Cross-linking, Immunoprecipitation and Proteomic Analysis to Identify Interacting Proteins in Cultured Cells

Extracellular expression is essential for the function of secreted and cell surface proteins. Proper intracellular trafficking depends on protein interactions in multiple subcellular compartments. Co-immunoprecipitation and the yeast two-hybrid system are commonly used to investigate protein-protein interactions. These methods, however, depend on high-affinity protein interactions. In many glycoproteins, glycans are important for protein intracellular trafficking and extracellular expression. If glycoprotein interactions are transient and relatively weak, it may be challenging to use co-immunoprecipitation or the two-hybrid system to identify glycoprotein-binding partners. To circumvent this problem, protein cross-linking can be applied first to immobilize the transient and/or low-affinity protein interactions. Here we describe a protocol of protein cross-linking, co-immunoprecipitation, and proteomic analysis, which was used to identify endoplasmic reticulum (ER) chaperones critical for the folding and ER exiting of N-glycosylated serine proteases in human embryonic kidney (HEK) 293 cells. This approach can be used to identify other protein interactions in a variety of cells.

. These results suggest that N-glycans on corin, particularly those in the protease domain, may interact with other ER proteins that are critical for corin intracellular trafficking . Identification of such ER proteins should help to understand the cellular mechanism in regulating corin expression and function.
Co-immunoprecipitation and the yeast two-hybrid system are commonly used to analyze proteinprotein binding and complex formation (Berggard et al., 2007;Kaboord and Perr, 2008). These methods are suitable mostly for studying stable and/or high-affinity protein-protein interactions. In many cases, however, protein interactions in specific subcellular compartments are transient and unstable. In glycoprotein synthesis, for example, transient N-glycan-protein interactions are essential for glycoprotein folding and subsequent ER exiting (Ellgaard and Frickel, 2003;Lamriben et al., 2016). The traditional methods such as protein co-immunoprecipitation and the two-hybrid system may not be suitable for studying such N-glycan-protein interactions. To circumvent this problem, protein crosslinking can be applied to immobilize the transient and/or weak protein interactions before coimmunoprecipitation proceeds.
In a recent study, we designed a protocol of protein cross-linking, co-immunoprecipitation, and proteomic analysis to examine the role of N-glycans in corin intracellular trafficking. We expressed corin wild-type (WT) and a mutant lacking the N-glycosylation site in the protease domain (N1022Q) in separate HEK293 cells. The cells were treated with dithiobis succinimidyl propionate (DSP), a cell membrane permeable cross-linker, which has an amine-reactive N-hydroxysuccinimide (NHS) ester at each end of a cleavable spacer (Mattson et al., 1993). NHS esters react with primary amines in the side chain of lysine residues and the N-termini of proteins, thereby forming stable amide bonds connecting co-localized proteins (Mattson et al., 1993). Proteins in HEK293 cells cross-linked to corin WT and the mutant were isolated by immunoprecipitation. After breaking the disulfide bond in the spacer of DSP under reducing conditions, proteins were separated by SDS-PAGE and analyzed by in-gel digestion and liquid chromatography-mass spectrum (LC-MS). By comparing proteins that were differentially bound to corin WT and the mutant, we identified calnexin as a key ER chaperone that mediates the N-glycandependent folding and ER exiting of corin and other N-glycosylated serine proteases such as enteropeptidase and prothrombin .

Materials and Reagents
Procedure Notes: 1. pcDNA 3.1/V5-His-based plasmids expressing human corin WT and the N1022Q mutant were described previously . The corin proteins encoded by these plasmids contain a C-terminal V5 tag that is recognized by an anti-V5 antibody.

The corin-expressing plasmids were transfected into HEK293 cells (authenticated by STR DNA
profiling, no mycoplasma contamination) using Fugene reagents. The cells were cultured in DMEM with 10% FBS and 400 μg/ml of G418 at 37 °C in a humidified incubator with 5% CO2 to select stable corin-expressing cell clones. The experimental procedures were described previously . 5 www.bio-protocol.org/e3258         2. Elute the peptides from the trapping and reversed-phase columns at a flow rate of 0.3 μl/min with a binary gradient starting at 95% A (0.1% formic acid) and 5% B (acetonitrile/0.1% formic acid) for 5 min followed by a linear increase from 2-40% B in 85 min ( Figure 5). The column is washed by ramping to 80% B and holding for 5 min prior to re-equilibration of the column at 2% B for 15 min.

Figure 5. Illustration of a binary gradient in HPLC analysis
Note: An example of peptide elution profile is shown in Figure 6. Mascot (dat files) search results into the program Scaffold. Utilize Scaffold to perform additional database searches using X!Tandem with the same parameters as described above. Perform False Discover Rate analysis by searching the data against a reversed UniProtKB database and filter identifications based on a peptide level FDR of 0.1% and protein level FDR of 1%.
Only consider proteins identified by two peptides, one of which is unique.

Data analysis
The spectral counts in the LC-MS proteomic analysis (Step E) were examined. Proteins with high spectral counts were possible bait-interacting candidates. All proteins with spectral counts ≥ 10 were included for further analysis. To identify the proteins that differentially interacted with corin WT and the mutant, a ratio of ≥ 2-fold difference in spectral counts between WT and the mutant was used as a selection criterion. The selected candidates were analyzed for their subcellular expression patterns by searching the human UniProtKB database (www.uniprot.org/uniprot), which includes information on protein subcellular locations. The proteins that are predominantly expressed in the targeted subcellular location, i.e., ER in our study, were selected and tested in additional biochemical and cellular experiments .
Note: The criterion for protein spectral counts and the ratio between the control and the targeted protein may be modified depending on experimental settings.