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

Summary

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.

Abstract

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.

Introduction

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 proteome^1,2,3,4,5. Capillary zone electrophoresis (CZE) is a powerful technique for the separation of biomolecules based on their size-to-charge ratios⁶. CZE is relat....

Protocol

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 with 50% (v/v) 3-(trimethoxysilyl)propyl methacrylate in methanol using a syringe pump. Seal both ends of the capillary with silica rubber and incubate ....

Results

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.......

Discussion

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.......

Materials

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