[Construction of a two-dimensional liquid chromatography separation system for high abundance proteins depletion in human plasma].
High abundance proteins existing in human plasma severely impede the detection of low abundance proteins. This is one of the most difficult problems encountered in plasma proteomics research. We developed a two-dimensional liquid chromatography system with strong anion exchange chromatography-reversed-phase liquid chromatography (SAX-RPLC) for the extensive separation of plasma proteins and selective depletion of high abundance proteins. TSKgel SuperQ-5PW was selected as the first dimensional separation column for crude human plasma fractionation and Jupiter C4 column was selected as the second dimensional separation column. Separation gradients of the two-dimensional liquid chromatography system were optimized to ensure an extensive separation of plasma proteins. Ten peaks with high signal intensities ( >20 mAU) at 215 nm during the second dimensional separation were collected and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). As a result, 32 proteins, all of which were reported to be high abundance proteins in plasma, including human serum albumin (HSA), immunoglobulin G (IgG) and so on were successfully identified. This system provides an effective method for future depletion of more high abundance proteins and in-depth research in human plasma proteomics.
Department of Chemistry, Fudan University, Shanghai 200433, China.
Shaochun
S
Zhu
Xueyang
X
Zhang
Mingxia
M
Gao
Guoquan
G
Yan
Xiangmin
X
Zhang
D001798:Blood Proteins / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D006801:Humans
D001798:Blood Proteins