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J Chromatogr B Analyt Technol Biomed Life Sci. 2017 Aug 15;1060:272-280. doi: 10.1016/j.jchromb.2017.06.023. Epub 2017 Jun 16.

Sample preparation for detection of low abundance proteins in human plasma using ultra-high performance liquid chromatography coupled with highly accurate mass spectrometry.

Author information

1
Molecular Recognition Research Center, Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea; Department of Biological Chemistry, University of Science and Technology, Daejeon 34113, Republic of Korea.
2
Molecular Recognition Research Center, Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea.
3
Molecular Recognition Research Center, Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea; Department of Biological Chemistry, University of Science and Technology, Daejeon 34113, Republic of Korea. Electronic address: mjkang1@kist.re.kr.

Abstract

Proteomics is a valuable approach to discover biomarkers in human plasma for early diagnosis. However, detection of biomarkers in the plasma is still challenging because of its large protein content. In our study, we established albumin/IgG depletion methods for identification of low abundance proteins using two commercial kits with additional buffer conditions and various concentrations of cold acetone. Trypsin digestion, desalting, and data-dependent acquisition were also optimized. More than 80% depletion of albumin/IgG was achieved with two commercial kits and 98% depletion of albumin was obtained with 70% cold acetone. Recovery of four reference proteins, BNP (47-76), insulin, cytochrome c, and ubiquitin was obtained in all optimized methods. The best recovery of reference proteins was obtained using the ProteoExtract albumin/IgG removal kit with buffer A (61%-106%). After cold acetone precipitation, three reference proteins were recovered more than 48% except ubiquitin (12%). The number of identified proteins by Mascot was 28, 35, 17, and 34 for ProteoExtract, ProteoPrep, 70%, and 50% cold acetone, respectively. Furthermore, optimized methods detected MS/MS fragmentation patterns of elevated BNP in patient samples with cardiac disease. Our study provides the conditions for efficient biomarker discovery by minimal removing of high abundant proteins.

KEYWORDS:

Biomarkers; Human plasma; LTQ orbitrap; Low abundance proteins; Sample preparation

PMID:
28649027
DOI:
10.1016/j.jchromb.2017.06.023
[Indexed for MEDLINE]

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