JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Chemistry

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry

Published: October 24th, 2018

DOI:

10.3791/58644

1Department of Chemistry, Michigan State University, 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, 3Center for Computational Biology and Bioinformatics, Indiana University School of Medicine
* These authors contributed equally

A detailed protocol is described for the separation, identification, and characterization of proteoforms in protein samples using capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS). The protocol can be used for the high-resolution characterization of proteoforms in simple protein samples and the large-scale identification of proteoforms in complex proteome samples.

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down proteomics that aims to characterize proteoforms in complex proteomes. However, the application of CZE-MS/MS for large-scale top-down proteomics has been impeded by the low sample-loading capacity and narrow separation window of CZE. Here, a protocol is described using CZE-MS/MS with a microliter-scale sample-loading volume and a 90-min separation window for large-scale top-down proteomics. The CZE-MS/MS platform is based on a linear polyacrylamide (LPA)-coated separation capillary with extremely low electroosmotic flow, a dynamic pH-junction-based online sample concentration method with a high efficiency for protein stacking, an electro-kinetically pumped sheath flow CE-MS interface with extremely high sensitivity, and an ion trap mass spectrometer with high mass resolution and scan speed. The platform can be used for the high-resolution characterization of simple intact protein samples and the large-scale characterization of proteoforms in various complex proteomes. As an example, a highly efficient separation of a standard protein mixture and a highly sensitive detection of many impurities using the platform is demonstrated. As another example, this platform can produce over 500 proteoform and 190 protein identifications from an Escherichia coli proteome in a single CZE-MS/MS run.

Top-down proteomics (TDP) aims for the large-scale characterization of proteoforms within a proteome. TDP relies on the effective liquid-phase separation of intact proteins before electrospray ionization-tandem mass spectrometry (ESI-MS/MS) analysis due to the high complexity and large concentration dynamic range of the proteome1,2,3,4,5. Capillary zone electrophoresis (CZE) is a powerful technique for the separation of biomolecules based on their size-to-charge ratios6. CZE is relat....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

1. Preparation of LPA Coating on the Inner Wall of the Separation Capillary

  1. Pretreatment of the capillary
    1. Flush a fused silica capillary (120 cm in length, 50 µm in inner diameter [i.d.], 360 µm in outer diameter [o.d.]) successively with 500 µL of 1 M sodium hydroxide, deionized water, 1 M hydrochloric acid, deionized water, and LC-MS grade methanol using a syringe pump.
    2. Dry the capillary with nitrogen gas (10 psi, ≥ 12 h) and fill the capillary wi.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Figure 1 shows a diagram of the dynamic pH-junction-based CZE-ESI-MS system used in the experiment. A long plug of the sample in a basic buffer is injected into an LPA-coated separation capillary filled with an acidic BGE. After applying high voltages I and II, the analytes in the sample zone will be concentrated via the dynamic pH junction method. To evaluate the performance of the CZE-MS system, a standard protein mixture (cytochrome c, lysozy.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Here we provide a detailed protocol to use CZE-MS/MS forthe high-resolution characterization of proteoforms in simple protein samples and for the large-scale identification of proteoforms in complex proteome samples. A diagram of the CZE-ESI-MS/MS system is shown in Figure 1. There are four critical steps in the protocol. First, the preparation of high-quality LPA coating on the inner wall of the separation capillary is extremely important. An LPA-coated separation capillary can reduce the E.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The authors thank Heedeok Hong's group at the Department of Chemistry, Michigan State University, for kindly providing the Escherichia coli cells for the experiments. The authors thank the support from the National Institute of General Medical Sciences, the National Institutes of Health (NIH) through Grant R01GM118470 (to X. Liu) and Grant R01GM125991 (to L. Sun and X. Liu).

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
Fused silica capillary Polymicro Technologies 1068150017 50 µm i.d. 360 µm o.d.
Sodium hydroxide pellets Macron Fine Chemicals 7708-10 Corrosive
LC-MS grade water Fisher Scientific W6-1
Hydrochloric acid Fisher Scientific SA48-1 Corrosive
Methanol Fisher Scientific A456-4 Toxic, Health Hazard
3-(Trimethoxysilyl)propyl methacrylate Sigma-Aldrich M6514 Moisture and heat sensitive
Hydrofluoric acid Acros Organics 423805000 Highy toxic
Acrylamide Acros Organics 164855000 Toxic, health hazard
Ammonium persulfate Sigma-Aldrich A3678 Health hazard, Oxidizer
lysozyme Sigma-Aldrich L6876
Cytochrome C Sigma-Aldrich C7752
Myoglobin Sigma-Aldrich M1882
ß-casein Sgma-Aldrich C6905
Carbonic anhydrase Sigma-Aldrich C3934
Bovine serum albumin Sigma-Aldrich A2153
Urea Alfa Aesar 36428-36
DL-Dithiothreitol Sigma-Aldrich D0632 Health Hazard
Iodoacetamide Fisher Scientific AC122270250 Health Hazard
Formic Acid Fisher Scientific A117-50 Corrosive, Health Hazard
C4 trap column Sepax Technologies 110043-4001C 3 µm particles, 300 Å pores, 4.0 mm i.d. 10 mm long
Acetonitrile Fisher Scientific A998SK-4 Toxic, Oxidizer
Ammonium bicarbonate Sigma-Aldrich 1066-33-7
Nalgene rapid-flow filters Thermo Scientific 126-0020 0.2 µm CN membrane, and 50 mm diameter
E. coli cells K-12 MG1655
Dulbecco's phosphate-buffered saline Sigma-Aldrich D8537
BCA assay Thermo Scientific 23250
Acetone Fisher Scientific A11-1
HPLC system for protein desalting Agilient 1260 Infinity II
Acetic Acid Fisher Scientific A38-212
CE autosampler CMP Scientific ECE-001
Electro-kinetically pumped sheath flow interface CMP Scientific
Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer Thermo Fisher Scientific
Sutter flaming/brown micropipette puller Sutter Instruments P-1000
Ultrasonic cell disruptor for cell lysis Branson 101063196 Model S-250A
Vaccum concentrator Thermo Fisher Scientific SPD131DDA-115

