An Alternative Approach: Top-Down Bioanalysis of Intact Large Molecules Can this be part of the future? Lecture 8, Page 27
Top-down HRAM Bioanalysis of Native Proteins/Molecules Relative Abundance 100 90 80 70 60 50 40 Relative Abundance 3294.767, 45+ 100 90 3154.638 3369.633 3223.124 3294.767 Denatured 80 3088.943 3219.647 3226.666 3291.188 3447.922 3298.404 70 3025.893 60 2965.343 50 2907.266 3530.056 3216.437 3230.197 3301.975 40 3287.947 2851.409 3616.137 30 2797.570 3209.727 3280.972 3233.765 3305.632 20 2695.873 3706.528 3801.425 10 2601.420 3180 3200 3220 3240 3260 3280 3300 3320 3340 4007.002 m/z 0 2000 2500 3000 3500 4000 4500 5924.917 m/z 26+ 25+ 5924.917 5696.948 5493.334 6171.751 Native 46+ 45+ 5716.809 5945.477 30 5679.038 5906.339 20 5295.972 6440.595 5866.915 10 5600 5650 5700 5750 5800 5850 5900 5950 6000 0 2000 3000 4000 5000 6000 7000 8000 9000 10000 m/z m/z Lecture 8, Page 28
The Following Data is Gratefully Acknowledged from the LC/GC Series Webinar on 29 January 2015 Lecture 8, Page 29
Lecture 8, Page 30
Lecture 8, Page 31
Lecture 8, Page 32
Lecture 8, Page 33
Chip-Based Nano ESI Infusion of Native Proteins Thermo Orbitrap ESI Chip inlet Advion TriVersa NanoMate Sample Conductive pipette tip Lecture 8, Page 34
Chip-Based Nano ESI Infusion of Native Proteins Lecture 8, Page 35
Lecture 8, Page 36
Lecture 8, Page 37
Lecture 8, Page 38
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/10.1021/ac201540t Anal. Chem. 2011, 83, 8937 8944
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/10.1021/ac201540t Anal. Chem. 2011, 83, 8937 8944
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/10.1021/ac201540t Anal. Chem. 2011, 83, 8937 8944
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
Liquid Chromatography- Mass Spectrometry: Summary Now HPLC/UHPLC 1.7µm - 5µm particle 2.1-4.6 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 0.05-0.5 mm column diameter 0.25-5 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
Relevant Publications 1. Ramagiri, S. and F. Garofolo, Large molecule bioanalysis using Q-TOF without predigestion and its data processing challenges. Bioanalysis, 2012. 4(5): p. 529-540. 2. 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, 2014. 00: p. 1 9. 3. Ruan, Q., et al., Strategy and Its Implications of Protein Bioanalysis Utilizing High-Resolution Mass Spectrometric Detection of Intact Protein. Analytical Chemistry, 2011. 83(23): p. 8937-8944. 4. 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, 2013. 86(1): p. 317-320. 5. Sun, L., et al., Fast Top-Down Intact Protein Characterization with Capillary Zone Electrophoresis Electrospray Ionization Tandem Mass Spectrometry. Analytical Chemistry, 2013. 85(12): p. 5989-5995. 6. Zhang, J., et al., Top-Down Mass Spectrometry on Tissue Extracts and Biofluids with Isoelectric Focusing and Superficially Porous Silica Liquid Chromatography. Analytical Chemistry, 2013. 85(21): p. 10377-10384. 7. Ref: Hongcheng Liu, Georgeen Gaza-Bulseco, and Chris ChumsaeJ Am Soc Mass Spectrom 2009, 20, 2258 2264. 8. R. Jenkins, et al., Recomendations for validation of LC/MS/MS bioanalytical methods for protein biotherapeutics, The AAPS Journal, 2014 Published online 13 Nov 2014. Lecture 8, Page 44