Name: large-scale top-down proteomics using capillary zone electrophoresis tandem mass spectrometry
Uploaded: 2018-10-24T15:00:00
Description: Watch this Scientific Journal Video about Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry at JoVE.com

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).

1. Preparation of LPA Coating on the Inner Wall of the Separation Capillary
<ol>
	<li>Pretreatment of the capillary
	<ol>
		<li>Flush a fused silica capillary (120 cm in length, 50 &#181;m in inner diameter [i.d.], 360 &#181;m in outer diameter [o.d.]) successively with 500 &#181;L of 1 M sodium hydroxide, deionized water, 1 M hydrochloric acid, deionized water, and LC-MS grade methanol using a syringe pump.</li>
		<li>Dry the capillary with nitrogen gas (10 psi, &#8805; 12 h) and fill the capillary wi...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> Analytical Chemistry. 87 (8), 4572-4577 (2015).</li><li>Sun, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Over+10,000+peptide+identifications+from+the+HeLa+proteome+by+using+single-shot+capillary+zone+electrophoresis+combined+with+tandem+mass+spectrometry.">Over 10,000 peptide identifications from the HeLa proteome by using single-shot capillary zone electrophoresis combined with tandem mass spectrometry.</a> Angewandte Chemie International Edition in English. 53 (50), 13931-13933 (2014).</li><li>Aebersold, R., Morrison, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+dilute+peptide+samples+by+capillary+zone+electrophoresis.">Analysis of dilute peptide samples by capillary zone electrophoresis.</a> Journal of Chromatography. 516 (1), 79-88 (1990).</li><li>Britz-McKibbin, P., Chen, D. D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+Focusing+of+Catecholamines+and+Weakly+Acidic+Compounds+by+Capillary+Electrophoresis+Using+a+Dynamic+pH+Junction.">Selective Focusing of Catecholamines and Weakly Acidic Compounds by Capillary Electrophoresis Using a Dynamic pH Junction.</a> Analytical Chemistry. 72 (6), 1242-1252 (2000).</li><li>Zhao, Y., Sun, L., Zhu, G., Dovichi, N. 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Using Dynamic pH Junction Preconcentration and Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry.</a> Analytical Chemistry. 86 (13), 6331-6336 (2014).</li><li>Lubeckyj, R. A., McCool, E. N., Shen, X., Kou, Q., Liu, X., Sun, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Shot+Top-Down+Proteomics+with+Capillary+Zone+Electrophoresis-Electrospray+Ionization-Tandem+Mass+Spectrometry+for+Identification+of+Nearly+600+Escherichia+coli+Proteoforms.">Single-Shot Top-Down Proteomics with Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry for Identification of Nearly 600 Escherichia coli Proteoforms.</a> Analytical Chemistry. 89 (22), 12059-12067 (2017).</li><li>Busnel, J. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> Analytical Chemistry. 89 (12), 6774-6780 (2017).</li><li>Zhu, G., Sun, L., Dovichi, N. 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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+Top-Down+Proteomics+Using+Capillary+Zone+Electrophoresis-Tandem+Mass+Spectrometry:+Identification+of+5700+Proteoforms+from+the+Eschericia+coli+Proteome.">Deep Top-Down Proteomics Using Capillary Zone Electrophoresis-Tandem Mass Spectrometry: Identification of 5700 Proteoforms from the Eschericia coli Proteome.</a> Analytical Chemistry. 90 (9), 5529-5533 (2018).</li><li>Kessner, D., Chambers, M., Burke, R., Agus, D., Mallick, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ProteoWizard:+open+source+software+for+rapid+proteomics+tools+development.">ProteoWizard: open source software for rapid proteomics tools development.</a> Bioinformatics. 24 (21), 2534-2536 (2008).</li><li>Kou, Q., Xun, L., Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TopPIC+:+a+software+tool+for+top-down+mass+spectrometry-based+proteoform+identification+and+characterization.">TopPIC : a software tool for top-down mass spectrometry-based proteoform identification and characterization.</a> Bioinformatics. 32 (22), 3495-3497 (2016).</li><li>Elias, J. 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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...

