Nanoflow Size Exclusion Chromatography–Native Mass Spectrometry of Intact Proteoforms and Protein Complexes

Abstract

High Resolution Image Download MS PowerPoint Slide Native size-exclusion chromatography (SEC) coupled with native mass spectrometry (nMS) enables the characterization of proteins and protein complexes by combining liquid-phase separation (SEC) and mass measurement (nMS). This approach allows for an increase in the throughput of nMS experiments, reduces the bias that may be present due to the co-ionization of oligomers, and facilitates online sample buffer exchange. Conventional SEC-nMS uses volatile buffers and relatively wide-diameter columns (e.g., ≥1 mm), with flow rates in the tens of μL/min. To ionize sample components under this flow regime, relatively harsh electrospray ionization (ESI) desolvation conditions are needed, potentially resulting in protein dissociation/denaturation. Additionally, relatively large amounts of samples are required (several μgs). Herein, we describe the development of a nanoflow SEC-nMS method using 200 μm I.D. columns, operated at 500 nL min –1 . This approach enables buffer exchange, oligomer separation, and mild ionization conditions (e.g., without the assistance of heated gas flow or temperature). Compared to microflow (1 mm I.D. column), the nanoflow method achieved a 4-fold increase in MS peak intensity, despite using a sample 20 times less concentrated (0.05 mg mL –1 for nanoflow vs 1 mg mL –1 for microflow). Furthermore, we evaluated the impact of three injection approaches on sensitivity and separation efficiency: large volume (1 μL), nanovolume (50 nL), and online mixed-bed ion-exchange capillary trap injection. To demonstrate its performance and applicability for sample-limited analysis, the final method using nanovolume injection was applied to several model proteins, protein complexes and a urine sample from a pregnant donor.

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