An Alternative Approach: Top-Down Bioanalysis of Intact Large Molecules Can this be part of the future? Lecture 8, Page 27

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1 An Alternative Approach: Top-Down Bioanalysis of Intact Large Molecules Can this be part of the future? Lecture 8, Page 27

2 Top-down HRAM Bioanalysis of Native Proteins/Molecules Relative Abundance Relative Abundance , Denatured m/z m/z Native m/z m/z Lecture 8, Page 28

3 The Following Data is Gratefully Acknowledged from the LC/GC Series Webinar on 29 January 2015 Lecture 8, Page 29

4 Lecture 8, Page 30

5 Lecture 8, Page 31

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7 Lecture 8, Page 33

8 Chip-Based Nano ESI Infusion of Native Proteins Thermo Orbitrap ESI Chip inlet Advion TriVersa NanoMate Sample Conductive pipette tip Lecture 8, Page 34

9 Chip-Based Nano ESI Infusion of Native Proteins Lecture 8, Page 35

10 Lecture 8, Page 36

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13 Strategy and Its Implications of Protein Bioanalysis Utilizing High-Resolution Mass Spectrometric Detection of Intact Protein Protein digestion followed by LC/MS/MS cannot distinguish between protein modifications such as PTM s or biotransformation (BTX) products: so why not measure the intact, native protein? Employ the XIC of an exact mass to gain selectivity! Lecture 8, Page 39 Qian Ruan, Qin C. Ji,*, Mark E. Arnold, W. Griffith Humphreys, and Mingshe Zhu dx.doi.org/ /ac201540t Anal. Chem. 2011, 83,

14 High Resolution MS is a Way to Measure Intact Proteins Lecture 8, Page 40 Qian Ruan, Qin C. Ji,*, Mark E. Arnold, W. Griffith Humphreys, and Mingshe Zhu dx.doi.org/ /ac201540t Anal. Chem. 2011, 83,

15 Chromatography of Proteins can be Tricky Elevated HPLC Column Temperatures can Help Lecture 8, Page 41 Qian Ruan, Qin C. Ji,*, Mark E. Arnold, W. Griffith Humphreys, and Mingshe Zhu dx.doi.org/ /ac201540t Anal. Chem. 2011, 83,

16 Data Analysis LIMS Huge data files result from full-scan HRMS LC/MS data acquisitions What is the preferred HRAM acquisition mode? How do we optimally monitor the preferred ion current for bioanalysis Bioanalysis software needs improved speed and data storage space Lecture 8, Page 42

17 Liquid Chromatography- Mass Spectrometry: Summary Now HPLC/UHPLC 1.7µm - 5µm particle mm column diameter 1-10 minute gradients Primarily 1-Dimensional separation Triple-quadrupole and Selected Reaction Monitoring (SRM) High flow rate heated ESI Future HPLC/UHPLC Sub-micron to 2µm particle mm column diameter minute gradients Online-extraction combined with multi-dimensional separation SRM and HRAMS Micro sampling Dried matrix spots Integrated nanoscale techniques Chip cube: Agilent Ion Key: Waters Ion Mobility coupled with MS Waters Agilent Sciex Lecture 8, Page 43

18 Relevant Publications 1. Ramagiri, S. and F. Garofolo, Large molecule bioanalysis using Q-TOF without predigestion and its data processing challenges. Bioanalysis, (5): p Zhang, J., et al., Proteoform analysis of lipocalin-type prostaglandin D-synthase from human cerebrospinal fluid by isoelectric focusing and superficially porous liquid chromatography with Fourier transform mass spectrometry. Proteomics, : p Ruan, Q., et al., Strategy and Its Implications of Protein Bioanalysis Utilizing High-Resolution Mass Spectrometric Detection of Intact Protein. Analytical Chemistry, (23): p Li, H., et al., Native Top-Down Electrospray Ionization-Mass Spectrometry of 158 kda Protein Complex by High-Resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Analytical Chemistry, (1): p Sun, L., et al., Fast Top-Down Intact Protein Characterization with Capillary Zone Electrophoresis Electrospray Ionization Tandem Mass Spectrometry. Analytical Chemistry, (12): p Zhang, J., et al., Top-Down Mass Spectrometry on Tissue Extracts and Biofluids with Isoelectric Focusing and Superficially Porous Silica Liquid Chromatography. Analytical Chemistry, (21): p Ref: Hongcheng Liu, Georgeen Gaza-Bulseco, and Chris ChumsaeJ Am Soc Mass Spectrom 2009, 20, R. Jenkins, et al., Recomendations for validation of LC/MS/MS bioanalytical methods for protein biotherapeutics, The AAPS Journal, 2014 Published online 13 Nov Lecture 8, Page 44