Identification of Extracellular Segments by Mass Spectrometry Improves Topology Prediction of Transmembrane Proteins

Abstract

Transmembrane proteins play crucial role in signaling, ion transport, nutrient uptake, as well as in maintaining the dynamic equilibrium between the internal and external environment of cells. Despite their important biological functions and abundance, less than 2% of all determined structures are transmembrane proteins. Given the persisting technical difficulties associated with high resolution structure determination of transmembrane proteins, additional methods, including computational and experimental techniques remain vital in promoting our understanding of their topologies, 3D structures, functions and interactions. Here we report a method for the high-throughput determination of extracellular segments of transmembrane proteins based on the identification of surface labeled and biotin captured peptide fragments by LC/MS/MS. We show that reliable identification of extracellular protein segments increases the accuracy and reliability of existing topology prediction algorithms. Using the experimental topology data as constraints, our improved prediction tool provides accurate and reliable topology models for hundreds of human transmembrane proteins.

A split ubiquitin system to reveal topology and released peptides of membrane proteins

“…Understanding of the topology of membrane proteins is the first step towards their structural elucidations and functional investigations, as well as developments of potential new drugs, as more than half of known drugs target membrane proteins [ 12 ]. Cell surface protein could be labeled by sulfo-NH-SS-Biotin and analyzed based on mass data to detect domains outside of the plasma membrane [ 13 , 14 ]. A cysteine-reactive molecule, maleimide polyethylene glycol (mPEG), is applied to determine the topology of a transmembrane protein by labeling the introduced cysteine in the cytosol [ 15 ].…”

Partial proteolysis improves the identification of the extracellular segments of transmembrane proteins by surface biotinylation

“…First, we used Sulfo-NHS-SS-biotin for modifying the side chains of lysine residues on the cell surface of red blood cells, HL60 and K562 cells. These experiments resulted in 730 extra-cytosolic labelled positions for almost 200 TM proteins 43 . Second, we targeted the carboxyl groups with a two-step reaction, first activating them with EDC and Sulfo-NHS, then labelling the activated carboxyl groups with biotinyl cystamine.…”

“…Despite these methods being more cost effective and quick, they also have some disadvantages. First of all, not every protein can be labelled on the cell surface, due to the lack or inaccessibility of carboxyl and primary amino groups in the extracellular domains, limiting the number of TMPs that can be characterized 43,44 . Additionally, we can only get topology data for those TMPs that are the most abundant and/or have a big extracellular domain with many targetable residues on the cell surface in these experiments.…”

CIRFESS: An interactive resource for querying the set of theoretically detectable peptides for cell surface and extracellular enrichment proteomic studies

“…As prediction strategies alone are insufficient to define the set of proteins localized to the cell surface and extracellular space, experimentation is required. To aid in the selection of proteomic strategies that are likely to produce the desired coverage of the cell surface proteome, the SP, TM, and SPC analyses described above were integrated with in silico analyses designed to predict which proteins would generate tryptic peptides likely to be detectable by electrospray MS, and of those, which are expected to be captured by application of commonly used biorthogonal enrichment strategies targeting N -glycans and lysines 23–25,27,49–54 . We also considered cysteines as they are enriched in surface proteins compared to nonsurface proteins 40 and numerous affinity reagents are available for targeting cysteines 55 , although this is not yet a widely described approach for cell surface proteins.…”