Martin Humphries
LAB

Integrin proteomics



Mass spectrometry–based proteomics


Proteomics is the study of the entire complement of proteins in a given system, such as a cell or an organism. The term “proteome” is a combination of the words “protein” and “genome”; proteomics is analogous to genomics, which is the study of an entire set of genes. Compared to genomics, proteomics is further complicated by protein modifications such as phosphorylation, ubiquitination and glycosylation, and the fact that proteins can be expressed as alternative splice forms or in distinct cell types at different times.



A number of strategies have commonly been employed to study the proteomes of particular cell types, tissues or disease states. These include two-dimensional–gel electrophoresis, antibody chips and, with increasing use recently, mass spectrometry (MS).


Peptide sequence and mass spectra

MS has a broad range of chemical and biological applications. In terms of its usefulness for proteomics, MS can be described as an analytical method that measures the mass-to-charge ratio of peptides generated from enzymatic digestion of proteins. From this mass-to-charge ratio data, the accurate measurement of the mass of peptides can be calculated using the charge-state of the peptide ions detected by MS. When two or more mass spectrometers are combined, a set-up commonly termed tandem-MS or MS/MS, sequence information regarding the parent peptide can be obtained. Sequence information results from the selection and fragmentation of peptides in one of the mass spectrometers, and the subsequent measurement of the mass-to-charge ratios of the peptide fragments in the other mass spectrometer. MS is often directly coupled to reverse phase liquid chromatography (LC), permitting the separation and analysis of complex mixtures of peptides, and hence proteins, as they are eluted from the LC column. A set-up such as this is called LC-MS/MS.


A number of recent technical and methodological advances have made a significant impact on the usefulness and usability of MS-based proteomics by biologists. These advances include the construction of high-resolution and sensitive mass spectrometers, the availability of complete genomic databases for a number of species and the generation of workflows for the quantification of peptides and proteins within complex mixtures. With these advances in MS, it is now not uncommon to see research articles containing quantitative data for hundreds or even thousands of proteins at a time. Thus, the global, non-candidate–based analysis of signalling events using MS is emerging as a powerful approach for investigating cellular responses to stimuli or genetic modifications.

Proteomics data as a heatmap

Proteomics of cell adhesion


Adhesion to the extracellular matrix is essential for a multicellular existence. Adhesion receptors on the cell surface transduce signals that control cell morphology, movement, survival and differentiation in various developmental, homeostatic and disease processes. Integrin adhesion complexes, like other receptor-associated signalling complexes, have been refractory to proteomic analysis. This is because integrin complexes consist of a complicated mixture of transmembrane, cytoskeletal and signalling molecules, which readily fall apart when they are isolated biochemically, especially when solubilised by detergent. The complexes are also difficult to isolate because they require ligand engagement to induce macromolecular assembly. Therefore, the global analysis of adhesion complex components has been restricted to the collation and interrogation of data derived from published experimental studies. Most MS-based studies have concentrated on the analysis of post-translational modifications and binding partners of certain relatively soluble cytoplasmic adhesion proteins. An overview of some recent proteomic studies of adhesion-related biology can be found at the Cell Migration Consortium.



We have developed a methodology for the affinity isolation and mass spectrometric analysis of integrin-associated complexes. Importantly, the technique (1) isolates ligand-engaged integrin adhesion complexes, (2) stabilises the protein complexes by chemical cross-linking and (3) fractionates the cell to permit the enrichment of insoluble integrin-associated cytoskeletal components.

Different integrin proteomes

We have used this approach to compare the proteomes of two receptor-ligand pairs: α4β1–vascular cell adhesion molecule–1 and α5β1–fibronectin. Numerous well-characterised components of integrin adhesion complexes were detected, along with many putative novel integrin-associated proteins, which demonstrated the effectiveness of this strategy. Moreover, this approach led to the identification of regulator of chromosome condensation–2 (RCC2) as a novel regulatory component of integrin-GTPase signalling pathways, orchestrating adhesion complex assembly, cell spreading and directional cell migration. (See our Integrin proteomics gallery for a selection of images.)


These findings represent the first experimentally defined integrin proteomes. Furthermore, the development of this workflow now allows the molecular composition of various adhesion complexes — and indeed other transmembrane receptor–ligand protein complexes — to be measured directly and presents an entry point for systems-level analyses of adhesion signalling.


Related galleries



Further reading


  • A Byron, JD Humphries, MD Bass, D Knight and MJ Humphries (2011) Proteomic analysis of integrin adhesion complexes. Sci. Signal. 4: pt2. Full text | PubMed entry


  • A Byron*, MR Morgan* and MJ Humphries (2010) Adhesion signalling complexes. Curr. Biol. 20: R1063-7. Full text | PubMed entry


  • JD Humphries*, A Byron*, MD Bass, SE Craig, JW Pinney, D Knight and MJ Humphries (2009) Proteomic analysis of integrin-associated complexes identifies RCC2 as a dual regulator of Rac1 and Arf6. Sci. Signal. 2: ra51. Full text | PubMed entry



  • *These authors contributed equally to this work.