High-performance liquid chromatography separation and intact mass analysis of detergent-solubilized integral membrane proteins

Abstract

We have developed a method for intact mass analysis of detergent-solubilized and purified integral membrane proteins using liquid chromatography-mass spectrometry (LC-MS) with methanol as the organic mobile phase. Membrane proteins and detergents are separated chromatographically during the isocratic stage of the gradient profile from a 150-mm C3 reversed-phase column. The mass accuracy is comparable to standard methods employed for soluble proteins; the sensitivity is 10-fold lower, requiring 0.2-5 μg of protein. The method is also compatible with our standard LC-MS method used for intact mass analysis of soluble proteins and may therefore be applied on a multiuser instrument or in a high-throughput environment.

Fig.1

Chromatogram and mass spectra of purified KCNJ12. (A) Total ion current (TIC) elution profile for KCNJ12 purified in the detergent Cymal-6, eluted with methanol. Dotted line: methanol concentration. Solid line: TIC (arbitrary units). The gray rectangles denote regions of the chromatogram (regions 1–4) referred to in the text. (B) m/z spectrum of region 4; combination of detergent signals (Cymal 6) and a protein-like spectrum. Inset: m/z spectrum of regions 2 + 3, identified as Cymal-6. (C) m/z spectrum of region 4, enlarged segment of the spectrum shown in (B), showing a typical protein ion series. (D) Mass spectrum of KCNJ12 after deconvolution.

Fig.2

Optimization of gradient and column lengths. (A) KCNJ12 elution profile: 50-mm column, 12 min gradient, detergent, Cymal-6. (B) SCD elution profile: 50-mm column, 16 min gradient/isocratic, detergent, DDM. (C) SCD elution profile: 150-mm column 22 min gradient/isocratic, detergent, DDM. (D) SCD elution profile: 200-mm column, 40 min gradient/isocratic, detergent, DM + lipids.

Fig.3

Detection of stripped protein and detergent adducts. (A) TIC elution profile of ABCB10. (B) m/z spectrum of region 2 of the chromatogram. The free detergent ions dominate the spectrum, dwarfing the protein ionization signal. (C) m/z spectrum of region 2, magnified segment of figure (B). (D) Deconvoluted mass spectrum of region 2 of the chromatogram, showing the accurate intact mass, as well as adducts with 1, 2, and 3 detergent molecules.

Fig.4

Detection of protein and lipids. (A) TIC elution profile of SCD purified in the presence of a lipid/detergent mix. (B) Chromatogram region 4 of the chromatogram: ionization of lipid species (regions 2 and 3 contain detergents and contaminants; not shown). (C) m/z spectrum of region 1, illustrating the separation of the protein from the lipids and detergents. Note that protein spectra are rarely as clean as this; compare Fig. 3B. (D) Deconvoluted mass spectrum.

Fig.5

Intact mass analysis of a mixed population of proteins. A mixture of the membrane protein KCNJ12, the soluble TEV protease used to cleave the fusion tag, and the membrane protein HVCN1, which occurred as a contaminant from an earlier analysis on the same LC column.