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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 relatively simple, requiring only an open tubular-fused silica capillary, a background electrolyte (BGE), and a power supply. A sample of intact proteins can be loaded into the capillary using pressure or voltage, and separation is initiated by immersing both ends of the capillary in the BGE and applying a high voltage. CZE can approach ultra-high separation efficiency (> one million theoretical plates) for the separation of biomolecules7. CZE-MS has a drastically higher sensitivity than widely used reversed-phase liquid chromatography (RPLC)-MS for the analysis of intact proteins8. Although CZE-MS has a great potential for large-scale top-down proteomics, its wide application in proteomics has been impeded by several issues, including a low sample-loading capacity and narrow separation window. The typical sample loading volume in CZE is about 1% of the total capillary volume, which usually corresponds to less than 100 nL9,10,11. The separation window of CZE is usually less than 30 min due to the strong electroosmotic flow (EOF)9,10. These issues limit the CZE-MS/MS for the identification of a large number of proteoforms and low abundant proteoforms from a complex proteome.
Much effort has been made to improve the sample loading volume of CZE via online sample concentration methods (e.g., solid-phase microextraction [SPME]12,13, field-enhanced sample stacking [FESS]9,11,14, and dynamic pH junction15,16,17,18). FESS and dynamic pH junction are simpler than SPME, only requiring a significant difference between the sample buffer and the BGE in conductivity and pH. FESS employs a sample buffer with much lower conductivity than the BGE, leading to a stacking of analytes on the boundary between the sample zone and the BGE zone in the capillary. Dynamic pH junction utilizes a basic sample plug (e.g., 50 mM ammonium bicarbonate, pH 8) and an acidic BGE (e.g., 5% [v/v] acetic acid, pH 2.4) on both sides of the sample plug. Upon application of a high positive voltage at the injection end of the capillary, titration of the basic sample plug occurs, focusing the analytes into a tight plug before undergoing a CZE separation. Recently, the Sun group systematically compared FESS and dynamic pH junction for the online stacking of intact proteins, demonstrating that dynamic pH junction could produce much better performance than FESS for the online concentration of intact proteins when the sample injection volume was 25% of the total capillary volume19.
Neutrally coated separation capillaries (e.g., linear polyacrylamide [LPA]) have been employed to reduce the EOF in the capillary, slowing down the CZE separation and widening the separation window20,21. Recently, the Dovichi group developed a simple procedure for the preparation of stable LPA coating on the inner wall of capillaries, utilizing ammonium persulfate (APS) as the initiator and temperature (50 °C) for free radical production and polymerization22. Very recently, the Sun group employed the LPA-coated separation capillary and the dynamic pH junction method for the CZE separation of intact proteins, reaching a microliter-scale sample loading volume and a 90-min separation window19. This CZE system opens the door to using CZE-MS/MS for large-scale top-down proteomics.
CZE-MS requires a highly robust and sensitive interface to couple CZE to MS. Three CE-MS interfaces have been well developed and commercialized in the history of CE-MS, and they are the co-axial sheath-flow interface23, the sheathless interface using a porous tip as the ESI emitter24, and the electro-kinetically pumped sheath flow interface25,26. The electro-kinetically pumped sheath-flow-interface-based CZE-MS/MS has reached a low zeptomole peptide detection limit9, over 10,000 peptide identifications (IDs) from the HeLa cell proteome in a single run14, a fast characterization of intact proteins11, and highly stable and reproducible analyses of biomolecules26. Recently, the LPA-coated separation capillary, the dynamic pH junction method, and the electro-kinetically pumped sheath flow interface were used for large-scale top-down proteomics of an Escherichia coli (E. coli) proteome19,27. The CZE-MS/MS platform approached over 500 proteoform IDs in a single run19 and nearly 6,000 proteoform IDs via coupling with size-exclusion chromatography (SEC)-RPLC fractionation27. The results clearly show the capability of CZE-MS/MS for large-scale top-down proteomics.
Herein, a detailed procedure of using CZE-MS/MS for large-scale top-down proteomics is described. The CZE-MS/MS system employs the LPA-coated capillary to reduce the EOF in the capillary, the dynamic pH junction method for the online concentration of proteins, the electro-kinetically pumped sheath flow interface for coupling CZE to MS, an orbitrap mass spectrometer for the collection of MS and MS/MS spectra of proteins, and a TopPIC (TOP-Down Mass Spectrometry-Based Proteoform Identification and Characterization) software for proteoform ID via database search.
1. Preparation of LPA Coating on the Inner Wall of the Separation Capillary
2. Etching of the Capillary with Hydrofluoric Acid
CAUTION: Use appropriate safety procedures while handling hydrofluoric acid (HF) solutions. All the HF-related operations need to be done in a chemical hood. Before any HF-related operation, make sure that 2.5% calcium gluconate gel is available for use in the case of exposure. Double gloves are required, a typical nitrile glove inside and a heavy neoprene glove outside. Wear a lab coat and chemical safety goggles. After the HF operations, keep liquid and solid hazardous waste separate. The liquid HF waste must be neutralized immediately with a high-concentration sodium hydroxide solution for temporary storage before waste pick-up. The solid HF waste needs to be temporarily stored in a plastic container that is lined with two thick plastic one-gallon Ziploc bags and a lid. Both the solid and liquid waste must be labeled properly.
3. Preparation of the Samples
4. Set-up of the CZE-MS/MS System and Analysis of the Samples
5. Database Search of the Collected Raw Files with the TopPIC Software
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...
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...
The authors have nothing to disclose.
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).
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 |
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