Evaluation of two high-abundance protein depletion kits and optimization of downstream isoelectric focusing
- Authors:
- Fanghua Qiu
- Tieying Hou
- Dehong Huang
- Zhifeng Xue
- Dongyan Liang
- Qiuming Li
- Weimiao Lin
View Affiliations
Affiliations: Department of Clinical Laboratory, Guangzhou Hospital of Traditional Chinese Medicine, Guangzhou, Guangdong 510130, P.R. China, Department of Laboratory Medicine, Guangdong Academy of Medicine Science and Guangdong General Hospital, Guangzhou, Guangdong 510000, P.R. China, Department of Neurology, Guangzhou Hospital of Traditional Chinese Medicine, Guangzhou, Guangdong 510130, P.R. China
- Published online on: October 1, 2015 https://doi.org/10.3892/mmr.2015.4417
-
Pages:
7749-7755
Metrics: Total
Views: 0 (Spandidos Publications: | PMC Statistics: )
Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )
This article is mentioned in:
Abstract
Disease biomarkers for diagnostic and prognostic purposes are most likely within an extremely low concentration range and are thus masked by the presence of high‑abundance proteins. Therefore, removing high‑abundance proteins is the main challenge for identifying disease biomarkers. In addition, the solution obtained from high‑abundance protein depletion kits contains a rich array of compounds, which interfere with isoelectric focusing (IEF). In the present study, the effect of two commercial kits was evaluated and the downstream IEF protocol was optimized. High‑resolution results could be obtained according to the following conditions: The ProteoPrep Blue Albumin and IgG Depletion kit depleted albumin and IgG; immobilized pH gradient strips (typically 18 cm) were rehydrated with sample buffer containing 250 µg serum proteins at 30 v for 6 h, 60 v for 6 h, 200 v for 2 h, 500 v for 2 h, 1,000 v for 2 h, 5,000 v for 2 h, 10,000 v for 2 h and then focusing at 10,000 v up to 110 k vhs. In addition, the protein spots identified by matrix‑assisted laser desorption ionization time‑of‑flight mass spectrometry demonstrated that all proteins had a low abundance. The present study not only provides a definite and effective method for removing high‑abundance proteins, but also provides a proper protocol (protocol C) for downstream IEF. The present study includes a comprehensive investigation of serum proteomics, which paves the way for serum protein research.
View Figures |
Figure 1
|
 |
Figure 2
|
 |
Figure 3
|
 |
Figure 4
|
 |
Figure 5
|
 |
Figure 6
|
View References
|
1
|
Ahmed N, Barker G, Oliva K, Garfin D,
Talmadge K, Georgiou H, Quinn M and Rice G: An approach to remove
albumin for the proteomic analysis of low abundance biomarkers in
human serum. Proteomics. 3:1980–1987. 2003. View Article : Google Scholar : PubMed/NCBI
|
|
2
|
Anderson NL and Anderson NG: The human
plasma proteome: History, character, and diagnostic prospects. Mol
Cell Proteomics. 1:845–867. 2002. View Article : Google Scholar : PubMed/NCBI
|
|
3
|
Bjorhall K, Miliotis T and Davidsson P:
Comparison of different depletion strategies for improved
resolution in proteomic analysis of human serum samples.
Proteomics. 5:307–317. 2005. View Article : Google Scholar
|
|
4
|
Desrosiers RR, Beaulieu E, Buchanan M and
Béliveau R: Proteomic analysis of human plasma proteins by
two-dimensional gel electrophoresis and by antibody arrays
following depletion of high-abundance proteins. Cell Biochem
Biophys. 49:182–195. 2007. View Article : Google Scholar : PubMed/NCBI
|
|
5
|
Echan LA, Tang HY, Ali-Khan N, Lee K and
Speicher DW: Depletion of multiple high-abundance proteins improves
protein profiling capacities of human serum and plasma. Proteomics.
5:3292–3303. 2005. View Article : Google Scholar : PubMed/NCBI
|
|
6
|
Greenough C, Jenkins RE, Kitteringham NR,
Pirmohamed M, Park BK and Pennington SR: A method for the rapid
depletion of albumin and immunoglobulin from human plasma.
Proteomics. 4:3107–3111. 2004. View Article : Google Scholar : PubMed/NCBI
|
|
7
|
Hoffman SA, Joo WA, Echan LA and Speicher
DW: Higher dimensional (Hi-D) separation strategies dramatically
improve the potential for cancer biomarker detection in serum and
plasma. J Chromatogr B Analyt Technol Biomed Life Sci. 849:43–52.
2007. View Article : Google Scholar
|
|
8
|
Kim MR and Kim CW: Human blood plasma
preparation for two-dimensional gel electrophoresis. J Chromatogr B
Analyt Technol Biomed Life Sci. 849:203–210. 2007. View Article : Google Scholar
|
|
9
|
Lescuyer P, Hochstrasser D and Rabilloud
T: How shall we use the proteomics toolbox for biomarker discovery?
J Proteome Res. 6:3371–3376. 2007. View Article : Google Scholar : PubMed/NCBI
|
|
10
|
Liu B, Qiu FH, Voss C, Xu Y, Zhao MZ, Wu
YX, Nie J and Wang ZL: Evaluation of three high abundance protein
depletion kits for umbilical cord serum proteomics. Proteome Sci.
9:242011. View Article : Google Scholar : PubMed/NCBI
|
|
11
|
Mahn A, Reyes A, Zamorano M, Cifuentes W
and Ismail M: Depletion of highly abundant proteins in blood plasma
by hydrophobic interaction chromatography for proteomic analysis. J
Chromatogr B Analyt Technol Biomed Life Sci. 878:1038–1044. 2010.
View Article : Google Scholar : PubMed/NCBI
|
|
12
|
Qiu F, Huang D, Xiao H, Qiu F, Lu L and
Nie J: Detection of tyrosine phosphorylated proteins in
hepatocellular carcinoma tissues using a combination of
GST-Nck1-SH2 pull-down and two-dimensional electrophoresis. Mol Med
Rep. 7:1209–1214. 2013.PubMed/NCBI
|
|
13
|
Thadikkaran L, Siegenthaler MA, Crettaz D,
Queloz PA, Schneider P and Tissot JD: Recent advances in
blood-related proteomics. Proteomics. 5:3019–3034. 2005. View Article : Google Scholar : PubMed/NCBI
|
