Periodic Reporting for period 1 - SystemDirectMS (A Native Mass Spectrometry Systemic View of Cellular Structural Biology)
Okres sprawozdawczy: 2023-05-01 do 2025-10-31
A central premise of the SystemDirectMS project is that protein structure—and thereby function—is dynamically shaped by the cellular environment. Splice variants, post-translational modifications, proteolytic events, and interactions with cofactors or therapeutics are all governed by context-dependent factors such as cell type, developmental stage, stress, disease, and aging. These influences give rise to a diverse array of protein forms that coexist within cells, forming a complex and dynamic structural landscape. Traditional structural biology approaches, which typically rely on extensive biochemical purification, often fail to preserve this native diversity and may distort biological context. Motivated by our recent discovery that native mass spectrometry (MS) can be applied directly to crude cellular lysates while maintaining structural integrity, this project aims to establish a novel methodology—termed SystemDirectMS—that enables the systemic and near-native structural analysis of protein complexes. This report summarizes the progress made toward this goal, focusing on the development of the underlying experimental systems and methodologies.
Aim 1: Development of a High-Throughput Platform for Probing Protein–Protein Interactions (PPIs)
To facilitate high-throughput analysis of protein–protein interactions (PPIs) under near-native conditions, we have invested in assembling a versatile and fully automated robotic system. After evaluating available technologies, we selected the Beckman Coulter Biomek i7 Hybrid Automated Workstation, known for its flexibility and capacity to handle complex workflows. This system supports the integration with various devices, including centrifuges, ultrasonicator and cell imaging multi-mode reader enhancing its capability to process both prokaryotic and eukaryotic samples.
The assembly of this integrated system is currently underway, with protocol development for full automation in progress. Once operational, this setup will enable standardized, scalable workflows for studying PPIs directly from complex biological samples.
Aim 2: Enable direct-MS of protein-protein interactions (PPIs) in human and other eukaryotic cells
The objective of Aim 2 is to facilitate direct MS investigations of PPIs within human and other eukaryotic cellular environments. To achieve this, we employed a multicistronic vector strategy that allows the translation of multiple genes from a single transcript, ensuring coordinated expression of protein complexes.
We engineered two sets of multicistronic plasmids based on the pCX backbone:
2A Peptide-Based Vectors: These vectors incorporate various self-cleaving 2A peptides (e.g. F2A, P2A, E2A, T2A) to facilitate the co-expression of proteins. Our evaluations revealed that while F2A and P2A sometimes resulted in covalently linked fusion proteins, T2A consistently yielded efficient cleavage, producing separate protein products.
IRES-Based Vectors: Utilizing internal ribosome entry sites (IRES), these vectors enable initiation of translation from internal mRNA regions. However, we observed that IRES elements generally led to lower expression levels compared to 2A peptides, aligning with previous findings that downstream genes in IRES constructs often exhibit reduced expression.
To validate these systems, we co-expressed wild-type and mutant forms of the homodimeric protein SOD1, associated with familial ALS. The T2A-based system demonstrated superior performance, enabling efficient co-expression and proper processing of SOD1 variants.
Building on these findings, we initiated a project investigating the response of SOD1 to specific drug treatments using the T2A-based expression system. Preliminary results are promising, and we are preparing a manuscript detailing these findings for publication. Additionally, we are drafting a methodological paper describing our co-expression system to
Aim 3: Establish a method for whole-organ direct-MS analysis
The objective of Aim 3 is to develop a robust whole-organ direct MS approach to investigate PPIs within the complex environment of intact tissues. To achieve this, we selected intrinsically disordered proteins (IDPs) as our model system, given their ubiquitous expression across human tissues and remarkable resistance to thermal aggregation. This property allows for their selective enrichment, facilitating the capture of tissue-specific IDP landscapes and the characterization of their diverse proteoform ensembles. We have meticulously developed and optimized a generic sample preparation protocol tailored for whole-organ direct-MS analysis of IDPs. This involved systematic evaluation and refinement of each procedural step, including the composition of protease inhibitor cocktails, ionic strength and pH of homogenization buffers, centrifugation speeds and durations, and boiling times for thermal enrichment. Additionally, we fine-tuned the mass spectrometry data acquisition parameters using the Direct Mass Technology (DMT) mode on the Q Exactive UHMR mass spectrometer. DMT enhances resolution and dynamic range by enabling simultaneous measurement of both mass-to-charge and charge for individual. Applying this optimized workflow, we analyzed full datasets from brain, kidney, and liver tissues obtained from three age groups of mice—young (6 weeks), mature (6 months), and old (18 months)—with ten mice per group (five males and five females).