  1. Aebersold, R., et al. How many proteoforms are there. Nature Chemical Biology. 14 (3), 206-214 (2018).
  2. Tran, J. C., et al. Mapping intact protein isoforms in discovery mode using top-down proteomics. Nature. 480 (7376), 254-258 (2011).
  3. Catherman, A. D., et al. Large-scale Top-down Proteomics of the Human Proteome: Membrane Proteins, Mitochondria, and Senescence. Molecular and Cellular Proteomics. 12 (12), 3465-3473 (2013).
  4. Cai, W., et al. Top-Down Proteomics of Large Proteins up to 223 kDa Enabled by Serial Size Exclusion Chromatography Strategy. Analytical Chemistry. 89 (10), 5467-5475 (2017).
  5. Toby, T. K., Fornelli, L., Kelleher, N. L. Progress in Top-Down Proteomics and the Analysis of Proteoforms. Annual Review of Analytical Chemistry. 9 (1), 499-519 (2016).
  6. Jorgenson, J. W., Lukacs, K. D. Capillary Zone Electrophoresis. Science. 222 (4621), 266-272 (1983).
  7. Keithley, R. B. Capillary electrophoresis with three-color fluorescence detection for the analysis of glycosphingolipid metabolism. Analyst. 138 (1), 164-170 (2013).
  8. Han, X., et al. In-Line Separation by Capillary Electrophoresis Prior to Analysis by Top-Down Mass Spectrometry Enables Sensitive Characterization of Protein Complexes. Journal of Proteome Research. 13 (12), 6078-6086 (2014).
  9. Sun, L., Zhu, G., Zhao, Y., Yan, X., Mou, S., Dovichi, N. J. Ultrasensitive and Fast Bottom-up Analysis of Femtogram Amounts of Complex Proteome Digests. Angewandte Chemie International Edition. 52 (51), 13661-13664 (2013).
  10. Choi, S. B., Lombard-Banek, C., Muñoz-LLancao, P., Manzini, M. C., Nemes, P. Enhanced Peptide Detection Toward Single-Neuron Proteomics by Reversed-Phase Fractionation Capillary Electrophoresis Mass Spectrometry. Journal of the American Society for Mass Spectrometry. 29 (5), 913-922 (2018).
  11. Sun, L., Knierman, M. D., Zhu, G., Dovichi, N. J. Fast Top-Down Intact Protein Characterization with Capillary Zone Electrophoresis-Electrospray Ionization Tandem Mass Spectrometry. Analytical Chemistry. 85 (12), 5989-5995 (2013).
  12. Wang, Y., Fonslow, B. R., Wong, C. C., Nakorchevsky, A., Yates, J. R. Improving the comprehensiveness and sensitivity of sheathless capillary electrophoresis-tandem mass spectrometry for proteomic analysis. Analytical Chemistry. 84 (20), 8505-8513 (2012).
  13. Zhang, Z., Yan, X., Sun, L., Zhu, G., Dovichi, N. J. Detachable strong cation exchange monolith, integrated with capillary zone electrophoresis and coupled with pH gradient elution, produces improved sensitivity and numbers of peptide identifications during bottom-up analysis of complex proteomes. Analytical Chemistry. 87 (8), 4572-4577 (2015).
  14. Sun, L., et al. Over 10,000 peptide identifications from the HeLa proteome by using single-shot capillary zone electrophoresis combined with tandem mass spectrometry. Angewandte Chemie International Edition in English. 53 (50), 13931-13933 (2014).
  15. Aebersold, R., Morrison, H. D. Analysis of dilute peptide samples by capillary zone electrophoresis. Journal of Chromatography. 516 (1), 79-88 (1990).
  16. Britz-McKibbin, P., Chen, D. D. Y. Selective Focusing of Catecholamines and Weakly Acidic Compounds by Capillary Electrophoresis Using a Dynamic pH Junction. Analytical Chemistry. 72 (6), 1242-1252 (2000).
  17. Zhao, Y., Sun, L., Zhu, G., Dovichi, N. J. Coupling Capillary Zone Electrophoresis to a Q Exactive HF Mass Spectrometer for Top-down Proteomics: 580 Proteoform Identifications from Yeast. Journal of Proteome Research. 15 (10), 3679-3685 (2016).
  18. Zhu, G., Sun, L., Yan, X., Dovichi, N. J. Bottom-Up Proteomics of Escherichia coli. Using Dynamic pH Junction Preconcentration and Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry. Analytical Chemistry. 86 (13), 6331-6336 (2014).
