Related Experiment Video
Updated: Apr 30, 2026
Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles
Published on: October 10, 2013
Decrease of dynamic range of proteins in human plasma by ampholine immobilized polymer microspheres
Nan Deng1, Guijie Zhu2, Yuanbo Chen1
1National Chromatographic Research and Analysis Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China; Graduate School of the Chinese Academy of Sciences, Beijing 100039, China.
Abstract:
A novel protein sample pretreatment method based on ampholine immobilized polymer microsphere (ampholine@PM) was developed for the fractionation of intact proteins prior to protein digestion and peptide analysis to reduce the dynamic range of human plasma proteome. After incubation with our prepared ampholine@PM, the captured plasma proteins were successively desorbed by 2M NaCl, 100mM glycine-hydrochloric acid, and 30% (v/v) acetonitrile with 0.1% (v/v) trifluoroacetic acid. The SDS-PAGE results showed the protein dynamic range in such three fractions was obviously reduced as compared with the native plasma. On-particle digestion was ultimately performed to release all proteins retained on ampholine@PM. Followed by MuPIT analysis, the number of identified proteins in plasma was improved by 75% after ampholine@PM treatment. Furthermore, the spectral count of 9 high abundance proteins was decreased by 37.6-97.2%, and the identified low abundance protein (<100ngmL(-1)) number was increased from 4 to 17. These results demonstrated that the fractionation by ampholine@PM could efficiently decrease the protein dynamic range in abundance, beneficial to achieve the deep coverage identification of human plasma proteome.
Insights
A new method using ampholine immobilized polymer microspheres (ampholine@PM) effectively fractionates human plasma proteins. This reduces the protein dynamic range, improving the identification of low-abundance proteins for deeper proteome coverage.
Area of Science:
- Proteomics
- Biochemistry
- Analytical Chemistry
Background:
- The human plasma proteome is characterized by a vast dynamic range of protein concentrations.
- This wide range poses significant challenges for identifying low-abundance proteins using mass spectrometry-based proteomics.
- Effective sample pretreatment methods are crucial for overcoming these limitations.
Purpose of the Study:
- To develop and evaluate a novel protein sample pretreatment method for fractionating intact proteins.
- To reduce the dynamic range of the human plasma proteome.
- To enhance the identification and coverage of low-abundance proteins in plasma.
Main Methods:
- Development of ampholine immobilized polymer microspheres (ampholine@PM) for protein fractionation.
- Incubation of human plasma with ampholine@PM, followed by sequential desorption using different solutions (2M NaCl, 100mM glycine-HCl, 30% acetonitrile/0.1% TFA).
- On-particle digestion of captured proteins and subsequent analysis using MuPIT (Mass-Unbiased Protein Identification Technology).
Main Results:
- SDS-PAGE confirmed a significant reduction in the protein dynamic range across the three fractions compared to native plasma.
- The ampholine@PM treatment resulted in a 75% increase in the number of identified plasma proteins.
- Spectral counts of high-abundance proteins decreased by 37.6-97.2%, while the number of identified low-abundance proteins (<100 ng/mL) increased from 4 to 17.
Conclusions:
- Ampholine@PM is an effective method for fractionating intact proteins in human plasma.
- This fractionation significantly reduces protein abundance dynamic range, enabling deeper proteome coverage.
- The developed method facilitates the identification of low-abundance proteins, advancing plasma proteomic research.
