absolute quantification of the proteins of interest. It finds applications in signaling pathway investigation, tumor biomarker research, post-translational modification analysis, and serves as an alternative protein targeting quantification technology beyond antibody-based protein quantification methods.
What are the Technologies of Targeted Quantitative Proteomics?
Targeted quantitative proteomics primarily encompasses MRM/SRM and PRM techniques. MRM (Multiple Reaction Monitoring), also known as SRM (Selected Reaction Monitoring), is a classical targeted protein profiling method that relies on triple quadrupole mass spectrometry. The more recent PRM (Parallel Reaction Monitoring) technique, on the other hand, is based on next-generation high-resolution Orbitrap mass spectrometry.
SRM, often referred to as the gold standard of targeted proteomics, serves as the foundation for PRM technology, a derivative technique. PRM relies on high-quality accurate/high-resolution mass spectrometry instruments, leading to results in targeted quantification that are more selective, sensitive, and reproducible compared to SRM. Additionally, PRM exhibits enhanced resistance to interference in complex backgrounds. In terms of practical implementation, PRM offers advantages over SRM with simpler procedures and lower costs, as follows:
No need for pre-designed precursor/fragment ion pairs for targeted proteins, saving experiment design and operational time.
Flexibility in choosing whether to include synthetic standard peptides, offering substantial cost savings.
In terms of application, PRM can replace traditional Western blot techniques. On one hand, it liberates protein validation from commercial antibodies and can be applied to various non-model organisms. On the other hand, it allows high-throughput protein validation on a large scale of biological samples, thereby enhancing experimental efficiency.
DIA Quantitative Proteomics
Protein quantification methods such as iTRAQ/TMT, SILAC, and label-free rely on Data-Dependent Acquisition (DDA) technology, where during precursor ion selection, mass spectrometry tends to scan high-abundance peptide segments. Data-Independent Acquisition (DIA) serves as an alternative to DDA. DIA is a targeted proteomics technique that involves fragmenting and performing second-stage mass spectrometry analysis on all ions within a selected mass-to-charge ratio (m/z) range. This approach enables the detection and quantification of target proteins.
DIA offers a significant advantage over DDA, particularly in efficiently measuring low-abundance protein molecules within complex samples. This greatly enhances the reliability of quantitative analysis, providing higher accuracy and reproducibility in quantification.
Comparison of Principles for DDA, DIA, and MRM
Targeted Proteomics Workflow
- Method Development and Establishment: The initial phase of targeted proteomics involves devising an appropriate analytical workflow for the target samples and constructing corresponding spectral databases using High-Resolution Mass Spectrometry (HRM). For the proteins of interest, a series of detection methods (also known as transitions or exclusion lists) need to be established and optimized. Pre-analysis involves gathering characteristic information about peptide peak profiles, such as fragment ion intensities and retention times (iRT), which serve as references for formal experimental data collection.
- Data Acquisition: Data acquisition involves employing Triple Quadrupole Mass Spectrometry (MRM) or High-Resolution Orbitrap Mass Spectrometry (PRM). Both approaches utilize precursor ion selection by the front-end quadrupole from the preliminary experiments, followed by introducing ions into the collision chamber. In MRM, only pre-selected ions are detected in the third quadrupole, whereas PRM employs its high-resolution detector to capture all fragment ions, enabling the quantification of a greater number of proteins in a single analysis.
- Data Analysis: Subsequent analysis of data from large-scale samples.
Applications and Significance of Targeted Proteomics
Targeted proteomics has had a profound impact on systems biology and biomedical research, with its utilization continuously expanding into clinical applications. The applications of targeted proteomics encompass a wide range of areas, including studies focusing on post-translational modification (such as phosphoproteomics), protein conformations, protein-protein interactions, dynamics, proteomics, as well as system-level investigations of metabolism and signaling pathways. Notably, the achievement of absolute quantification of proteins involved in metabolism and signaling pathways has enabled precise pathway modeling and engineering. In the clinical realm, targeted proteomics finds applications in preclinical validation of candidate protein biomarkers and pharmacokinetic (PK) studies of monoclonal antibody (mAb) therapeutics. Targeted proteomics aids in enhancing our understanding of the phenotypic significance of biological networks and specific network states, driving the translation of biomarkers into clinical practice.
Advantages of Targeted Proteomics
Targeted proteomics techniques represent a powerful tool for protein quantification, enabling selective and sensitive detection as well as quantitative analysis of any protein of interest within complex proteomes. In comparison to untargeted proteomics, targeted proteomics offers superior accuracy, sensitivity, and reproducibility. Employing QQQ-MS or QQ-Orbitrap-MS, targeted proteomics monitors specific proteins or peptides. In contrast to other MS methods, targeted proteomics exhibits higher sensitivity for low-abundance peptide fragments and delivers superior quantitative precision.
References
- Gallien S, Kim S Y, Domon B. Large-scale targeted proteomics using internal standard triggered-parallel reaction monitoring (IS-PRM). Molecular & Cellular Proteomics, 2015, 14(6): 1630-1644.
- Lin L. et al., High throughput and accurate serum proteome profiling by integrated sample preparation technology and single-run data independent mass spectrometry. 2018, J Proteomics.