Profiling of integral membrane proteins and their post translational modifications using high-resolution mass spectrometry

Abstract

Integral membrane proteins pose challenges to traditional proteomics approaches due to unique physicochemical properties including hydrophobic transmembrane domains that limit solubility in aqueous solvents. A well resolved intact protein molecular mass profile defines a protein's native covalent state including post-translational modifications, and is thus a vital measurement toward full structure determination. Both soluble loop regions and transmembrane regions potentially contain post-translational modifications that must be characterized if the covalent primary structure of a membrane protein is to be defined. This goal has been achieved using electrospray-ionization mass spectrometry (ESI-MS) with low-resolution mass analyzers for intact protein profiling, and high-resolution instruments for top-down experiments, toward complete covalent primary structure information. In top-down, the intact protein profile is supplemented by gas-phase fragmentation of the intact protein, including its transmembrane regions, using collisionally activated and/or electron-capture dissociation (CAD/ECD) to yield sequence-dependent high-resolution MS information. Dedicated liquid chromatography systems with aqueous/organic solvent mixtures were developed allowing us to demonstrate that polytopic integral membrane proteins are amenable to ESI-MS analysis, including top-down measurements. Covalent post-translational modifications are localized regardless of their position in transmembrane domains. Top-down measurements provide a more detail oriented high-resolution description of post-transcriptional and post-translational diversity for enhanced understanding beyond genomic translation.

Fig. 1

Zero-charge intact protein molecular mass profile (MMP) of bovine major intrinsic protein (MIP). The data was collected using a low-resolution triple quad mass spectrometer and transformed to obtain the zero-charge molecular mass profile shown. The molecular heterogeneity of different protein species is clearly visible with phosphorylation (80) at 28303.8, cysteinylation (119) at 28,343.3 and also C-terminal processing by removal of Ala and Leu/Ile residues. Mass calculated from the MIP gene sequence was 28,223.1 which reflects a delta of 2.1 or 0.0075%, coincident within experimental error. Bovine MIP is one of very few eukaryotic proteins whose mature primary sequence is exactly as predicted from the genomic translation without post-translation processing.

Fig. 2

Schematic workflow for integral protein mass spectrometry. The technique employs a flow splitter between HPLC and mass spectrometer to facilitate collection of fractions for later use in downstream experiments for protein identification and PTM characterization on high-resolution Fourier transform mass spectrometers.

Fig. 3

Top-down mass spectrometry of bacteriorhodopsin holoprotein. (A) A typical charge state distribution of bacteriorhodopsin after purification by size exclusion chromatography (SEC) in chloroform/methanol/aqueous formic acid. Paired signals are generated as a result of partial hydrolysis of the retinal chromophore in acidic conditions. (B) Zero change molecular mass profile obtained after deconvolution of selected ion monitoring experiment (m/z 100 width; 40 transients averaged) on the 11-charge ion shows both forms differing by the mass of retinal (266 Da) as well as mild oxidation (+16 Da) and formylation (+28 Da). (C) CAD of the holoprotein is shown with ion isolation of the 11-charge precursor (2460, inset top-left) and its CAD tandem mass spectrum. Unit resolution was achieved on all product ions by operating the instrument at 750,000 resolution at 400 m/z. Note that the ion isolation experimental window mass kept wide enough that there was some contamination of the desired molecular ion with adducts of higher mass, reflective of the ever-present pressure to maximize signal strength for better sequence coverage in the dissociation experiment.

Fig. 3

Top-down mass spectrometry of bacteriorhodopsin holoprotein. (A) A typical charge state distribution of bacteriorhodopsin after purification by size exclusion chromatography (SEC) in chloroform/methanol/aqueous formic acid. Paired signals are generated as a result of partial hydrolysis of the retinal chromophore in acidic conditions. (B) Zero change molecular mass profile obtained after deconvolution of selected ion monitoring experiment (m/z 100 width; 40 transients averaged) on the 11-charge ion shows both forms differing by the mass of retinal (266 Da) as well as mild oxidation (+16 Da) and formylation (+28 Da). (C) CAD of the holoprotein is shown with ion isolation of the 11-charge precursor (2460, inset top-left) and its CAD tandem mass spectrum. Unit resolution was achieved on all product ions by operating the instrument at 750,000 resolution at 400 m/z. Note that the ion isolation experimental window mass kept wide enough that there was some contamination of the desired molecular ion with adducts of higher mass, reflective of the ever-present pressure to maximize signal strength for better sequence coverage in the dissociation experiment.

Fig. 4

Ion assignments for the bacteriorhodopsin holoprotein. Matched peak lists from CAD experiments on 6 different precursor ions were pooled, and the composite list was matched to the structure to give the ion assignments shown. 67 b- and 55 y-ions were matched, giving coverage of 79 of 247 peptide bonds (32%) and a P Score of 3.9e−150. The experiment confirms proteolytic processing that removes N-terminal residues 1–13 and the C-terminal Asp262 residue, as well as cyclization of the N-terminus to pyroglutamate and the retinal chromophore between residues 225 – 248, at Lys 229. Transmembrane domains are boxed. Note that while the experiment completely defines the native covalent state of mature bacteriorhodopsin, the regions where bonds were not cleaved rely upon genomic translation for sequence assignment.