Search for a high-mass calibrant for NALIM (Native Liquid MALDI MS): the case of mouse IgA
Résumé
Native mass spectrometry (native MS) is a set of methods to analyze biomolecular assemblies directly in an instrument. To this end, biomolecular objects or complexes are exposed only to non-denaturing conditions to preserve their three-dimensional structure intact from the sample solution to the gas phase.
In pharmacology, membrane proteins represent two-thirds of therapeutic targets, yet only 10% have been targeted so far. The characterization of membrane proteins by native MS can open many paths for biology and pre-clinical studies.
To this day, the go-to method for native MS is based on electrospray ionization (ESI). However, membrane protein analysis by native ESI-MS is still a challenge. This is because these proteins necessitate detergents for solubilization in MS-compatible buffers. Detergents in turn can give rise to ion suppression, adduction, and degraded instrument performance. These effects can be somewhat mitigated by working above the detergent's critical micellar concentration [1].
MALDI (Matrix-assisted laser desorption-ionization) ionization presents numerous advantages in this context. It is highly tolerant to contaminants and consumes little quantities of sample, making it de facto attractive for the native MS analysis of membrane proteins. Using these unique properties, we established a new native MS MALDI method using liquid spots called Native Liquid MALDI (NALIM) [2]. The success of this method relies on: (i) a matrix mixture that can be used in native conditions (pH 5), (ii) a liquid matrix that enables to gently transfer complexes in the gas phase while avoiding the transition through the solid state, which is the basis of classical solid-spot deposition methods.
We are currently extending applications of the NALIM method to the observation of membrane protein complexes. In NALIM, we mainly observe low charge states (1+ and 2+), the monocharged state being largely predominant. Thus, the application of NALIM to large membrane protein-containing assemblies requires calibrant standards at high m/z ratios. In search of such calibrants, we investigated the use of an immunoglobulin A.
Here we report on the suitability and usability of an IgA for calibration over a wide mass range. Optimizations include biochemical preparation, control of the degree of oligomerization and instrumental setup for NaLiM.
[1] Barrera, N.P., Bartolo, N.D., Booth, P.J., and Robinson, C.V. (2008). Micelles Protect Membrane Complexes from Solution to Vacuum. Science 321, 243–246.
[2] Beaufour, M., Ginguené, D., Le Meur, R., Castaing, B., and Cadene, M. (2018). Liquid Native MALDI Mass Spectrometry for the Detection of Protein-Protein Complexes. J. Am. Soc. Mass Spectrom. 29, 1981–1994.
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