title: Three dimensional liquid chromatography coupling ion exchange chromatography/hydrophobic interaction chromatography/reverse phase chromatography for effective protein separation in top-down proteomics.
abstract: To address the complexity of the proteome in mass spectrometry (MS)-based top-down proteomics, multidimensional liquid chromatography (MDLC) strategies that can effectively separate proteins with high resolution and automation are highly desirable. Although various MDLC methods that can effectively separate peptides from protein digests exist, very few MDLC strategies, primarily consisting of 2DLC, are available for intact protein separation, which is insufficient to address the complexity of the proteome. We recently demonstrated that hydrophobic interaction chromatography (HIC) utilizing a MS-compatible salt can provide high resolution separation of intact proteins for top-down proteomics. Herein, we have developed a novel 3DLC strategy by coupling HIC with ion exchange chromatography (IEC) and reverse phase chromatography (RPC) for intact protein separation. We demonstrated that a 3D (IEC-HIC-RPC) approach greatly outperformed the conventional 2D IEC-RPC approach. For the same IEC fraction (out of 35 fractions) from a crude HEK 293 cell lysate, a total of 640 proteins were identified in the 3D approach (corresponding to 201 nonredundant proteins) as compared to 47 in the 2D approach, whereas simply prolonging the gradients in RPC in the 2D approach only led to minimal improvement in protein separation and identifications. Therefore, this novel 3DLC method has great potential for effective separation of intact proteins to achieve deep proteome coverage in top-down proteomics.
authors: Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors: Santosh G
authors: SG
authors: Valeja
authors: Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors: Lichen
authors: L
authors: Xiu
authors: Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, Wisconsin, USA.|Molecular and Cellular Pharmacology Training Program, University of Wisconsin-Madison, Wisconsin, USA.
authors: Zachery R
authors: ZR
authors: Gregorich
authors: Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, Wisconsin, USA.|Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors: Huseyin
authors: H
authors: Guner
authors: Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors: Song
authors: S
authors: Jin
authors: Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, Wisconsin, USA.|Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.|Molecular and Cellular Pharmacology Training Program, University of Wisconsin-Madison, Wisconsin, USA.|Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors: Ying
authors: Y
authors: Ge
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D020543:Proteome / Q000032:analysis / Q000302:isolation & purification*; D040901:Proteomics; D053719:Tandem Mass Spectrometry
chemical_list: D020543:Proteome