MassIVE MSV000096079

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Nanoflow Size Exclusion Chromatography_Native Mass Spectrometry of Intact Proteoforms and Protein Complexes

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Description

Native size-exclusion chromatography (SEC) and native mass spectrometry (nMS) are techniques used to characterize oligomeric states of protein complexes and their proteoform distributions. Coupling SEC with nMS combines the advantages of liquid-phase separation of oligomeric states (SEC) with molecular measurement of the complex (nMS), reducing bias that may be present in nMS due to ion suppression when oligomers are ionized together and additionally allowing buffer exchange of the sample. SEC-nMS uses volatile buffers and relatively wide-diameter columns (e.g., 1 mm), with flow rates in the tens of microliter per minute. To ionize sample components under this flow regime, relatively harsh electrospray ionization (ESI) desolvation conditions, including nebulization gas heating, electrospray, and in-source dissociation voltages, are needed, which may result in protein dissociation or denaturation. Also, relatively large amounts of samples are required to provide sufficient sensitivity for detection. Herein, we describe the development of a nanoflow SEC-nMS method and its application to the analysis of model proteins and complexes. We studied the influence of several parameters on the separation performance of capillary SEC columns with a 200 um I.D., such as frits and packing solvents. NanoSEC-MS was performed at a flow rate of 0.5 uL min-1, allowing for buffer exchange and oligomer separation. The nanoflow regime utilizes coated fused silica emitters to perform ionization without the assistance of heated gas flow or temperature and reduces the voltages applied (about 1.8 kV). This resulted in a 10-fold increased MS peak intensity with respect to a microflow method using a 1 mm I.D. SEC column. Furthermore, we evaluated the performance of three injection approaches for sensitivity and separation: large-volume injection (1 uL), nano-volume injection (50 nL), and an online mix-bed ion-exchange capillary trap column. The proposed setup and workflow provide opportunities for native top-down analysis of proteins in sample-limited applications. [doi:10.25345/C59S1KX4P] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: nanoflow rate ; size-exclusion chromatography ; native mass spectrometry ; protein and protein complexes ; intact analysis ; DatasetType:Proteomics

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Principal Investigators:
(in alphabetical order) 		Ziran Zhai, University of Amsterdam, Netherlands
Submitting User: ZiranZhai
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