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 (&#62; 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 &#176;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.

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	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">Fused silica capillary</td>
			<td style="width:117px;">Polymicro Technologies</td>
			<td style="width:147px;">1068150017</td>
			<td style="width:355px;">50 &#181;m i.d. 360 &#181;m o.d.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">Sodium hydroxide pellets</td>
			<td style="width:117px;">Macron Fine Chemicals</td>
			<td style="width:147px;">7708-10</td>
			<td>Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">LC-MS grade water</td>
			<td style="width:117px;">Fisher Scientific</td>
			<td>W6-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Hydrochloric acid</td>
			<td style="width:117px;">Fisher Scientific</td>
			<td style="width:147px;">SA48-1</td>
			<td>Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Methanol</td>
			<td style="width:117px;">Fisher Scientific</td>
			<td style="width:147px;">A456-4</td>
			<td>Toxic, Health Hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">3-(Trimethoxysilyl)propyl methacrylate</td>
			<td style="width:117px;">Sigma-Aldrich</td>
			<td style="width:147px;">M6514</td>
			<td>Moisture and heat sensitive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Hydrofluoric acid</td>
			<td style="width:117px;">Acros Organics</td>
			<td style="width:147px;">423805000</td>
			<td>Highy toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Acrylamide</td>
			<td style="width:117px;">Acros Organics</td>
			<td style="width:147px;">164855000</td>
			<td>Toxic, health hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Ammonium persulfate</td>
			<td style="width:117px;">Sigma-Aldrich</td>
			<td style="width:147px;">A3678</td>
			<td>Health hazard, Oxidizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">lysozyme</td>
			<td style="width:117px;">Sigma-Aldrich</td>
			<td style="width:147px;">L6876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Cytochrome C</td>
			<td style="width:117px;">Sigma-Aldrich</td>
			<td style="width:147px;">C7752</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Myoglobin</td>
			<td style="width:117px;">Sigma-Aldrich</td>
			<td style="width:147px;">M1882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">&#223;-casein</td>
			<td style="width:117px;">Sgma-Aldrich</td>
			<td style="width:147px;">C6905</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Carbonic anhydrase</td>
			<td style="width:117px;">Sigma-Aldrich</td>
			<td style="width:147px;">C3934</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Bovine serum albumin</td>
			<td style="width:117px;">Sigma-Aldrich</td>
			<td style="width:147px;">A2153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Urea</td>
			<td style="width:117px;">Alfa Aesar</td>
			<td style="width:147px;">36428-36</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">DL-Dithiothreitol</td>
			<td style="width:117px;">Sigma-Aldrich</td>
			<td style="width:147px;">D0632</td>
			<td>Health Hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Iodoacetamide</td>
			<td style="width:117px;">Fisher Scientific</td>
			<td style="width:147px;">AC122270250</td>
			<td>Health Hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Formic Acid</td>
			<td style="width:117px;">Fisher Scientific</td>
			<td style="width:147px;">A117-50</td>
			<td>Corrosive, Health Hazard</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">C4 trap column</td>
			<td style="width:117px;">Sepax Technologies</td>
			<td style="width:147px;">110043-4001C</td>
			<td>3 &#181;m particles, 300 &#197; pores, 4.0 mm i.d. 10 mm long</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Acetonitrile</td>
			<td style="width:117px;">Fisher Scientific</td>
			<td style="width:147px;">A998SK-4</td>
			<td>Toxic, Oxidizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Ammonium bicarbonate</td>
			<td style="width:117px;">Sigma-Aldrich</td>
			<td style="width:147px;">1066-33-7</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">Nalgene rapid-flow filters</td>
			<td style="width:117px;">Thermo Scientific</td>
			<td style="width:147px;">126-0020</td>
			<td>0.2 &#181;m CN membrane, and 50 mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">E. coli cells</td>
			<td style="width:117px;"></td>
			<td style="width:147px;"></td>
			<td>K-12 MG1655</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Dulbecco&#39;s phosphate-buffered saline</td>
			<td style="width:117px;">Sigma-Aldrich</td>
			<td style="width:147px;">D8537</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">BCA assay</td>
			<td style="width:117px;">Thermo Scientific</td>
			<td style="width:147px;">23250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Acetone</td>
			<td style="width:117px;">Fisher Scientific</td>
			<td style="width:147px;">A11-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">HPLC system for protein desalting</td>
			<td style="width:117px;">Agilient</td>
			<td style="width:147px;">1260 Infinity II</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Acetic Acid</td>
			<td style="width:117px;">Fisher Scientific</td>
			<td style="width:147px;">A38-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">CE autosampler</td>
			<td style="width:117px;">CMP Scientific</td>
			<td style="width:147px;">ECE-001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">Electro-kinetically pumped sheath flow interface</td>
			<td style="width:117px;">CMP Scientific</td>
			<td style="width:147px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer</td>
			<td style="width:117px;">Thermo Fisher Scientific</td>
			<td style="width:147px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">Sutter flaming/brown micropipette puller</td>
			<td style="width:117px;">Sutter Instruments</td>
			<td style="width:147px;">P-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Ultrasonic cell disruptor for cell lysis</td>
			<td style="width:117px;">Branson</td>
			<td align="right" style="width:147px;">101063196</td>
			<td>Model S-250A</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">Vaccum concentrator</td>
			<td style="width:117px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">SPD131DDA-115</td>
			<td></td>
		</tr>
	</tbody>
</table>