  19. Lubeckyj, R. A., McCool, E. N., Shen, X., Kou, Q., Liu, X., Sun, L. Single-Shot Top-Down Proteomics with Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry for Identification of Nearly 600 Escherichia coli Proteoforms. Analytical Chemistry. 89 (22), 12059-12067 (2017).
  20. Busnel, J. M. High capacity capillary electrophoresis-electrospray ionization mass spectrometry: coupling a porous sheathless interface with transient-isotachophoresis. Analytical Chemistry. 82 (22), 9476-9483 (2010).
  21. Zhang, Z., Peuchen, E. H., Dovichi, N. J. Surface-Confined Aqueous Reversible Addition-Fragmentation Chain Transfer (SCARAFT) Polymerization Method for Preparation of Coated Capillary Leads to over 10 000 Peptides Identified from 25 ng HeLa Digest by Using Capillary Zone Electrophoresis-Tandem Mass Spectrometry. Analytical Chemistry. 89 (12), 6774-6780 (2017).
  22. Zhu, G., Sun, L., Dovichi, N. J. Thermally-initiated free radical polymerization for reproducible production of stable linear polyacrylamide coated capillaries, and their application to proteomic analysis using capillary zone electrophoresis-mass spectrometry. Talanta. 146, 839-843 (2016).
  23. Smith, R. D., Barinaga, C. J., Udseth, H. R. Improved Electrospray Ionization Interface for Capillary Zone Electrophoresis-Mass Spectrometry. Analytical Chemistry. 60 (16), 1948-1952 (1988).
  24. Moini, M. Simplifying CE-MS Operation. 2. Interfacing Low-Flow Separation Techniques to Mass Spectrometry Using a Porous Tip. Analytical Chemistry. 79 (11), 4241-4246 (2007).
  25. Wojcik, R., Dada, O. O., Sadilek, M., Dovichi, N. J. Simplified capillary electrophoresis nanospray sheath-flow interface for high efficiency and sensitive peptide analysis. Rapid Communications in Mass Spectrometry. 24 (17), 2554-2560 (2010).
  26. Sun, L., Zhu, G., Zhang, Z., Mou, S., Dovichi, N. J. Third-Generation Electrokinetically Pumped Sheath-Flow Nanospray Interface with Improved Stability and Sensitivity for Automated Capillary Zone Electrophoresis-Mass Spectrometry Analysis of Complex Proteome Digests. Journal of Proteome Research. 14 (5), 2312-2321 (2015).
  27. McCool, E. N., et al. Deep Top-Down Proteomics Using Capillary Zone Electrophoresis-Tandem Mass Spectrometry: Identification of 5700 Proteoforms from the Eschericia coli Proteome. Analytical Chemistry. 90 (9), 5529-5533 (2018).
  28. Kessner, D., Chambers, M., Burke, R., Agus, D., Mallick, P. ProteoWizard: open source software for rapid proteomics tools development. Bioinformatics. 24 (21), 2534-2536 (2008).
  29. Kou, Q., Xun, L., Liu, X. TopPIC : a software tool for top-down mass spectrometry-based proteoform identification and characterization. Bioinformatics. 32 (22), 3495-3497 (2016).
  30. Elias, J. E., Gygi, S. P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nature Methods. 4 (3), 207-214 (2007).
  31. Ansong, C., et al. Top-down proteomics reveals a unique protein S-thiolation switch in Salmonella Typhimurium in response to infection-like conditions. Proceedings of the National Academy of Sciences of the United States of America. 110 (25), 10153-10158 (2013).
  32. Shen, Y., et al. High-resolution ultrahigh-pressure long column reversed-phase liquid chromatography for top-down proteomics. Journal of Chromatography A. 1498, 99-110 (2017).
  33. Olsen, J. V., Macek, B., Lange, O., Makarov, A., Horning, S., Mann, M. Higher-energy C-trap dissociation for peptide modification analysis. Nature Methods. 4 (9), 709-712 (2007).
  34. Syka, J. E., Coon, J. J., Schroeder, M. J., Shabanowitz, J., Hunt, D. F. Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proceedings of the National Academy of Sciences of the United States of America. 101 (26), 9528-9533 (2004).
  35. Shaw, J. B., et al. Complete Protein Characterization Using Top-Down Mass Spectrometry and Ultraviolet Photodissociation. Journal of the American Chemical Society. 135 (34), 12646-12651 (2013).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved