[2-25]Development of a 2D Top-Down Mass Spectrometry Platform and Its Application in Clinical Proteomics

报告题目：Development of a 2D Top-Down Mass Spectrometry Platform and Its Application in Clinical Proteomics

报 告 人：Dr. Junmei Zhang (University of Texas Southwestern Medical Center)

报告时间：2014年2月25日（星期二）上午10:00

报告地点：独墅湖校区二期云轩楼2301室

Introduction     

Dr. Junmei Zhang was graduated from Beijing Medical University with a Master’s Degree in Pharmaceutical Chemistry. She received her Ph.D. Degree in Analytical Chemistry from the University of Texas at Austin. The title of her Ph.D. thesis was “Structural Characterization and Enhanced Detection of Flavonoids by Electrospray Ionization Mass Spectrometry and Molecular Modeling”. After joining the University of Texas Southwestern Medical Center, She has been conducting proteomics research using both top-down and bottom-up mass spectrometry approaches. 

Abstract

Comprehensive characterization of various proteoforms for a given gene in a high throughput manner is critical for clinical proteomics. However, the commonly used 2D gel electrophoresis has limited resolving power and poor quantitative reliability, and peptide-based approaches are not designed to differentiate proteoforms of the same protein due to the digestion step.  Top-down mass spectrometry (MS) has the potential but has had limited application to clinical proteomics investigations due to the complexity and poor dynamic range of chromatography used to separate intact proteins from tissue and biofluids. To address these limitations we developed a 2D chromatography platform that includes isoelectric focusing (IEF) through immobilized pH gradient (IPG) and superficially porous liquid chromatography (SPLC). As demonstrated by standard proteins, and proteins from heart myofibrils and cerebrospinal fluid (CSF), the 2D platform provides ~7.0 femtomole detection limits, 4 orders of magnitude linear dynamic range, ~4× improved mass range, 5-6× increase in number of unique monoisotopic masses, label-free quantitation of proteins and their post-translational modifications (PTMs), as well as enables prospective protein identification and proteoform analysis investigations by complementary top-down and bottom-up strategies.

Among those heavily modified proteins in CSF, lipocalin-type prostaglandin D synthase (L-PGDS) is a glycoprotein known to contribute to the maturation and maintenance of the central nervous system, and thus considered a potential biomarker for neurological diseases. Across 3D physiochemical space (pI, hydrophobicity, and mass) 217 putative proteoforms were observed, among which 208 could be assigned with varied PTM positional occupants, including 15 structurally-related N-glycans at N29 and N56, a core-1 HexHexNAc-O-glycan at S7, acetylation at K16 and K138, sulfonation at S41 and T142, and dioxidation at C43 and C145. Most of the proteoforms were shared between the control and patient samples, however, their relatively abundances were different. The control sample had higher levels of proteoforms at the higher mass side while the patient sample seemed to have higher abundance for the lower mass proteoforms. Overall, the IEF-SPLC-MS platform presented provides the resolution, sensitivity, quantitative reliability, and speed necessary to identify and characterize proteoform-based biomarkers.