large-scale top-down proteomics using capillary zone electrophoresis tandem mass spectrometry

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.

Figure 1&#160;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...

Watch this Scientific Journal Video about Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry at JoVE.com

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

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 ...

This method can help improve large scale and high resolution characterization of protein isoforms and protein post-translational modifications in cells. The main advantage of this technique is that capillary zone electrophoresis mass spectrometry offers highly efficient separation and highly sensitive detection of intact proteins. To pre-treat the capillary, dry it with nitrogen gas at 10 psi for at least 12 hours.Then, fill the capillary with 50%volume-by-volume 3-propyl methacrylate in methanol using a syringe pump. Seal both ends of the capillary with silica rubber. After incubating at room temperature for at least 24 hours, continue with a linear polyacrylamide coating on the inner wall of the separation capillary, as described in the text protocol.Burn a portion of the capillary using a gentle flame to remove the polyamide outer coating around four centimeters away from one end of the capillary. Gently clean the burnt portion of the capillary by wiping to remove the polyamide coating completely. Drill a small hole at the end of a 200 microliter tube around the same size as the capillary outer diameter to sufficiently hold the capillary in place once it is threaded through this hole.Thread the end of the capillary that is close to the burnt portion through the hole until the burnt portion is in the tube. Then, add approximately 150 microliters of HF to the 200 microliter tube so that the HF solution is about halfway up the burnt portion on the capillary. Incubate the capillary in the HF solution at room temperature for 90 to 100 minutes.Prepare the standard protein mixture and the E.coli sample as described in the text protocol. To set up the capillary zone electrophoresis, or CZE system, load the sample, the background electrolyte, and the separation capillary into CZE auto-sampler. Select sample load in the manual control page of the CZE auto-sampler.Load the background electrolyte vial into the buffer tray of the auto-sampler, noting its position in the buffer tray. Then, load the sample vial into the sample tray of the auto-sampler, noting its position in the sample tray. Put the auto-sampler to the alignment position using manual control.Now, load the separation capillary into CZE auto-sampler using the non-etched end of the capillary. Adjust the height of the injection end of the capillary if the sample volume is lower than 50 microliters. Switch the auto-sampler to standby using manual control.Type the sample position in the system and move the capillary to the sample. Push the injection end of the capillary further down to reach the bottom of the sample vial. Continuing to use the manual control, flush the capillary with the background electrolyte for 20 minutes using a pressure of 20 psi.Now, mount a commercial electrokinetically pumped sheath flow interface to the front of the mass spectrometer. Fill the sheath buffer reservoir with a buffer containing 10%volume-volume methanol and 0.2%volume-volume formic acid in LCMS-grade water. Flush the T in the interface with the sheath buffer via applying pressure manually with a syringe.Thread the one millimeter outer diameter end of an electrospray emitter through a sleeve tubing and connect the emitter with one port of the T via a fitting. Simply flush the T manually again with a syringe to fill the emitter with the sheath buffer. Adjust the distance between the orifice of the emitter and the entrance of the mass spectrometer to approximately two millimeters with the help of the camera that came with the interface.Apply a two to 2.2 kilovolt voltage at the sheath buffer vial for electrospray. Then, adjust the spray voltage to reach a stable electrospray. Apply a low pressure at the injection end of the capillary during this process to make sure there are no bubbles in the capillary.Turn off the spray voltage and gently thread the etched end of the separation capillary through the T into