Scalable Multiplex Co-fractionation/mass Spectrometry Platform for Accelerated Protein Interactome Discovery

Overview

Journal Nat Commun

Specialty Biology

Date 2022 Jul 13

PMID 35831314

Authors

Pierre C Havugimana

Raghuveera Kumar Goel

Sadhna Phanse

Ahmed Youssef

Dzmitry Padhorny

Sergei Kotelnikov

Dima Kozakov

Andrew Emili

Affiliations

Soon will be listed here.

Abstract

Co-fractionation/mass spectrometry (CF/MS) enables the mapping of endogenous macromolecular networks on a proteome scale, but current methods are experimentally laborious, resource intensive and afford lesser quantitative accuracy. Here, we present a technically efficient, cost-effective and reproducible multiplex CF/MS (mCF/MS) platform for measuring and comparing, simultaneously, multi-protein assemblies across different experimental samples at a rate that is up to an order of magnitude faster than previous approaches. We apply mCF/MS to map the protein interaction landscape of non-transformed mammary epithelia versus breast cancer cells in parallel, revealing large-scale differences in protein-protein interactions and the relative abundance of associated macromolecules connected with cancer-related pathways and altered cellular processes. The integration of multiplexing capability within an optimized workflow renders mCF/MS as a powerful tool for systematically exploring physical interaction networks in a comparative manner.

Citing Articles

SWI/SNF-type complexes-transcription factor interplay: a key regulatory interaction.

Maassen A, Steciuk J, Wilga M, Szurmak J, Garbicz D, Sarnowska E Cell Mol Biol Lett. 2025; 30(1):30.

PMID: 40065228 PMC: 11895388. DOI: 10.1186/s11658-025-00704-y.

Comprehensive dataset of interactors for the entire PARP family using TurboID proximity labeling.

Zheng J, Deng Y, Fang C, Xiong S, Zhu X, Wu W Sci Data. 2025; 12(1):405.

PMID: 40057523 PMC: 11890743. DOI: 10.1038/s41597-025-04722-5.

SEC-MX: an approach to systematically study the interplay between protein assembly states and phosphorylation.

Doron-Mandel E, Bokor B, Ma Y, Street L, Tang L, Abdou A Nat Commun. 2025; 16(1):1176.

PMID: 39885126 PMC: 11782603. DOI: 10.1038/s41467-025-56303-0.

Decoding the functional impact of the cancer genome through protein-protein interactions.

Fu H, Mo X, Ivanov A Nat Rev Cancer. 2025; 25(3):189-208.

PMID: 39810024 DOI: 10.1038/s41568-024-00784-6.

Recent Advances in Mass Spectrometry-Based Protein Interactome Studies.

Wu S, Zhang S, Liu C, Fernie A, Yan S Mol Cell Proteomics. 2024; 24(1):100887.

PMID: 39608603 PMC: 11745815. DOI: 10.1016/j.mcpro.2024.100887.

References

Habib J, OShaughnessy J . The hedgehog pathway in triple-negative breast cancer. Cancer Med. 2016; 5(10):2989-3006. PMC: 5083752. DOI: 10.1002/cam4.833. View

Siddharth S, Parida S, Muniraj N, Hercules S, Lim D, Nagalingam A . Concomitant activation of GLI1 and Notch1 contributes to racial disparity of human triple negative breast cancer progression. Elife. 2021; 10. PMC: 8664295. DOI: 10.7554/eLife.70729. View

Nepusz T, Yu H, Paccanaro A . Detecting overlapping protein complexes in protein-protein interaction networks. Nat Methods. 2012; 9(5):471-2. PMC: 3543700. DOI: 10.1038/nmeth.1938. View

Drew K, Wallingford J, Marcotte E . hu.MAP 2.0: integration of over 15,000 proteomic experiments builds a global compendium of human multiprotein assemblies. Mol Syst Biol. 2021; 17(5):e10016. PMC: 8111494. DOI: 10.15252/msb.202010016. View

Mallam A, Sae-Lee W, Schaub J, Tu F, Battenhouse A, Jang Y . Systematic Discovery of Endogenous Human Ribonucleoprotein Complexes. Cell Rep. 2019; 29(5):1351-1368.e5. PMC: 6873818. DOI: 10.1016/j.celrep.2019.09.060. View

