Journal
ANALYTICA CHIMICA ACTA
Volume 826, Issue -, Pages 43-50Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.aca.2014.04.004
Keywords
Ampholine; Fractionation; Human plasma; Protein dynamic range
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Funding
- National Basic Research Program of China [2012CB910601]
- National Natural Science Foundation [21175131, 21190043, 21005078]
- National High Technology Research and Development Program of China [2012AA020202]
- NSFC [21321064]
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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 2 M NaCl, 100 mM 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 (< 100 ng mL(-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. (c) 2014 Elsevier B. V. All rights reserved.
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