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

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive Orbitrap Mass Spectrometer

ORGANISM(S): Myoglobin Bsa Ovalbumin Catalase L-asparaginase Proteinase K Human Chorionic Gonadotropin Alcohol Dehydrogenase Enolase Concanavalin A Trastuzumab Ovitrelle

SUBMITTER: Ziran Zhai

PROVIDER: MSV000096079 | MassIVE | Mon Oct 14 06:35:00 BST 2024

REPOSITORIES: MassIVE

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Project description:Datasets part of manuscript submitted at analytica chimica acta Size-exclusion chromatography (SEC) employing aqueous mobile phases with volatile salts at neutral pH combined with native mass spectrometry (nMS) is a valuable tool to characterize proteins and protein aggregates in their native state. However, the liquid-phase conditions (high salt concentrations) frequently used in SEC-nMS hinder the analysis of labile protein complexes in the gas phase, necessitating higher desolvation-gas flow and source temperature, leading to protein fragmentation/dissociation. To overcome this issue, we investigated narrow SEC columns (1.0 mm internal diameter, I.D.) operated at 15-uL/min flow rates and their coupling to nMS for the characterization of proteins, protein complexes and higher-order structures (HOS). The reduced flow rate resulted in a significant increase in the protein-ionization efficiency, facilitating the detection of low-abundant impurities and HOS up to 230 kDa (i.e., the upper limit of the Orbitrap-MS instrument used). More-efficient solvent evaporation and lower desolvation energies allowed for softer ionization conditions (e.g., lower gas temperatures), ensuring little or no structural alterations of proteins and their HOS during transfer into the gas phase. Furthermore, ionization suppression by eluent salts was decreased, permitting the use of volatile-salt concentrations up to 400 mM. Band broadening and loss of resolution resulting from the introduction of injection volumes exceeding 3% of the column volume could be circumvented by incorporating an online trap-column containing a mixed-bed ion-exchange (IEX) material. The online IEX-based solid-phase extraction (SPE) or trap-and-elute set-up provided on-column focusing (sample preconcentration). This allowed the injection of large sample volumes on the 1-mm I.D. SEC column without compromising the separation. The enhanced sensitivity attained by the micro-flow SEC-MS, along with the on-column focusing achieved by the IEX precolumn, provided picogram detection limits for proteins.

Project description:Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processes by shuttling material out of and between cells. Tissue EVs may thus lend insights into disease mechanisms and also betray disease when released into easily accessed biological fluids. Since brain-derived EVs (bdEVs) and their cargo may serve as biomarkers of neurodegenerative diseases, we evaluated modifications to a published, rigorous protocol for separation of EVs from brain tissue and studied effects of processing variables on quantitative and qualitative outcomes. To this end, size exclusion chromatography (SEC) and sucrose density gradient ultracentrifugation were compared as final separation steps in protocols involving stepped ultracentrifugation. bdEVs were separated from brain tissues of human, macaque. Effects of post-mortem interval (PMI) before final bdEV separation were probed. MISEV2018-compliant EV characterization was performed, and both small RNA and protein profiling were done. We conclude that the modified, SEC-employing protocol achieves EV separation efficiency roughly similar to a protocol using gradient density ultracentrifugation, while decreasing operator time and, potentially, variability. The protocol appears to yield bdEVs of higher purity for human tissues compared with macaque, suggesting opportunities for optimization. The interval between death/tissue storage/processing and final bdEV separation can also affect bdEV populations and composition and should thus be recorded for rigorous reporting. Different populations of EVs obtained through the modified method reported herein display characteristic RNA and protein content that hint at biomarker potential. To conclude, this study finds that the automatable and increasingly employed technique of SEC can be applied to tissue EV separation, and also reveals more about the importance of species-specific and technical considerations when working with tissue EVs. These results are expected to enhance the use of bdEVs in revealing and understanding brain disease.

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

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