Development of gel-filter method for high enrichment of low-molecular weight proteins from serum.
The human serum proteome has been extensively screened for biomarkers. However, the large dynamic range of protein concentrations in serum and the presence of highly abundant and large molecular weight proteins, make identification and detection changes in the amount of low-molecular weight proteins (LMW, molecular weight ≤ 30kDa) difficult. Here, we developed a gel-filter method including four layers of different concentration of tricine SDS-PAGE-based gels to block high-molecular weight proteins and enrich LMW proteins. By utilizing this method, we identified 1,576 proteins (n = 2) from 10 μL serum. Among them, 559 (n = 2) proteins belonged to LMW proteins. Furthermore, this gel-filter method could identify 67.4% and 39.8% more LMW proteins than that in representative methods of glycine SDS-PAGE and optimized-DS, respectively. By utilizing SILAC-AQUA approach with labeled recombinant protein as internal standard, the recovery rate for GST spiked in serum during the treatment of gel-filter, optimized-DS, and ProteoMiner was 33.1 ± 0.01%, 18.7 ± 0.01% and 9.6 ± 0.03%, respectively. These results demonstrate that the gel-filter method offers a rapid, highly reproducible and efficient approach for screening biomarkers from serum through proteomic analyses.
State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing, 102206, P. R. China; State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, 530005, P. R. China.
Lingsheng
L
Chen
State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing, 102206, P. R. China.
Linhui
L
Zhai
State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing, 102206, P. R. China.
Yanchang
Y
Li
State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing, 102206, P. R. China.
Ning
N
Li
State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing, 102206, P. R. China.
Chengpu
C
Zhang
State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing, 102206, P. R. China.
Lingyan
L
Ping
State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing, 102206, P. R. China.
Lei
L
Chang
Department of Biochemistry, School of Medicine, Wuhan University, Wuhan, 430071, P. R. China.
Junzhu
J
Wu
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, 530005, P. R. China.
Xiangping
X
Li
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, 530005, P. R. China.
Deshun
D
Shi
State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing, 102206, P. R. China; Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan, 430071, P. R. China.
Ping
P
Xu
D001798:Blood Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D013058:Mass Spectrometry; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D020543:Proteome; D040901:Proteomics / Q000379:methods
D001798:Blood Proteins; D010455:Peptides; D020543:Proteome