Dataset Information

Self-packed size-exclusion columns enable versatile high-throughput native, top-down, and ion-mobility-mass spectrometry studies on proteins and complexes.

ABSTRACT: Native MS (nMS) is a key structural biology technique that makes it possible to study intact proteins and their interactions. Unfortunately, non-volatile salts are incompatiblewith nMS, which demands a laborious desalting procedure. Non-denaturing size exclusion chromatography (SEC) allows both rapid desalting and separation and has previously been explored for nMS automation. However, SEC at conventional scale requires rather large sample amounts as well as harsh ESI conditions, which can cause protein unfolding. Capillary LC allows softer conditions; however, the few commercially available SEC columns appropriate for this flow rate are prohibitively expensive for many laboratories. Existing protocols for packing buffer exchange columns rely on specialized equipment and/or result in columns that have limited capacity for size-based protein separation. Here, we present self-packed miniaturized SEC columns with different stationary phases and customizable dimensions. The columns, produced via slurry packing with an ordinary LC pump were used across a range of samples in several applications including nMS, top-down MS (TDMS), ligand screening, and ion mobility (IM)-MS. Native separation allowed acquisition of data from samples containing more than one protein. We acquired native TDMS data of 3 proteins in 12 minutes, with up to 47% sequence coverage. IM-MS of alpha-synuclein at different charge states was measured in ca. 60 minutes (including calibrants), with results that match the literature. Finally, we used SEC-nMS to rapidly screen proteolysis-targeting chimera candidates and performed collision-induced unfolding (CIU) of a PROTAC-induced ternary complex. Through this work, we highlight the potential of SEC to support developments in structural MS.

INSTRUMENT(S): Synapt MS, nanoACQUITY UPLC

ORGANISM(S): Equus Caballus (horse) Homo Sapiens (human) Bos Taurus (bovine) Escherichia Coli Gallus Gallus (chicken) Candida Albicans (yeast)

SUBMITTER: Edvaldo Maciiel

LAB HEAD: Frederik Lermyte

PROVIDER: PXD061452 | Pride | 2025-06-29

REPOSITORIES: Pride

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	j.talanta.2025.127868.xlsx	Xlsx
	j.talanta.2025.127868.zip	Other

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Self-packed size-exclusion columns enable versatile high-throughput native, top-down, and ion mobility-mass spectrometry studies on proteins and complexes.

Maciel Edvaldo Vasconcelos Soares EVS Habeck Tanja T Meyners Christian C Lermyte Frederik F

Talanta 20250301

Native MS (nMS) is a key structural biology technique that makes it possible to study intact proteins and their interactions. Unfortunately, non-volatile salts are incompatible with nMS, which demands a laborious desalting procedure. Non-denaturing size-exclusion chromatography (SEC) allows both rapid desalting and separation and has previously been explored for nMS automation. However, SEC at conventional scale requires rather large sample amounts as well as harsh ESI conditions, which can caus ...[more]

PMID: 40056653

Similar Datasets

Project 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.

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.