We present a methodology using
in vivo crosslinking combined with HPLC-MS for the global analysis of endogenous protein complexes by protein correlation profiling. Formaldehyde crosslinked protein complexes were extracted with high yield using denaturing buffers that maintained complex solubility during chromatographic separation. We show this efficiently detects both integral membrane and membrane-associated protein complexes,in addition to soluble complexes, allowing identification and analysis of complexes not accessible in native extracts. We compare the protein complexes detected by HPLC-MS protein correlation profiling in both native and formaldehyde crosslinked U2OS cell extracts. These proteome-wide data sets of both
in vivo crosslinked and native protein complexes from U2OS cells are freely available via a searchable online database (
www.peptracker.com/epd). Raw data are also available via ProteomeXchange (identifier PXD003754).Proteins rarely work as monomers to carry out all the biological processes needed for cells to function. An estimate of the total number of protein-protein interactions within the human proteome, based on currently available data sets, is ∼650,000 (
1). This is likely an underestimate, given that many proteins form either transient, or weak interactions within intact cells that may not yet have been detected. This suggests that the majority of human proteins can participate in protein complex formation, at least under some conditions. This includes the many well-studied soluble protein complexes in the cytoplasm, exemplified by the proteasome, ribosomes and cytoskeletal network. It also includes many membrane-associated complexes, for example receptor tyrosine kinase signaling complexes, integrin networks and transmembrane transporters (
2). To characterize the many roles of multi-protein complexes in biological regulatory mechanisms, it is important to have convenient methods for the rapid and efficient analysis of their composition and dynamics (
3). Ideally, such methods should be applicable to system-wide studies and allow the analysis of endogenous proteins, rather than exclusively use tagged and/or over-expressed baits.The methods available for the proteome-wide analysis of protein interactions have developed swiftly over the last ten years. This field is dominated by affinity-enrichment based approaches, using either tagged constructs, or antibodies specific for endogenous proteins. Another approach is
in vivo proximity labeling, based, for example, on the exogenous expression of a protein of interest, fused either to a promiscuous biotin-ligase (BioID) (
4), or to a peroxidase enzyme that activates biotin-phenol (APEX) (
5). While these data sets have proved very useful, there are some downsides. For example, a large expense in terms of both time and money to generate the thousands of individual “bait” proteins required for global interaction analyses. In addition, each of these affinity enrichments will be performed in only one type of buffer system, which is unlikely to be compatible with the maintenance of all protein-protein interactions. Another dimension to the analytical problem is that many proteins are expressed as different sized isoforms and/or in different post-translationally modified forms, resulting in formation of multiple, related, but functionally distinct complexes, with different combinations of interaction partners (
6). Using affinity-enrichment/pull-down methods alone makes it difficult to resolve such mixtures of different forms of related protein complexes, complicating a detailed understanding of biological response mechanisms.An alternative strategy involves protein correlation profiling-MS,
i.e. correlating similarities in the fractionation profiles of proteins detected by mass spectrometry, assuming that proteins in a common complex will cofractionate. This approach was previously applied to the analysis of subcellular organelle proteomes (
7,
8), and subsequently extended to analyze soluble protein complexes. Thus, recent studies have shown that chromatography-based separation of soluble protein complexes, combined with fraction collection and high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS)
1, facilitates analysis of many hundreds of soluble complexes from a single experiment (
6,
9–
11). A limitation of all of these studies, however, is that the native extraction conditions used to preserve protein-protein interactions isolates predominantly stable, soluble complexes. For example, many proteins that are integral to membranes are not recovered (
12). Similarly, soluble protein complexes that have weakly bound protein subunits can dissociate upon cell lysis and the inevitable dilution associated with extraction. Thus, the potential value of this approach for the system-wide analysis of protein complexes is limited without a covalent tether to hold protein-protein interactions intact during extraction and subsequent chromatographic separation (
13).Covalent protein crosslinking has been used extensively to stabilize protein complexes, cultured cells and tissues for subsequent analysis, either by microscopy, nucleotide sequencing or mass spectrometry. The agents employed to crosslink proteins to each other include various chemical groups able to react with the side-chains of either amino acids, nucleotides, carbohydrates or lipids (
14). These crosslinking agents vary in the efficiency with which they perfuse into unbroken cells/tissues and the speed of their reaction when in proximity to a suitable chemical group. One of the most widely used crosslinkers is formaldehyde, which can reversibly form a covalent crosslink to stabilize both protein-protein and protein-nucleotide interactions (
15–
21). One of the main benefits of using formaldehyde is that because of its small size, it readily permeates intact cells and tissues. Another benefit of using formaldehyde is the easy reversal of the crosslinks by heating and subsequent compatibility with mass spectrometry-based proteome analysis.Here, we describe a mass spectrometry-based proteomic approach for the efficient global analysis of protein complexes, including membrane proteins, using
in vivo protein crosslinking combined with denaturing extraction. Using high-resolution, size-exclusion chromatography (SEC) to separate crosslinked complexes under denaturing conditions and MS analysis of fractionated proteins, we could identify membrane bound and membrane associated complexes not accessible in native extracts. We present a detailed comparison of the sets of protein complexes that can be identified using protein correlation profiling MS analysis in conjunction with both formaldehyde crosslinked and native extracts from U2OS cells. We provide access to the entire proteome-wide data sets of both
in vivo crosslinked and native U2OScell protein complexes via a searchable online database (
http://www.peptracker.com/epd/).
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