How to Integrate Shotgun Proteomics and Targeted Proteomics for Joint Analysis?
- Compare protein expression changes under different conditions (e.g., disease group vs normal group)
- Construct protein functional networks
- Identify potential differentially expressed proteins or candidate biomarkers
- Select signature peptides and ion pairs (transitions).
- Optimize chromatographic and mass spectrometric parameters.
- Introduce internal standard peptides to improve quantitative accuracy.
- Access to high-resolution mass spectrometers (e.g., Orbitrap Exploris 480, Q Exactive HF-X)
- Extensive experience in targeted protein measurement (>1000 target panels/libraries)
- Customized services integrating peptide selection, internal standard synthesis, and data analysis
In life science research, proteins are central executors of biological function. Their expression levels, modification states, and interaction networks often directly shape cellular fate and disease progression. To comprehensively characterize these dynamic changes, a range of mass spectrometry–based approaches has been developed. Among the most widely used are Shotgun proteomics (data-dependent acquisition, DDA) and targeted proteomics (e.g., PRM/MRM). Nevertheless, each approach has notable limitations when applied to complex biological systems. Shotgun strategies offer broad coverage for protein identification, but their quantitative accuracy and cross-run reproducibility can be limited. In contrast, targeted strategies provide high sensitivity and specificity for confirming predefined targets, yet they are not designed for large-scale, discovery-oriented profiling. Accordingly, integrating Shotgun and targeted proteomics is widely adopted as a practical strategy to address both discovery and verification within proteomics studies.
Shotgun Proteomics: A Discovery Tool from 0 to 1
Shotgun proteomics, also referred to as the DDA mode, performs untargeted surveying of complex samples using high-resolution mass spectrometers and can identify hundreds to thousands of proteins in a single analysis. This strategy is commonly used to:
1. Advantages
(1) High throughput
(2) Suitable for screening previously unknown proteins
(3) Can be combined with enrichment strategies (e.g., phosphorylation, acetylation) to study post-translational modifications.
2. Limitations
(1) Quantification accuracy can be constrained, and measurements may be susceptible to abundance-related effects in complex mixtures, particularly for low-abundance species.
(2) Reproducibility across batches can be relatively limited, making it less suitable for long-term monitoring or clinical validation.
Targeted Proteomics: A Validation Tool from 1 to ∞
Targeted proteomics mainly includes methods such as MRM (multiple reaction monitoring) and PRM (parallel reaction monitoring). These approaches leverage prior knowledge of peptide targets to selectively measure predefined peptides, offering high quantitative precision and robust analytical performance.
1. Applicable Scenarios
(1) Validate candidate proteins identified through Shotgun screening.
(2) Develop clinical-grade assays for protein biomarker measurement.
(3) Validate drug targets and confirm mechanisms of action.
2. Advantages
(1) High sensitivity (enabling detection of low-abundance proteins)
(2) High reproducibility (suitable for multi-batch and long-term studies)
(3) High specificity (reducing background interference)
Joint Analysis Strategy: An Integrated Path from Discovery to Verification
Step 1: Shotgun Mining Candidate Proteins
First, Shotgun proteomics is used to identify proteins and perform quantitative comparisons between experimental and control groups, thereby prioritizing candidate proteins or peptides with statistically significant expression differences. For example, in a breast cancer study, DDA profiling revealed multiple signaling-pathway proteins upregulated in tumor tissues (e.g., STAT3, AKT1, MMP9).
Step 2: Bioinformatics Analysis Assists in Screening Targets
GO functional enrichment, KEGG pathway analysis, and PPI network construction can then be integrated to prioritize candidates with stronger biological relevance, narrowing the list of targets for subsequent targeted validation.
Step 3: Design Targeted Proteomics Methods (PRM/MRM)
Based on proteotypic peptide information for the candidate proteins, PRM or MRM assays can be designed using tools such as Skyline, including:
Step 4: Batch Validation in Multiple Samples
Targeted validation is then performed in larger cohorts (e.g., 30 pairs, 50 pairs) to confirm the reproducibility of observed expression differences and to facilitate downstream translation toward biomarker development or drug-target research.
The Advantages of Joint Analysis of MtoZ Biolabs
At MtoZ Biolabs, we have established an end-to-end platform spanning shotgun proteomics discovery through PRM/MRM validation, supporting full workflows from trace tissue samples to clinical blood specimens:
Our goal is to support research teams in completing the discovery-to-validation cycle more efficiently and accurately, thereby facilitating the translation of proteomics findings.
The integration of shotgun proteomics and targeted proteomics is not merely a 1+1 combination, but rather a coordinated strategy that can be applied across the full research workflow. From mechanistic exploration to clinical biomarker development, this “broad-to-focused” approach is increasingly adopted as a practical paradigm in life science research. If you are conducting proteomics-related projects, you are welcome to contact MtoZ Biolabs for a tailored solution. With a dedicated technical platform and analysis team, we aim to support your research progress and downstream applications.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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