Top-down characterization of endogenous protein complexes with native proteomics

Abstract

Protein complexes exhibit great diversity in protein membership, post-translational modifications and noncovalent cofactors, enabling them to function as the actuators of many important biological processes. The exposition of these molecular features using current methods lacks either throughput or molecular specificity, ultimately limiting the use of protein complexes as direct analytical targets in a wide range of applications. Here, we apply native proteomics, enabled by a multistage tandem MS approach, to characterize 125 intact endogenous complexes and 217 distinct proteoforms derived from mouse heart and human cancer cell lines in discovery mode. The native conditions preserved soluble protein-protein interactions, high-stoichiometry noncovalent cofactors, covalent modifications to cysteines, and, remarkably, superoxide ligands bound to the metal cofactor of superoxide dismutase 2. These data enable precise compositional analysis of protein complexes as they exist in the cell and demonstrate a new approach that uses MS as a bridge to structural biology.

Figure 1. Native proteomics implemented in untargeted mode

Samples are produced via native cell lysis or tissue homogenization methods and are fractionated with either native GELFrEE or ion exchange chromatography. The resulting fractions are analyzed using a three-tiered approach to native top-down mass spectrometry (nTDMS) which maintains many of the fragile noncovalent interactions prior to controlled fragmentation of protein complexes into subunits and their backbone fragment ions. Finally, the nTDMS data are analyzed with ProSight PC 4.0 and a Search Engine for Multi-Proteoform Complexes to identify and characterize the complexes and proteoforms formed endogenously within the cell or tissue samples.

Figure 2. A summary of results from performing native proteomics

(a) The molecular weight distribution of proteoforms and their assemblies characterized in this study. (b) The distribution of biological processes of the identified gene products. (c) The observed distribution of stoichiometries of identified homomeric multi-proteoform complexes (MPCs).

Figure 3. Direct readout of metal cofactors by native proteomics

(a) An intact mass spectrum of murine aconitase showing the distribution of Zn²⁺ binding in addition to a mass shift consistent to within 0.3 Da with the 2+ charge state of its 3Fe4S cluster. (b) The crystal structure of porcine aconitase (98% sequence identity, PDB: 5acn) with the tandem MS-localized Zn²⁺ binding site indicated in purple. (c) The intact mass spectrum of the enolase dimer bound to zero and one copy (but not two) of its di-Mg²⁺ cofactors. (d) A crystal structure of the enolase dimer indicating the di-Mg²⁺ binding pocket that is only occupied on one monomer. (e) One of two iron-binding pockets of human serotransferrin (72% sequence identity) is located near two probable sites of N-linked glycosylation. (f) The normalized relative abundance of species with zero, one, and two bound Fe³⁺ cofactors indicates a 52% increase in the efficiency of binding two irons with a non-fucosylated glycan. See Supplementary Figure 9 for more details.

Figure 4. Detection of labile superoxide bound to mitochondrial superoxide dismutase

(a–c) The intact mass spectrum of the tetrameric human mitochondrial superoxide dismutase bound to between zero and four copies of its superoxide substrate in addition to four copies of its Mn²⁺ cofactor. Slightly increasing the amount of ion activation in the electrospray source region, from 5 V (a) to 20 V (b) to 30 V (c), progressively ejected more of the weakly bound substrates while retaining the intact tetramer and all four of the Mn²⁺ cofactors. (d) A comparison of the observed fourth-highest mass peak from a (in blue) with theoretical isotopic distributions for the intact tetramer, its four Mn²⁺ cofactors, and three copies of either O₂, H₂O₂, and O₂⁻. The arrow above each peak indicates the centroid, with the dotted lines corresponding to shifts of ±10 ppm; thus, the results strongly indicate the presence of superoxide and not either of the products. The difference in mass between O₂⁻ and O₂ is due to the difference in formal charge, changing the number of protons on the ions required to produce a net charge state of 20+.