Buffer 4-Ethylmorpholinium/Acetate: Exploring a New Alternative Buffer for Native Mass Spectrometry

Abstract

Rationale

To perform native mass spectrometry (MS) studies, there are a limited number of volatile and electrospray ionization (ESI)-MS compatible solutions, such as ammonium bicarbonate and ammonium acetate (AA). These solutions could induce the unfolding of proteins due to the formation of CO₂ bubbles or induced acidification during ESI. Hence, it was important to introduce a buffer suitable to preserve the native form of proteins while simulating physiological conditions.

Methods

The 4-ethylmorpholinium/acetate (4EM/A) buffer was compared to AA for the analysis of proteins and protein complexes with mass ranges from 5 to 103 kDa and isoelectric points (pI) between 3 and 11. The evaluations were conducted by comparing the native-MS profiles, CCS values, arrival time distributions (ATDs), and proteins bioactivities. The human cardiac troponin complex (cTn complex) and its subunit cardiac troponin T (cTnT) were analyzed as proof of the applicability of this buffer for challenging proteins and protein complexes.

Results

4EM/A led to lower charge states compared to AA, supporting the likelihood of preserving protein folding during nano-ESI and in a high vacuum environment of MS. Ion mobility measurements revealed that proteins in 4EM/A exhibit a lower degree of conformational variation compared to AA, suggesting enhanced conformational stability and potential retention of natural-like compactness. Additionally, testing the impact of 4EM/A on bioactivity, lysozyme showed increased biological activity in 4EM/A relative to AA, highlighting the buffer's potential for real-time assessment of protein interaction kinetics and bioactivity. The 4EM/A buffer enabled native-MS analysis of cTnT for the first time.

Conclusion

We introduced 4EM/A, with pK_a of 7.72/4.76, as a promising buffer for native-MS studies to maintain protein and protein complex bioactivity and conformational integrity.