Heusel M, Bludau I, Rosenberger G, Hafen R, Frank M, Banaei-Esfahani A . Complex-centric proteome profiling by SEC-SWATH-MS. Mol Syst Biol. 2019; 15(1):e8438. PMC: 6346213. DOI: 10.15252/msb.20188438. View

Kerrien S, Aranda B, Breuza L, Bridge A, Broackes-Carter F, Chen C . The IntAct molecular interaction database in 2012. Nucleic Acids Res. 2011; 40(Database issue):D841-6. PMC: 3245075. DOI: 10.1093/nar/gkr1088. View

Deutsch E, Bandeira N, Sharma V, Perez-Riverol Y, Carver J, Kundu D . The ProteomeXchange consortium in 2020: enabling 'big data' approaches in proteomics. Nucleic Acids Res. 2019; 48(D1):D1145-D1152. PMC: 7145525. DOI: 10.1093/nar/gkz984. View

Hwang S, Kim C, Yang S, Kim E, Hart T, Marcotte E . HumanNet v2: human gene networks for disease research. Nucleic Acids Res. 2018; 47(D1):D573-D580. PMC: 6323914. DOI: 10.1093/nar/gky1126. View

10.

Giurgiu M, Reinhard J, Brauner B, Dunger-Kaltenbach I, Fobo G, Frishman G . CORUM: the comprehensive resource of mammalian protein complexes-2019. Nucleic Acids Res. 2018; 47(D1):D559-D563. PMC: 6323970. DOI: 10.1093/nar/gky973. View

11.

Itzhak D, Tyanova S, Cox J, Borner G . Global, quantitative and dynamic mapping of protein subcellular localization. Elife. 2016; 5. PMC: 4959882. DOI: 10.7554/eLife.16950. View

12.

Geraldes P, Hiraoka-Yamamoto J, Matsumoto M, Clermont A, Leitges M, Marette A . Activation of PKC-delta and SHP-1 by hyperglycemia causes vascular cell apoptosis and diabetic retinopathy. Nat Med. 2009; 15(11):1298-306. PMC: 3290906. DOI: 10.1038/nm.2052. View

13.

Havugimana P, Hart G, Nepusz T, Yang H, Turinsky A, Li Z . A census of human soluble protein complexes. Cell. 2012; 150(5):1068-81. PMC: 3477804. DOI: 10.1016/j.cell.2012.08.011. View

14.

Huttlin E, Bruckner R, Navarrete-Perea J, Cannon J, Baltier K, Gebreab F . Dual proteome-scale networks reveal cell-specific remodeling of the human interactome. Cell. 2021; 184(11):3022-3040.e28. PMC: 8165030. DOI: 10.1016/j.cell.2021.04.011. View

15.

McWhite C, Papoulas O, Drew K, Cox R, June V, Dong O . A Pan-plant Protein Complex Map Reveals Deep Conservation and Novel Assemblies. Cell. 2020; 181(2):460-474.e14. PMC: 7297045. DOI: 10.1016/j.cell.2020.02.049. View

16.

Porter K, Padhorny D, Desta I, Ignatov M, Beglov D, Kotelnikov S . Template-based modeling by ClusPro in CASP13 and the potential for using co-evolutionary information in docking. Proteins. 2019; 87(12):1241-1248. PMC: 8200321. DOI: 10.1002/prot.25808. View

17.

Leutert M, Rodriguez-Mias R, Fukuda N, Villen J . R2-P2 rapid-robotic phosphoproteomics enables multidimensional cell signaling studies. Mol Syst Biol. 2019; 15(12):e9021. PMC: 6920700. DOI: 10.15252/msb.20199021. View

18.

Liu X, Zhao B, Sun L, Bhuripanyo K, Wang Y, Bi Y . Orthogonal ubiquitin transfer identifies ubiquitination substrates under differential control by the two ubiquitin activating enzymes. Nat Commun. 2017; 8:14286. PMC: 5290280. DOI: 10.1038/ncomms14286. View

19.

Tai Y, Chu P, Lai I, Wang M, Tseng H, Guan J . An EGFR/Src-dependent β4 integrin/FAK complex contributes to malignancy of breast cancer. Sci Rep. 2015; 5:16408. PMC: 4637903. DOI: 10.1038/srep16408. View

20.

Steinegger M, Soding J . MMseqs2 enables sensitive protein sequence searching for the analysis of massive data sets. Nat Biotechnol. 2017; 35(11):1026-1028. DOI: 10.1038/nbt.3988. View