Quantitative MRM analysis of target proteins refers to multiple reaction monitoring (MRM) based on mass spectrometry. This technology is essentially a mass spectrometry scanning mode, based on the specific precursor and product ion pairs of the target protein, selectively collect signals that conform to the target ion rules, remove signal interference that does not conform to the rules, and perform highly sensitive, accurate, and specific targeted protein quantitation. MRM mass spectrometry analysis mainly consists of three stages: (1) primary mass spectrometry scanning to screen out precursor ions that are consistent with the specificity of the target molecule;(2) Collision fragmentation of precursor ions to remove interfering ions;(3) Acquire only the mass spectrometry signal from the selected specific ions. Precursor transitions can be selected based on theoretical** or real-world experimental results.
Parallel Reaction Monitoring (PRM) is a derivative of MRM that can also be used to quantify multiple proteins of interest simultaneously or absolutely, in complex biological samples. PRM collects high-resolution MS2 mass spectra of the target peptide, and uses software to extract the peak area of the target ions at the ppm level, effectively excluding the interference of other ions. Compared with MRM, it has a wider dynamic range, higher accuracy, stronger sensitivity, better repeatability, stronger anti-background interference ability, simpler practical operation and lower cost. PRM does not require precursor and product ion pairing based on the protein of interest, enabling full scan of product ions. PRM can be used as an alternative to Western blot technology to validate antibodies in large biosample volumes at high throughput, and can be applied to a variety of non-model organisms.
PRM consists of five main experimental procedures: (1) selection of targeted peptides from Shotgun experiments or theoretical speculations(2) Prepare a mixture of samples;(3) Pre-experimental adjustment of parameters and targeted peptides;(4) PRM detection;(5) Data analysis. The disadvantage of the PRM method is that when the number of peptides to be analyzed is too large, the mass spectrometry acquisition parameters need to be fine-tuned, otherwise the accuracy and precision of the quantitative data will be greatly affected.
Figure PRM Experiment Flow Chart.
Customer** orders - order Confirmation of experimental materials - proteolysis - DDA mode library construction - general library optimization - data collection - data analysis - delivery results.
Our advantages
1.Compared with other types of samples, plant samples are readily available.
2.From sample preparation, on-board testing, experimental reports to professional after-sales service, we provide one-stop proteomics services.
3.The use of advanced mass spectrometers provides high-sensitivity, high-throughput analysis to ensure high-quality data.
4.Targeted validation experiments can be performed on proteins of interest to customers.
Applicable scenarios
1. Verification of target protein after large-scale proteome detection;
2. Quantitative study of homologous proteins or mutant proteins;
3. Verification of non-targeted proteome research results such as label-free;
4. Absolute quantitative research on multiple protein peptides at the same time
5. Study the changes of protein families with high homology but lack of specific recognition antibodies
6. Quantitative study of protein post-translational modifications;
7. Absolute quantitative research on biological disease targets;
8. Develop clinical diagnostic kits.
Experimental cycle
30 working days from the time of sample delivery.
Sample Acceptance Criteria
Customer orders and fill in project information
To register or log in to our official website, please fill in the project name, select the project type, fill in the personal information and the specific information required to submit the project according to the page prompts, including:
1. Sample**, content, status and other basic information;
2. Abundant relevant materials and literature as much as possible.
Experiment information
During the experiment, the customer can log in to the management system at any time to view the real-time progress of the project. When the experiment is completed, the system will automatically notify the customer via SMS and send the experiment report to view**. After the experiment is completed, the experimental report and test results can be viewed or printed, and permanently retained.
Experimental deliverables
1. An analysis report: including the experimental process, instrument model, parameters, reagent consumables, and quantitative result statistics
2. Quantitative result file: generally excel**;
3. Mass spectrometry raw file: the original data of the optimized spectral library and the WIFF format collected by PRM.
A classic case
Journal: Bioresource Technology
Impact factor: 114
Year of publication: 202009
1. Background.
Astaxanthin is a lutein carotenoid, which is red in color and has been used in many industries such as food and cosmetics, and has broad application prospects. One of the most advanced pathways of natural astaxanthin is Phaffia rhodozyma, and the production of astaxanthin will increase when external conditions are unfavorable, but the existing data cannot fully explain the stress response mechanism of astaxanthin synthesis by red yeast. Previous experimental results have shown that 500 mg ml TiO2 can significantly increase the production of carotenoids such as astaxanthin. Based on these results, this study analyzed the effect of TiO2 on astaxanthin production and the molecular mechanism of stress conditions promoting the synthesis of astaxanthin by Saccharomyces rubrum.
2. Experimental methods.
The authors took wild-type Red Fife Yeast PR106 as the research object, and cultured yeast cells with or without 500 mg ml TiO2 in Yepd medium to the middle stage, and took the strains C1, C2 and C3 cultured with 500 mg ml TiO2 as the control group, and the strains S1, S2 and S3 cultured without 500 mg ml TiO2 as the experimental group, extracted the total protein, labeled with TMT chemical reagent, and waited for detection by mass spectrometry. Combined with the mass spectrometry data, 15 differentially expressed proteins in the metaphase of Saccharomyces rubrum were selected for mass spectrometry analysis by PRM-targeted quantitative proteomics.
3. Experimental results.
In this study, a total of 3193 differentially expressed proteins of Y. rubrum were quantified, of which 5 protein expressions were up-regulated and 56 protein expressions were down-regulated in the experimental group compared with the control group. Upregulated proteins are mainly localized in the plasma membrane, nucleus, or extracellular, and downregulated proteins are mainly localized in the nucleus, mitochondria, plasma membrane, cytoplasm, and extracellular. The results of GO and KEGG enrichment analysis showed that the differentially expressed proteins were mainly related to the overall composition of the non-membrane binder and the membrane, and were mainly enriched in the RNA degradation pathway and almost all of them were down-regulated. The results of 15 differentially expressed proteins verified by PRM-targeted proteomics showed that 500 mg mL TiO2 could significantly promote the synthesis of astaxanthin in yeast metaphase.
Fig.1 Volcano diagram of differentially expressed proteins.
Fig.2 Functional enrichment analysis of differentially expressed proteins of red yeast based on GO and GGG under TiO2 stress.