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23rd Human Proteome Organization World Congress

Decoding the protein dance

Professor Dr

Keynote speaker

Keynote lecture

Prof.

Prof

Large scale cross-linking to discover, evaluate, and validate the human cell structural proteome and interactome

Structural Proteomics

Recent advances in structural biology have expanded our ability to create experimental structures for proteins and complexes, but many proteins remain refractory to these approaches or have not yet been analysed. Machine-learning based structure modellers have enabled access to highly accurate protein structure predictions for entire proteomes. These modellers are trained on experimental structures available in the Protein Data Bank (PDB), which constitute a relatively small subset of proteins, with many structures solved using non-native conditions or sequences. Therefore, a critical question is whether predicted (and PDB) structures reflect the bona fide structures and complexes formed by proteins in their native environment. We investigate this question using a large-scale cross-linking mass spectrometry (XL-MS) resource generated for the human cell.To generate a high density and high depth XL-MS dataset for human HEK293 cells, we utilised a multipronged approach. Briefly, we cross-linked four subcellular fractions (nucleus, endoplasmic reticulum, mitochondria, cytosol) using three different cross-linkers with orthogonal chemistries (DHSO, DSSO, DMTMM). Then, we enriched cross-linked peptides using offline size-exclusion chromatography followed by further fractionation by high pH reverse phase HPLC. Mass spectrometry was performed on concatenated fractions using hybrid-MS2-MS3, or MS/MS with EThcD or HCD, fragmentation strategies. Cross-linked peptides were identified using XlinkX 2.3 or pLink2, using stringent search parameters and post-hoc filtering to control the false discovery rate to &lt;2% at the unique residue pair (URP) or PPI levels.We identified 28,910 URPs representing 4,084 unique proteins and 2,110 unique putative PPIs. Subcellular fractionation before cross-linking resulted in significantly improved proteome coverage, whilst orthogonal reactivities (D/E-D/E, K-K and K-D/E) improved the density of cross-linking per protein, especially in intra-protein links. We demonstrate that our resource of URPs confirm and rediscover existing experimental structures, capturing proteoforms and complexes within their approximate subcellular niches and range of conformations. Remarkably, our intra-molecular URPs also largely corroborate thousands of new structures predicted by next-generation modeller AlphaFold2, including those involving proteins (or regions of proteins) without existing resolution, and those lacking any structural precedent. Furthermore, our inter-protein crosslinks recapture the topology of well-described complexes and PPIs, whilst supporting or discovering poorly characterised PPIs. Finally, the inter-protein crosslinks also help localise PPI interfaces, and we use this information to assess quaternary protein structures modelled in AlphaFold-Multimer.

Oral presentation

Advancing stability proteomics for functional characterization of intact proteoforms

IntroductionHigh-throughput top-down proteomics has significantly advanced the characterization of intact proteoforms in complex biological systems. However, linking proteoform structure with function remains challenging. Stability proteomics methods assess protein functionality by analyzing stability under a denaturation gradient, but bottom-up proteomics techniques limit their ability to characterize intact proteoforms with modifications. To address this, we developed top-down stability proteomics methods, including Top-Down Thermal Proteome Profiling (TD-TPP) and Methionine Oxidative Footprinting in Intact Proteins (MOFIP), using thermal and chemical denaturant gradients to probe protein stability. These methods were employed to study the effect of protein modification on proteoform stability in complex samples, aiming to bridge the gap between structure and function.MethodsWe developed TD-TPP and MOFIP to probe proteoform stability in E. coli, mouse brain, and HeLa lysates. For TD-TPP, aliquots were heated (37-56 °C) and centrifuged to remove aggregates. For MOFIP, lysates were treated with H2O2 and quenched with L-methionine. They were then incubated with increasing concentrations of guanidinium hydrochloride (GdmCl) followed by H2O2 incubation and quenching. Label-free quantitative TD proteomics was performed using our one-dimensional ultra-high-pressure liquid chromatography top-down mass spectrometry (1D UPLC-TDMS) platform for deep characterization of intact proteoforms.ResultsOur top-down platform enabled the characterization and profiling of hundreds of proteoforms in complex biological samples. Using TD-TPP, we analyzed the standard proteins β lactoglobulin A&amp;B. βLG B was slightly destabilized as a result of an amino acid substitution (D80G) to a more nonpolar residue on an exposed turn as well as the exchange to a more polar residue (V134A) internal beta-sheet. TD-TPP was further applied to HeLa lysate and determined the average melting point of the intact proteoforms was 59.3 °C for cytosolic proteoforms, consistent with previous reports.The MOFIP was applied to evaluate solvent-accessible Met residues based on their secondary structure. Interestingly, 26 proteoforms were partially oxidized while natively folded indicating multiple populations may co-exist in cell lysate. Furthermore, proteoform stability was examined and one protein, the Ribose import binding protein (RbsB), contains 1 solvent-accessible methionine residue on domain 1 and 3 solvent-inaccessible methionine residues on domain 2. We found that the three residues on domain 2 were simultaneously oxidized upon denaturation demonstrating the potential of MOFIP to obtain domain-specific information.ConclusionsOur novel top-down stability proteomics platforms have shown great potential for deep proteoform characterization and stability evaluation at the proteoform level.

Maintenance of Mitochondrial Complexes Ensures Bioenergetic Function in Differentiated Cells

The assembly sequence of mitochondrial complexes has been intensively investigated in proliferating cells, primarily focusing on de novo assembly. However, these studies provide limited insights into the dynamics of protein complexes in differentiated cells and tissues. In postmitotic tissues, the state of protein complexes is likely maintained through a balance of biosynthesis and degradation. This raises an important question: are protein complexes continuously assembled de novo, or do remodeling and repair mechanisms play a crucial role in maintaining mitochondrial function?To address this, we utilized complexome profiling, a technique combining blue native electrophoresis with quantitative mass spectrometry, able to identify scarce sub-complexes, assembly intermediates, and complex remodeling events. Furthermore, we integrated complexome profiling with pulse stable isotope labeling of amino acids in cell culture (Pulsed-SILAC) to investigate the turnover and half-life of individual proteins within protein complexes in differentiated post-mitotic C2C12-myotubes.Our findings provide a comprehensive dataset on the dynamics of stable mitochondrial protein complexes. We determined that the complete replacement of all complexes within the oxidative phosphorylation system (OXPHOS) requires approximately one month in mouse myotubes. Notably, we observed higher turnover rates among the interface subunits of dimeric and oligomeric ATP synthase and supercomplexes, indicating more dynamic interactions between complexes rather than within them.We further explored the maintenance mechanisms of complex I. Newly synthesized proteins are incorporated as spare parts, replacing modules within complex I. Applying this method to patients with mitochondrial disorders and mouse models revealed specific service factors involved in the repair process. In particular, we identified complex I subunits with higher turnover rates in the electron transport modules, suggesting that quality control and repair mechanisms within assembled complexes are essential for maintaining full bioenergetic function in postmitotic tissues.

Conference room 1-2

Session

Structural proteomics and protein structure prediction

Session
Structural proteomics and protein structure prediction

Appointment

Chairs

Program

SessionStructural proteomics and protein structure prediction

Appointment

Chairs

Program

Session
Structural proteomics and protein structure prediction