Tandem mass spectrometry of integral membrane proteins for top-down proteomics

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Abstract

Transmembrane domains of integral membrane proteins present a formidable analytical challenge due to their general lack of polarity; recovery is enhanced by maintaining their association with polar-loop regions. Multi-dimensional separations of intact integral membrane proteins coupled with high-resolution electrospray-ionization mass spectrometers enable “top-down” proteomics, with coverage of bilayer domains.

Introduction

Biological systems abound with lipid/protein bilayer membranes that play critical roles in life. Membrane proteins fall into two general classes: the integral or intrinsic proteins that form part of the membrane and must be extracted by its solubilization; and, the peripheral or extrinsic proteins that are more loosely associated with the membrane and can be displaced without its destruction. The integral proteins can be divided into two general structural classes: those exhibiting the transmembrane β-barrel porin-type fold; and, those with one or more transmembrane alpha helices. Transmembrane helix domains can be quite accurately predicted and analysis of assigned genomic open reading frames (ORFs) suggests that proteins with this motif constitute around 30% of the proteome [1]. When one considers the transmembrane porins, short transmembrane unassigned ORFs and peripheral membrane proteins, it is clear that membrane-associated proteins make up as much as 50% of the proteome. The critical role of membrane proteins in cellular processes is further emphasized by the estimate that 70% of drug targets fall in this category [2]. Consequently, it is essential that proteomics technologies address the bilayer proteome [3]. Unfortunately, integral membrane proteins tend to have awkward physico-chemical properties that result in their under-representation in general proteomics experiments. The origin of this discrepancy is probably the tendency of the hydrophobic transmembrane domain to aggregate/precipitate when removed from the bilayer, exacerbated by the sporadic presence of cysteine residues with reactive thiols. Consequently, sample-preparation protocols must be specially developed.

The bulk of the work in proteomics has fallen to mass spectrometry (MS) and the significance of the discovery of soft ionization techniques for large biological macromolecules was recognized with the award of the Chemistry Nobel Prize to Fenn and Tanaka in 2002. Since proteins can be matched to genomic sequence data by short, unique internal sequences, early proteomics protocols have broken up intact proteins into sets of peptides for MS. Data from one or more peptides is then matched to the genome to identify the parent gene. Integral membrane proteins are generally amenable to this type of analysis because loop regions yield soluble peptides convenient for MS [4]. Intact mass [5] or “top-down” [6] proteomics aims to include intact protein-mass measurements along with primary structure information to describe the entire protein, including transmembrane domains. The realization of this goal for integral membrane proteins will allow us to investigate post-translational modification within the bilayer domain.

Section snippets

The separation challenge in proteomics

Typical genomes contain thousands of ORFs such that the living organism contains several thousand different proteins of varying size, sequence and chemical properties. The first large-scale separation technology applied in proteomics was the two-dimensional gel (2D gel) that provided a useful visual representation of the abundant components of a complex mixture [7]. The first dimension of isoelectric focusing is limited to non-ionic or neutral zwitterionic detergents for solubilizing proteins

Intact mass measurement of integral membrane proteins

For intact mass and “top-down” proteomics, it is necessary to obtain intact proteins in a reasonably pure state in solvents compatible with electrospray ionization (ESI)-MS. In the case of integral membrane proteins, a suite of technologies has been developed for this purpose, allowing mass measurements of proteins with 1–15 transmembrane domains and masses to over 100 kDa [20], [21]. Liquid chromatography (LC) is used to purify the proteins in aqueous/organic solvent mixtures and protein-mass

2D chromatography

The goal of providing liquid samples for ESI-MS makes LC technologies preferable to gel-based technologies because recovery of intact proteins from gels is challenging and covalent side-reactions can modify the protein. 2D chromatography is therefore being evaluated for dissection of the membrane proteome.

Since we have demonstrated the ability to interface our LC systems to ESI-MS for successful analysis of integral membrane protein complexes, it is logical to reserve these for the second

Top-down proteomics – technology

Once the integral membrane proteins are solubilized in the aqueous organic solvent mixtures used for RPC and SEC, ESI is routine. The recorded mass spectrum yields protein-mass measurements of resolution and accuracy comparable to similar measurements on water-soluble proteins [5], so integral membrane proteins are amenable to Fourier-transform-ion cyclotron resonance MS (FT-ICR-MS) with the associated benefits to resolution, accuracy and so on [22]. Collision-activated dissociation (CAD) can

Ion sources for tandem MS of hydrophobic peptides

While ESI has covered our needs to date, alternatives are welcome in order to expand versatility. Photoionization has recently been applied to a collection of hydrophobic peptides, providing a potential alternative to ESI-MS. Interestingly, c- and z-type ions were present in the mass spectrum, suggesting some ECD/ETD-type chemistry occurring in the ionization source [39]. Extension of photoionization to proteins may be limited by the observation that singly charged ions dominate the mass

Summary. An integrated solution

Integral membrane proteins can be analyzed by ESI-tandem MS, once they are purified in aqueous/organic solvent mixtures compatible with their solubility. The latest combination of linear ion-trap technology with Fourier-transform MS represents a particularly exciting development in this respect. Sample-preparation and separation technologies for reproducible, quantitative simplification of the bilayer proteome present the biggest challenge for an integrated solution.

Acknowledgements

Richard LeDuc, Yong-Bin Kim, Andrew Forbes and the rest of the software development team in Neil Kelleher’s Laboratory are thanked.

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