Toward the bilayer proteome, electrospray ionization-mass spectrometry of large, intact transmembrane proteins

Abstract

Genes encoding membrane proteins comprise a substantial proportion of genomes sequenced to date, but ability to perform structural studies on this portion of the proteome is limited. Electrospray ionization-MS (ESI-MS) of an intact protein generates a profile defining the native covalent state of the gene product and its heterogeneity. Here we apply ESI-MS technology with accuracy exceeding 0.01% to a hydrophobic membrane protein with 12-transmembrane alpha-helices, the full-length lactose permease from Escherichia coli. Furthermore, ESI-MS is used to titrate reactive thiols with N-ethylmaleimide. Treatment of the native protein solubilized in detergent micelles reveals only two reactive thiols, and both are protected by a substrate analog.

Figure 1

Chromatographic purification of lactose permease by size-exclusion chromatography in aqueous organic solvent. Purified lactose permease (200 μg protein) was separated from nearly all noncovalently associated lipid and detergent to perform effective ESI-MS. The permease/detergent micelle was disrupted by chloroform/methanol phase separation with the protein precipitating at the interface. Aggregated protein was recovered by centrifugation and dissolved in 90% aqueous formic acid (50 μl), but was still accompanied by residual detergent and lipid. Size-exclusion chromatography in chloroform/methanol/1% aqueous formic acid (4:4:1, vol/vol) was used to separate the permease from these contaminants by using a silica column (G2000SW, 7.8 × 300 mm, TosoHaas) at 40°C and 0.5 ml/min. (A) Elution profile measured at 280 nm (mAU). Permease eluted at 600 sec as a single peak. The eluent is directed to the mass spectrometer, scanning the m/z range 600 to 2,400 every 6 sec. (B) Total ion chromatogram showing the signal detected for each scan versus time. Both chromatograms show separation from residual UV absorbing and ion-generating material.

Figure 2

ESI mass spectrum of lac permease collected upon elution of the 600-sec UV peak. ESI generates multiply charged ions, each with different mass/charge (m/z) ratios through the addition of variable numbers of protons. In the case of lactose permease, it was necessary to generate ions with at least 20 protons such that their m/z was in the scanning range of the mass spectrometer. Spectra from several scans across the peak were added to generate the spectrum shown. Typically ions carrying from 20 to 50 positive charges were detected, appearing to fall into at least three distributions, suggestive of different tertiary structures in the gas phase. The measured mass is calculated from the m/z of these ions after assigning the charge states. A computer-generated reconstruction of the zero-charged protein (Hypermass, PE Sciex, Toronto) is shown (Inset).

Figure 3

NEM modification and substrate protection by d-galactopyranosyl β-d-thio-galactopyranoside (TDG). ESI-MS is used to quantitate the addition of NEM groups to Cys residues of the solubilized protein. Each NEM addition increases the mass of the protein by 125 Da. (A) Unmodified permease. (B) Ten-minute labeling with NEM on ice. (C) Ten-minute labeling with NEM at room temperature. (D) same as C after preincubation with a saturating concentration (50 mM) of the substrate analog TDG. Of eight thiol groups in lactose permease only two are modified under the conditions used. Preincubation with substrate reveals differential protection of these Cys residues against alkylation with NEM (see text).