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 with 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 mass spectrometry 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. The data enable precise compositional analysis of protein complexes as they exist in the cell and demonstrate a new approach that uses mass spectrometry as a bridge to structural biology.

FLASHDeconv: Ultrafast, high-quality feature deconvolution for top-down proteomics

“…Let mi be the maximum number of intense charges that continuously increment by i. For example, if charges 3, 5, 6, 7, 9, 10 are intense, m1 = 3 (charges 5, 6, 7) while m2 = 4 (charges 3,5,7,9). A peak group is filtered out if m1 < max (3, m2,m3,...,m7).…”

“…Top-down (TD) proteomics has gained a lot of momentum for in-depth protein characterization and protein species analytics [1][2][3][4][5][6][7] . In contrast to bottom-up (BU) proteomics, where proteins are enzymatically digested and actually peptides are analyzed, the TD approach allows for the analysis of intact proteoforms (protein species arising from the same gene product via splice variants, genomic variation, post-translational modifications, degradation, etc.)…”

Computational Approaches in Proteomics

“…In TD data analysis, Proteome Discoverer is commonly used to process raw data files, and through its ProSight tools, as well as through MascotTD, identification and characterization of intact proteins can be achieved (46,47). A database search using ProSight against specific databases (UniProt, SwissProt and RefSEQ) leads to top-down data interpretation and also identifies PTMs within a protein sample (48,49). Furthermore, deconvolution is crucial for data interpretation, and it is commonly achieved through Xtract, MS-Deconv and YADA (within ProLuCID), among other tools (50).…”

Native mass spectrometry can effectively predict PROTAC efficacy

“…With the use of nano-electrospray ionisation (nESI) [14], protein complexes can retain their native topology and stoichiometry during transfer from solution into the gas phase, making protein-protein and protein-ligand interactions amenable to MS analysis [15]. Key advantages of this "native MS" approach [16] include the label-free measurement of protein complexes and its capability to report on multiple binding stoichiometries present in dynamic protein mixtures, including molecular species populated to a low extent [15,[17][18][19][20]. For these reasons, we anticipated that nMS would be particularly applicable for the characterisation of PROTAC systems.…”