Direct ionization of large proteins and protein complexes by desorption electrospray ionization-mass spectrometry

Abstract

Desorption electrospray ionization-mass spectrometry (DESI-MS) has advantages for rapid sample analysis with little or no sample pretreatment, but performance for large biomolecules has not been demonstrated. In this study, liquid sample DESI, an extended version of DESI used for analysis of liquid samples, was shown to have capabilities for direct ionization of large noncovalent protein complexes (>45 kDa) and proteins (up to 150 kDa). Protein complex ions (e.g., superoxide dismutase, enolase, and hemoglobin) desorbed from solution by liquid sample DESI were measured intact, indicating the capability of DESI for preserving weak noncovalent interactions. Doping the DESI spray solvent with supercharging reagents resulted in protein complex ions having increased multiple charging without complex dissociation. Ion mobility measurements of model protein cytochrome c showed that the supercharging reagent favored the more compact conformation for the lower charged protein ions. Liquid sample DESI of hydrophobic peptide gramicidin D suggests that the ionization mechanism involves a droplet pick-up mixing process. Measurement of liquid samples significantly extends the mass range of DESI-MS, allowing the analysis of high-mass proteins such as 150 kDa immunoglobulin G (IgG) and thus represents the largest protein successfully ionized by DESI to date.

Figure 1

Mass spectra showing liquid sample DESI analysis of 30 μM MnSOD. (A) MnSOD in 20 mM NH₄OAc sampled by a DESI solvent composed of 20 mM NH₄OAc aqueous solution. (B) MnSOD in 20 mM NH₄OAc sampled by a DESI solvent of ACN/H₂O/FA. (C) MnSOD (15 μM) in 1% HOAc sampled by a DESI solvent of 1:1 ACN/H₂O with 1% FA.

Figure 2

Liquid sample DESI mass spectra of enolase (10 μM in water) with the DESI solvent composed of (A) 20 mM NH₄OAc (aq), (B) 1:1 ACN/H₂O with 1% FA, and (C) 1:1 ACN/H₂O with 1% FA and 40 mM m-NBA supercharging reagent.

Figure 3

Liquid sample DESI mass spectra of human hemoglobin (50 μM) in 20 mM NH₄OAc and a DESI spray solvent of (A) 20 mM NH₄OAc and (B) 20 mM NH₄OAc with 40 mM m-NBA. The “α_hβ_h” notation refers to the heme-bound alpha and beta polypeptide chains. The free heme-bound alpha-chain is represented by the blue circle symbol (), and the red triangles () denote the heme-bound beta-chain.

Figure 4

Supercharging liquid sample DESI mass spectra of 50 μM cytochrome c in water with DESI spray solvents of (A) 1:1 ACN/H₂O and 0.1% FA, (B) 1:1 ACN/H₂O and 0.1% FA with 40 mM m-NBA and (C) 1:1 ACN/H₂O and 0.1% FA with 200 mM sulfolane.

Figure 5

Ion mobility drift time plots of the (A) 7+, (B) 8+, and (C) 9+ charged ions of cytochrome c (50 μM). The green () and blue () traces represent the ion mobility of cytc in 4% and 0% acetic acid (in water), respectively, and the DESI solvent of 1:1 ACN/H₂O with 0.1% FA. The red line () represents the data from cytc in water and the DESI solvent of 1:1 ACN/H₂O with 0.1% FA and 40 mM m-NBA.

Figure 6

Liquid sample DESI mass spectrum of IgG (6 μM) in H₂O/ACN/FA (90/10/0.1 by volume) with a DESI spray solvent composition the same as the analyte solvent.