To facilitate high-throughput analysis of protein–protein interactions (PPIs) under near-native conditions, we have invested in assembling a versatile and fully automated robotic system. After evaluating available technologies, we selected the Beckman Coulter Biomek i7 Hybrid Automated Workstation, known for its flexibility and capacity to handle complex workflows. This system supports the integration with various devices, including centrifuges, ultrasonicator and cell imaging multi-mode reader enhancing its capability to process both prokaryotic and eukaryotic samples.
The assembly of this integrated system is currently underway, with protocol development for full automation in progress. Once operational, this setup will enable standardized, scalable workflows for studying PPIs directly from complex biological samples.
Aim 2: Enable direct-MS of protein-protein interactions (PPIs) in human and other eukaryotic cells
The objective of Aim 2 is to facilitate direct MS investigations of PPIs within human and other eukaryotic cellular environments. To achieve this, we employed a multicistronic vector strategy that allows the translation of multiple genes from a single transcript, ensuring coordinated expression of protein complexes.
We engineered two sets of multicistronic plasmids based on the pCX backbone:
2A Peptide-Based Vectors: These vectors incorporate various self-cleaving 2A peptides (e.g. F2A, P2A, E2A, T2A) to facilitate the co-expression of proteins. Our evaluations revealed that while F2A and P2A sometimes resulted in covalently linked fusion proteins, T2A consistently yielded efficient cleavage, producing separate protein products.
IRES-Based Vectors: Utilizing internal ribosome entry sites (IRES), these vectors enable initiation of translation from internal mRNA regions. However, we observed that IRES elements generally led to lower expression levels compared to 2A peptides, aligning with previous findings that downstream genes in IRES constructs often exhibit reduced expression.
To validate these systems, we co-expressed wild-type and mutant forms of the homodimeric protein SOD1, associated with familial ALS. The T2A-based system demonstrated superior performance, enabling efficient co-expression and proper processing of SOD1 variants.
Building on these findings, we initiated a project investigating the response of SOD1 to specific drug treatments using the T2A-based expression system. Preliminary results are promising, and we are preparing a manuscript detailing these findings for publication. Additionally, we are drafting a methodological paper describing our co-expression system to
Aim 3: Establish a method for whole-organ direct-MS analysis
The objective of Aim 3 is to develop a robust whole-organ direct MS approach to investigate PPIs within the complex environment of intact tissues. To achieve this, we selected intrinsically disordered proteins (IDPs) as our model system, given their ubiquitous expression across human tissues and remarkable resistance to thermal aggregation. This property allows for their selective enrichment, facilitating the capture of tissue-specific IDP landscapes and the characterization of their diverse proteoform ensembles. We have meticulously developed and optimized a generic sample preparation protocol tailored for whole-organ direct-MS analysis of IDPs. This involved systematic evaluation and refinement of each procedural step, including the composition of protease inhibitor cocktails, ionic strength and pH of homogenization buffers, centrifugation speeds and durations, and boiling times for thermal enrichment. Additionally, we fine-tuned the mass spectrometry data acquisition parameters using the Direct Mass Technology (DMT) mode on the Q Exactive UHMR mass spectrometer. DMT enhances resolution and dynamic range by enabling simultaneous measurement of both mass-to-charge and charge for individual. Applying this optimized workflow, we analyzed full datasets from brain, kidney, and liver tissues obtained from three age groups of mice—young (6 weeks), mature (6 months), and old (18 months)—with ten mice per group (five males and five females).
A significant advancement of our project was the comprehensive characterization of the blood-circulating 20S proteasome, challenging the traditional view of proteasomes as solely intracellular complexes. Our study revealed that proteasomes are not only present in the extracellular space but also exhibit unique molecular characteristics distinct from their intracellular counterparts. Through advanced MS techniques, we identified specific PTMs, such as cysteinylation and glutathionylation, which may confer specialized functions to these extracellular proteasomes. These insights were made possible by the sensitivity and precision of advanced mass spectrometry techniques. Our findings highlight the limitations of traditional cell-line-based models and emphasize the importance of studying protein complexes within physiologically relevant extracellular contexts.
In addition, using murine models, we showed that conditions such as ovarian cancer, chronic oxidative stress, and aging result in altered levels of circulating 20S proteasomes—changes that parallel observations in humans. This establishes mice as a relevant and practical model system for investigating extracellular proteasomes in both normal and disease states, laying the groundwork for addressing critical unanswered questions in the field.
In addition, using murine models, we showed that conditions such as ovarian cancer, chronic oxidative stress, and aging result in altered levels of circulating 20S proteasomes—changes that parallel observations in humans. This establishes mice as a relevant and practical model system for investigating extracellular proteasomes in both normal and disease states, laying the groundwork for addressing critical unanswered questions in the field.