the emitter until it cannot be pushed further. After stopping the low pressure at the injection end of the capillary, flush the emitter a little bit with sheath buffer. Again, apply two to 2.2 kilovolts of spray voltage to test the spray.Select new method under the file dropdown menu on the main instrument screen. There, select inlet as starting sample vial, ending sample vial, tray as sample, pressure as five psi, kilovolts as zero, and duration as 95 seconds for a sample injection. Then, set inlet as inlet vial, tray as buffer, pressure as zero psi, kilovolts as 30, and duration as 4, 200 seconds for the separation of the standard protein mixture sample.For the separation of the E.coli proteome sample, set inlet as inlet vial, tray as buffer, pressure as zero psi, kilovolts as 20, and duration as 6, 600 seconds. Finally, select inlet as inlet vial, tray as buffer, pressure as 10 psi, kilovolts as 30, and duration as 600 seconds for capillary flushing. Now, setup the MS and MS/MS parameters for intact protein analysis using a quadruple ion trap mask spectrometer.To adjust the tune file settings, turn on intact protein mode and use a trapping pressure of 0.2. Set the ion transfer capillary temperature to 320 degrees Celsius and the S-lens RF level to 55. To perform the CZE MS/MS experiment, start the data acquisition method on the mass spectrometer computer, first by selecting run sequence.Then, start the capillary electrophoresis sequence on the capillary electrophoresis auto-sampler computer. Perform a database search with TopPIC in the TopPIC suite and in the TopPIC graphical user interface. Click on database file and select an appropriate database for searching.Click on spectrum file and select the generated ms2. msalign file generated as the input. Select the MS1 feature file and choose the generated feature file.Set cysteine carbamidomethyl C57 as a fixed modification due to the iodoacetamide treatment. Select the decoy database feature and under cutoff settings, select false discovery rate in the dropdown menu next to spectrum level. Set the false discovery rate to 0.01 at the spectrum level.Leave generating function unselected and set the error tolerance to 15 parts per million. Set the false discovery rate to 0.05 at the proteoform level. Under advanced parameters, select two for the maximum number of mass shifts in the dropdown menu.Set the maximum mass shift of unknown modifications to 500 daltons. Leave all other parameters at their default values and click the start button at the bottom right of the graphical user interface. The representative electropherogram for the standard protein mixture is shown.The standard protein mixture is typically run at least in duplicate to evaluate the separation efficiency and the reproducibility of the system. The separation efficiency can be evaluated with the number of theoretical plates of some proteins. The reproducibility can be evaluated by the relative standard deviations of protein intensity and migration time.The CZE MS/MS platform can be used for large-scale characterization of proteoforms in various complex proteomes, identifying over 500 proteoforms and 190 proteins from an E.coli proteome in a single run with high confidence. Here, a zoomed in view of the electropherogram can be used to assess the separation window of the system. The very low E-value and spectral FDR suggests the high confidence of the proteoform ID.The high number of matched fragment ions further indicates the high confidence of the ID.While attempting this procedure, it's important to remember to thread the etched separation capillary into the electrospray emitter slowly and gently.Stable isotope labeling or label-free methods can be incorporated into the CZE MS procedure to determine how proteoform abundance changes in cells across various conditions. After its development, this technique paved the way for researchers in the field of top-down proteomics to explore the roles of proteoforms in regulating various biological processes in cells. Don't forget that working with hydrofluoric acid can be extremely hazardous and precautions such as proper personal protective equipment and having emergency procedures in place should always be taken when performing this procedure.

Research

JoVE Journal

Chemistry

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