How Does PRM Technology Facilitate the Analysis of Low-Abundance Proteins?
- Advanced instrumentation: Equipped with high-resolution platforms such as the Orbitrap Exploris 480, enabling highly sensitive and high-throughput PRM analyses.
- Customized method development: Tailored peptide design, parameter optimization, and SIL internal standard synthesis based on specific client targets.
- Comprehensive quality control: Stringent quality checkpoints are implemented throughout the workflow, from sample preparation and peptide synthesis to data analysis and reporting.
- Adaptability to complex biological samples: Specialized enrichment and pretreatment solutions are available for matrices such as plasma, cerebrospinal fluid, and cell lysates.
In proteomics research and clinical translational applications, the quantitative detection of low-abundance proteins remains a major technical challenge. Although these proteins are expressed at very low levels, they often possess high biological significance, such as cytokines, transcription factors, inflammatory mediators, and early disease biomarkers. The conventional Data-Dependent Acquisition (DDA) approach is limited by acquisition depth and dynamic range, making it difficult to achieve consistent detection of such low-abundance targets. Parallel Reaction Monitoring (PRM), characterized by its high selectivity and sensitivity, offers an effective solution for the quantitative analysis of low-abundance proteins. This article provides an in-depth discussion of the key advantages, underlying mechanisms, and technological capabilities of MtoZ Biolabs’s PRM platform.
Challenges in Low-Abundance Proteins Analysis
1. Extensive Dynamic Range
Protein concentrations in plasma span more than 12 orders of magnitude, causing low-abundance signals to be easily masked by high-abundance components.
2. Low Signal-to-Noise Ratio
Weak target protein signals are easily overwhelmed by background ions in complex biological matrices.
3. Limited Reproducibility
Traditional proteomic approaches often exhibit poor quantitative stability near detection limits, leading to significant inter-sample variability.
4. Lack of Robust Internal Standards
The absence of reliable reference markers for low-abundance targets increases susceptibility to instrumental drift and sample preparation inconsistencies.
How Does PRM Overcome These Challenges?
PRM is a targeted mass spectrometry technique implemented on high-resolution instruments such as Orbitrap analyzers. It selectively fragments precursor ions and performs full-scan acquisition of all product ions, thereby enhancing detection sensitivity and specificity across multiple dimensions.
Major advantages include:
1. High Resolution to Reduce Background Interference
(1) Orbitrap instruments used in PRM typically offer resolutions exceeding 30,000, effectively separating ions with closely related mass-to-charge ratios.
(2) Narrow mass selection windows (±5 ppm) substantially improve the signal-to-noise ratio and enhance the detectability of low-abundance species.
2. Comprehensive Fragment Ion Acquisition Enhances Quantitative Specificity
(1) Unlike SRM, which collects only one or two transitions, PRM simultaneously records all fragment ions, allowing post-acquisition selection of optimal transitions for quantification.
(2) This multi-dimensional validation significantly strengthens the confidence of low-abundance signal identification.
3. Flexible Ion Selection Improves Reproducibility
Researchers can select the most stable and informative ion pairs for quantification in each run, mitigating fluctuations in signal intensity.
4. Accurate Quantification with Stable Isotope-Labeled Internal Standards
Synthetic SIL peptides share identical physicochemical and ionization behaviors with target peptides, effectively compensating for sample injection and ionization variability.
Key Factors Influencing PRM-Based Detection of Low-Abundance Proteins
1. Peptide Selection Quality
(1) Select peptides with strong ion responses, free of modifications and sequence ambiguity.
(2) Utilize DIA data and database prediction tools such as PeptideAtlas and Prosit for informed peptide screening.
2. Sample Pretreatment and Enrichment Strategies
Techniques such as desalting, solid-phase extraction (SPE), and immuno-enrichment (e.g., SISCAPA) effectively remove high-abundance proteins and enhance target signal representation.
3. Optimization of Chromatographic Separation
(1) Improve retention time stability to avoid signal masking during complex elution.
(2) Employ multi-dimensional chromatography or extended gradients to enhance separation resolution.
4. Optimization of Mass Spectrometry Parameters
Adjust isolation window width, injection time (IT), and automatic gain control (AGC) settings to ensure sufficient ion accumulation for low-abundance targets.
PRM Technology Platform of MtoZ Biolabs
MtoZ Biolabs has established a robust technical foundation for targeted quantitative proteomics, featuring the following advantages:
PRM technology, with its exceptional selectivity, sensitivity, and quantitative consistency, is emerging as a mainstream approach for the analysis of low-abundance proteins. In applications such as biomarker validation and drug target discovery, PRM provides researchers with reliable and high-quality quantitative data. MtoZ Biolabs will continue to enhance its PRM platform to deliver more precise and reproducible quantification services for low-abundance proteins.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
Related Services
How to order?
