Biofunctionalized Dissolvable Hydrogel Microbeads Enable Efficient Characterization of Native Protein Complexes

Overview

Journal Nat Commun

Specialty Biology

Date 2024 Oct 4

PMID 39366952

Authors

Xinyang Shao

Meng Tian

Junlong Yin

Haifeng Duan

Ye Tian

Hui Wang

Changsheng Xia

Ziwei Wang

Yanxi Zhu

Yifan Wang

Lingxiao Chaihu

Minjie Tan

Hongwei Wang

Yanyi Huang

Jianbin Wang

Guanbo Wang

Affiliations

Soon will be listed here.

Abstract

The characterization of protein complex is vital for unraveling biological mechanisms in various life processes. Despite advancements in biophysical tools, the capture of non-covalent complexes and deciphering of their biochemical composition continue to present challenges for low-input samples. Here we introduce SNAP-MS, a Stationary-phase-dissolvable Native Affinity Purification and Mass Spectrometric characterization strategy. It allows for highly efficient purification and characterization from inputs at the pico-mole level. SNAP-MS replaces traditional elution with matrix dissolving during the recovery of captured targets, enabling the use of high-affinity bait-target pairs and eliminates interstitial voids. The purified intact protein complexes are compatible with native MS, which provides structural information including stoichiometry, topology, and distribution of proteoforms, size variants and interaction states. An algorithm utilizes the bait as a charge remover and mass corrector significantly enhances the accuracy of analyzing heterogeneously glycosylated complexes. With a sample-to-data time as brief as 2 hours, SNAP-MS demonstrates considerable versatility in characterizing native complexes from biological samples, including blood samples.

References

Leney A, Heck A . Native Mass Spectrometry: What is in the Name?. J Am Soc Mass Spectrom. 2020; 28(1):5-13. DOI: 10.1021/jasms.8b05378. View

Heck A . Native mass spectrometry: a bridge between interactomics and structural biology. Nat Methods. 2008; 5(11):927-33. DOI: 10.1038/nmeth.1265. View

Tong W, Wang G . How can native mass spectrometry contribute to characterization of biomacromolecular higher-order structure and interactions?. Methods. 2018; 144:3-13. DOI: 10.1016/j.ymeth.2018.04.025. View

Parker M . Protein structure from x-ray diffraction. J Biol Phys. 2013; 29(4):341-62. PMC: 3456177. DOI: 10.1023/A:1027310719146. View

Wang H . Cryo-electron microscopy for structural biology: current status and future perspectives. Sci China Life Sci. 2015; 58(8):750-6. DOI: 10.1007/s11427-015-4851-2. View

Busch F, VanAernum Z, Lai S, Gopalan V, Wysocki V . Analysis of Tagged Proteins Using Tandem Affinity-Buffer Exchange Chromatography Online with Native Mass Spectrometry. Biochemistry. 2021; 60(24):1876-1884. PMC: 9080447. DOI: 10.1021/acs.biochem.1c00138. View

Lai S, Tamara S, Heck A . Single-particle mass analysis of intact ribosomes by mass photometry and Orbitrap-based charge detection mass spectrometry. iScience. 2021; 24(11):103211. PMC: 8529500. DOI: 10.1016/j.isci.2021.103211. View

Li H, Nguyen H, Ogorzalek Loo R, Campuzano I, Loo J . An integrated native mass spectrometry and top-down proteomics method that connects sequence to structure and function of macromolecular complexes. Nat Chem. 2018; 10(2):139-148. PMC: 5784781. DOI: 10.1038/nchem.2908. View

Wang G, Chaihu L, Tian M, Shao X, Dai R, de Jong R . Releasing Nonperipheral Subunits from Protein Complexes in the Gas Phase. Anal Chem. 2020; 92(24):15799-15805. DOI: 10.1021/acs.analchem.0c02845. View

10.

Vimer S, Ben-Nissan G, Morgenstern D, Kumar-Deshmukh F, Polkinghorn C, Quintyn R . Comparative Structural Analysis of 20S Proteasome Ortholog Protein Complexes by Native Mass Spectrometry. ACS Cent Sci. 2020; 6(4):573-588. PMC: 7181328. DOI: 10.1021/acscentsci.0c00080. View

11.

Zhou M, Lantz C, Brown K, Ge Y, Pasa-Tolic L, Loo J . Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry. Chem Sci. 2021; 11(48):12918-12936. PMC: 8163214. DOI: 10.1039/d0sc04392c. View

12.

Campuzano I, Nshanian M, Spahr C, Lantz C, Netirojjanakul C, Li H . High Mass Analysis with a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer: From Inorganic Salt Clusters to Antibody Conjugates and Beyond. J Am Soc Mass Spectrom. 2020; 31(5):1155-1162. PMC: 7261417. DOI: 10.1021/jasms.0c00030. View

13.

VanAernum Z, Busch F, Jones B, Jia M, Chen Z, Boyken S . Rapid online buffer exchange for screening of proteins, protein complexes and cell lysates by native mass spectrometry. Nat Protoc. 2020; 15(3):1132-1157. PMC: 7203678. DOI: 10.1038/s41596-019-0281-0. View

14.

Young C, Britton Z, Robinson A . Recombinant protein expression and purification: a comprehensive review of affinity tags and microbial applications. Biotechnol J. 2012; 7(5):620-34. DOI: 10.1002/biot.201100155. View

15.

Pedro L, Quinn R . Native Mass Spectrometry in Fragment-Based Drug Discovery. Molecules. 2016; 21(8). PMC: 6274484. DOI: 10.3390/molecules21080984. View

16.

Zhang Y, Liu L, Daneshfar R, Kitova E, Li C, Jia F . Protein-glycosphingolipid interactions revealed using catch-and-release mass spectrometry. Anal Chem. 2012; 84(18):7618-21. DOI: 10.1021/ac3023857. View

17.

Leitner A, Dorn G, Allain F . Combining Mass Spectrometry (MS) and Nuclear Magnetic Resonance (NMR) Spectroscopy for Integrative Structural Biology of Protein-RNA Complexes. Cold Spring Harb Perspect Biol. 2019; 11(7). PMC: 6601463. DOI: 10.1101/cshperspect.a032359. View

18.

Mehmood S, Allison T, Robinson C . Mass spectrometry of protein complexes: from origins to applications. Annu Rev Phys Chem. 2015; 66:453-74. DOI: 10.1146/annurev-physchem-040214-121732. View

19.

Kuzmanov U, Emili A . Protein-protein interaction networks: probing disease mechanisms using model systems. Genome Med. 2013; 5(4):37. PMC: 3706760. DOI: 10.1186/gm441. View

20.

Kaltashov I, Pawlowski J, Yang W, Muneeruddin K, Yao H, Bobst C . LC/MS at the whole protein level: Studies of biomolecular structure and interactions using native LC/MS and cross-path reactive chromatography (XP-RC) MS. Methods. 2018; 144:14-26. DOI: 10.1016/j.ymeth.2018.04.019. View