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What Is Parallel Reaction Monitoring (PRM)
Parallel reaction monitoring (PRM) is an ion monitoring technique based on high-resolution and high-precision mass spectrometry. The principle of this technique is comparable to SRM/MRM, but it is more convenient in assay development for absolute quantification of proteins and peptides. It is most suitable for quantification of multiple proteins in complex sample with an attomole-level detection. PRM technology is a novel technology for verification of antibody-free proteins, which is used for verification of relative quantitative proteomic data such as label free, iTRAQ, TMT, SILAC, or fortargeted quantitative analysis of target proteins in complex samples.
PRM Analysis Principle
PRM is based on Q-Orbitrap as the representative quadrupole-high resolution mass spectrum platform. Unlike the SRM, which performs one transition at a time, the PRM performs a full scan of each transition by a precursor ion, that is, parallel monitoring of all fragments from the precursor ion. First, the PRM uses the quadrupole (Q1) to select the precursor ion, and the selection window is usually m/z≤2; then, the precursor ion is fragmented in the collision cell (Q2); finally, Orbitrap replaces Q3, scans all product ions with high resolution and high accuracy. Therefore, PRM technology not only has the SRM/MRM target quantitative analysis capabilities, but also have the qualitative ability. (1) The mass accuracy can reach to ppm level, which can eliminate the background interference and false positive better than SRM / MRM, and improve the detection limit and sensitivity in complex background effectively; (2) Full scan of product ions, without the need to select the ion pair and optimize the fragmentation energy, easier to establish the assay; (3) a wider linear range: increased to 5-6 orders of magnitude.
Analytical Capabilities of PRM vs. SRM/MRM
|Technique||SRM/MRM (Selected Reaction Monitoring)||PRM (Parallel Reaction Monitoring)|
|Method Development (Relative Quantitation)||Dependent on standards, complex method development||Independent of standards, efficient method development|
|Quantitative Ability||Greater than non-targeted proteomics||Greater than SRM/MRM|
Highlight of PRM technique:
In high-throughput detection processes, it ensures quantitative sensitivity and specificity.
Compared to SRM, it does not require precursor/fragment ion pairs, only precursor ion information, making experimental design simpler.
It enables qualitative and quantitative analysis through secondary full scans, exhibiting strong resistance to interference and high resolution in complex backgrounds.
Synthetic standard peptides can be selectively incorporated as needed, saving experimental costs.
PRM technology boasts excellent reproducibility, ensuring result stability across multiple repeated tests.
PRM Analysis Services
PRM Analysis Services at Creative Proteomics offers you a state-of-the-art strategy of proteins analysis. The workflow is based on the following sections:
- The PRM assay development. This is the core of the approach. First, 2 or 3 targeted peptides should be selected from a spectral library generation or Skyline software. The selected peptides should be unique to the protein of interest and easily detected by LC-MS. They also require no missed cleavage sites and no frequently modified amino acids. Second, a working linear curve was built with the corresponding concentration ratios on the x-axis and peak area ratios on the y-axis.
- Analysis of the sample. After digestion, the sample is performed on the Q-Oribtrap mass spectrometer, such as Q Exactive or Fusion mass spectrometer.
- Data analysis. Data will be analyzed with Skyline software.
- Verification of iTRAQ differential protein;
- Verification of label-free differential protein follow-up;
- Protein and peptide absolute quantification;
- Quantification of disease markers, the establishment of diagnostic models;
- Phosphorylation protein quantification, methylation protein quantification;
- Other post-translationally modified protein quantification;
- Quantitative analysis of pathways.
Thermo Scientific Q Exactive HF-X
Thermo Scientific Orbitrap Fusion Lumos
Thermo Scientific Q Exactive Plus
Thermo Scientific Orbitrap Fusion
Thermo Scientific Q Exactive
For protein solutions, the total protein amount should be ≥300mg.
For tissue samples, the total sample amount should be ≥0.1g. For cell samples, the cell count should be ≥10^7 cells.
For fluid samples, serum should be ≥200ml, cerebrospinal fluid (CSF) should be ≥200ml, urine should be ≥1ml, and tears should be ≥1ml.
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Identification of serum biomarkers to predict pemetrexed/platinum chemotherapy efficacy for advanced lung adenocarcinoma patients by data-independent acquisition (DIA) mass spectrometry analysis with parallel reaction monitoring (PRM) verification.
Journal: Transl Lung Cancer Res
In order to discover novel serum biomarkers for predicting the efficacy of pemetrexed/cisplatin chemotherapy, researchers employed Data-Independent Acquisition (DIA) quantitative mass spectrometry (MS) analysis on 20 serum samples from advanced lung adenocarcinoma patients undergoing pemetrexed/cisplatin treatment.
DIA Proteomics and PRM Analysis
DIA-based quantitative proteomic approach was utilized to explore potential prognostic biomarkers in serum. A total of 23 significantly differentially expressed proteins were identified between groups with varying chemotherapy responses. Enrichment analysis highlighted the most significant pathway as the thyroid hormone synthesis pathway, including glutathione peroxidase (GPX) among others. Among the 23 significantly different proteins, 7 had AUC values greater than 0.8. The highest AUC protein was GPX (A0A087X1J7, AUC 0.885, encoded by GPX3 gene). GPX3 belongs to the oxidoreductase protein family and plays a crucial role in antioxidation, being associated with tumorigenesis and chemotherapy response in various cancers. Upregulation of GPX3 has been found to predict poor prognosis in oral squamous cell carcinoma, hepatocellular carcinoma, and gastric cancer. Additionally, GPX3 expression has been linked to the development of chemotherapy resistance in breast, ovarian, and colorectal cancers. Using the PRM method, protein level differences of discovered candidate biomarkers were verified, with 10 proteins exhibiting similar up- or down-regulation trends as observed by the DIA method. Both DIA and PRM approaches employed in the study consistently indicated significant upregulation of GPX3 in the poor response group compared to the good response group.
Differential protein expression levels in tumors and the external environment may be closely associated with chemotherapy efficacy, although serum protein biomarkers have not yet been successfully applied in clinical settings. The combined approach of DIA and targeted PRM holds immense potential for comprehensively revealing and validating predictive and prognostic candidate biomarkers in cancer patients.
- Jia B, Zhao X, Wu D, Dong Z, Chi Y, Zhao J, Wu M, An T, Wang Y, Zhuo M, Li J, Chen X, Tian G, Long J, Yang X, Chen H, Wang J, Zhai X, Li S, Li J, Ma M, He Y, Kong L, Brcic L, Fang J, Wang Z. Identification of serum biomarkers to predict pemetrexed/platinum chemotherapy efficacy for advanced lung adenocarcinoma patients by data-independent acquisition (DIA) mass spectrometry analysis with parallel reaction monitoring (PRM) verification. Transl Lung Cancer Res. 2021 Feb;10(2):981-994. doi: 10.21037/tlcr-21-153. PMID: 33718037; PMCID: PMC7947410.