Analysis Of Human Membrane Protein Complexes By Using Formaldehyde Cross-linking And Protein Correlation Profiling

Membrane proteins are essential to many biological processes,and their functions are often dependent on multimolecular complexes.Studying the intact membrane-protein complexes assembly will be helpful to understand their biological functions.However,the proportion of membrane protein complexes in the PDB database is only 0.6%,and most of the membrane protein structures are in the form of monomers or oligomers,and a large number of unknown membrane protein complexes are yet to be discovered and identified.But,the purification and analysis of large-scale membrane protein complexes remains a challenge.We applied an approach that combined cross-linking mass spectrometry(XL-MS)and protein correlation profiling(PCP)to analysis the endogenous membrane protein complexes in HeLa cell line.Protein correlation profiling with liquid chromatography fractionation and mass spectrometry quantification,has been approved to be an efficient method to identify global protein complexes.Meanwhile,cross-linking mass spectrometry can effectively capture transient and weak interactions and provide interface information between interacting proteins,which has become an important means to study the structure of protein complexes.Firstly,we optimized the method to determine the optimal concentration of formaldehyde and the optimal membrane protein enrichment strategy,and then investigated the method with known protein complexes.The elution spectra of the selected known protein complexes showed that most of the proteins in these complexes were elution in the same fraction after HPLC separation,demonstrating the effectiveness and reliability of the method.And the reliability and reproducibility of the data were verified by comparing the results of multiple biological replications.Subsequently,we used this method to analyze the membrane protein-related complexes of human Hela cell line.After machine learning and clustering analysis,we finally obtained 312 membrane protein complexes.The membrane protein complex network contains 2,333 proteins with 31,095(Protein-protein interactions,PPIs),of which approximately 12%PPIs have been reported in public databases,and the rest PPIs have been reported for the first time in this study.Functional enrichment analysis revealed that these protein complexes are associated with core biological processes such as translation,intracellular transport,metabolism and catabolism.Analysis of the 312 protein complexes associated with human disease and organelle interactions revealed that 73 of them were associated with more than 160 human disease,particularly alzheimer's disease,cancer and endocarditis,and almost half of them were associated with these diseases.In addition,939 proteins with major subcellular localization participated in the communication between endoplasmic reticulum and mitochondria,plasma membrane and mitochondria,endoplasmic reticulum and vesicles and other different organelles.They involve both indirect communication through the cytoplasm and direct interaction by membrane contact site on different organelles.In addition to identifying the complex protein composition,we also analyzed the possible interaction sites between the proteins.Due to the diversity of formaldehyde cross-linking sites and the uncertainty of its mass spectrometric fragmentation mode,there is a lack of software to analyze formaldehyde cross-linked peptides.In this study,we indirectly inferred the information of cross-linked peptides by using the difference of chromatographic behavior between cross-linked peptides and non-cross-linked peptides.Finally,we screened 16,165 possible cross-linked peptides in 270 compounds,including cross-linked peptides reported by other researchers using other cross-linking agents,providing further support for the reliability of our predicted cross-linked peptide data.It also provides further evidence for the prediction of protein complexes and the possible sites of interaction between proteins.In combination with the c cross-linking mass spectrometry and protein correlation profiling,a large number of new human membrane protein complexes were found in this study,and the related properties of the complexes were analyzed.Meanwhile,the interaction peptides between proteins in the complexes were predicted.The results of this study provide a basis for the further study of the functions of these membrane protein complexes,which will be beneficial to the study of diseases,and provide a new perspective for the interaction of organelles.