Increasing Proteome Coverage Through a Reduction in Analyte Complexity in Single-Cell Equivalent Samples.

The advancement of sophisticated instrumentation in mass spectrometry has catalyzed an in-depth exploration of complex proteomes. This exploration necessitates a nuanced balance in experimental design, particularly between quantitative precision and the enumeration of analytes detected. In bottom-up proteomics, a key challenge is that oversampling of abundant proteins can adversely affect the identification of a diverse array of unique proteins. This issue is especially pronounced in samples with limited analytes, such as small tissue biopsies or single-cell samples. Methods such as depletion and fractionation are suboptimal to reduce oversampling in single cell samples, and other improvements on LC and mass spectrometry technologies and methods have been developed to address the trade-off between precision and enumeration. We demonstrate that by using a monosubstrate protease for proteomic analysis of single-cell equivalent digest samples, an improvement in quantitative accuracy can be achieved, while maintaining high proteome coverage established by trypsin. This improvement is particularly vital for the field of single-cell proteomics, where single-cell samples with limited number of protein copies, especially in the context of low-abundance proteins, can benefit from considering analyte complexity. Considerations about analyte complexity, alongside chromatographic complexity, integration with data acquisition methods, and other factors such as those involving enzyme kinetics, will be crucial in the design of future single-cell workflows.

Selective targeting of Plasmodium falciparum Hsp90 disrupts the 26S proteasome.

The molecular chaperone heat shock protein 90 (Hsp90) has an essential but largely undefined role in maintaining proteostasis in Plasmodium falciparum, the most lethal malaria parasite. Herein, we identify BX-2819 and XL888 as potent P. falciparum (Pf)Hsp90 inhibitors. Derivatization of XL888's scaffold led to the development of Tropane 1, as a PfHsp90-selective binder with nanomolar affinity. Hsp90 inhibitors exhibit anti-Plasmodium activity against the liver, asexual blood, and early gametocyte life stages. Thermal proteome profiling was implemented to assess PfHsp90-dependent proteome stability, and the proteasome-the main site of cellular protein recycling-was enriched among proteins with perturbed stability upon PfHsp90 inhibition. Subsequent biochemical and cellular studies suggest that PfHsp90 directly promotes proteasome hydrolysis by chaperoning the active 26S complex. These findings expand our knowledge of the PfHsp90-dependent proteome and protein quality control mechanisms in these pathogenic parasites, as well as further characterize this chaperone as a potential antimalarial drug target.

Impact of Germline Depletion of Bonus on Chromatin State in Drosophila Ovaries.

Gene expression is controlled via complex regulatory mechanisms involving transcription factors, chromatin modifications, and chromatin regulatory factors. Histone modifications, such as H3K27me3, H3K9ac, and H3K27ac, play an important role in controlling chromatin accessibility and transcriptional output. In vertebrates, the Transcriptional Intermediary Factor 1 (TIF1) family of proteins play essential roles in transcription, cell differentiation, DNA repair, and mitosis. Our study focused on Bonus, the sole member of the TIF1 family in Drosophila, to investigate its role in organizing epigenetic modifications. Our findings demonstrated that depleting Bonus in ovaries leads to a mild reduction in the H3K27me3 level over transposon regions and alters the distribution of active H3K9ac marks on specific protein-coding genes. Additionally, through mass spectrometry analysis, we identified novel interacting partners of Bonus in ovaries, such as PolQ, providing a comprehensive understanding of the associated molecular pathways. Furthermore, our research revealed Bonus's interactions with the Polycomb Repressive Complex 2 and its co-purification with select histone acetyltransferases, shedding light on the underlying mechanisms behind these changes in chromatin modifications.

Protein Folding Stability Profiling of Colorectal Cancer Chemoresistance Identifies Functionally Relevant Biomarkers.

Reported here is the application of three protein folding stability profiling techniques (including the stability of proteins from rates of oxidation, thermal protein profiling, and limited proteolysis approaches) to identify differentially stabilized proteins in six patient-derived colorectal cancer (CRC) cell lines with different oxaliplatin sensitivities and eight CRC patient-derived xenografts (PDXs) derived from two of the patient derived cell lines with different oxaliplatin sensitivities. Compared to conventional protein expression level analyses, which were also performed here, the stability profiling techniques identified both unique and novel proteins and cellular components that differentiated the sensitive and resistant samples including 36 proteins that were differentially stabilized in at least two techniques in both the cell line and PDX studies of oxaliplatin resistance. These 36 differentially stabilized proteins included 10 proteins previously connected to cancer chemoresistance. Two differentially stabilized proteins, fatty acid synthase and elongation factor 2, were functionally validated in vitro and found to be druggable protein targets with biological functions that can be modulated to improve the efficacy of CRC chemotherapy. These results add to our understanding of CRC oxaliplatin resistance, suggest biomarker candidates for predicting oxaliplatin sensitivity in CRC, and inform new strategies for overcoming chemoresistance in CRC.

Quantitative profiling of PTM stoichiometry by resolvable mass tags.

Post-translational modifications (PTMs) play important roles in modulating the biological functions of proteins. Stoichiometry, which quantifies the modification percentage, is a critical factor for any given PTM. In this work, we developed a chemoproteomic strategy called "STO-MS" to systematically quantify the PTM stoichiometry in complex biological samples. This strategy employs a resolvable mass tag to differentiate proteoforms with different numbers of modifications and utilizes liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) techniques to measure PTM stoichiometry at the proteomic level. As a proof-of-concept, we successfully determined the stoichiometry of 197 proteins modified by 4-hydroxynonenal (HNE), a well-characterized lipid-derived electrophile and biomarker for oxidative stress. Our work expands the toolbox for quantification of PTM stoichiometry and sheds light on understanding the biological significance of PTMs in oxidative stress.

Chemoproteomic-enabled characterization of small GTPase Rab1a as a target of an N-arylbenzimidazole ligand's rescue of Parkinson's-associated cell toxicity.

The development of phenotypic models of Parkinson's disease (PD) has enabled screening and identification of phenotypically active small molecules that restore complex biological pathways affected by PD toxicity. While these phenotypic screening platforms are powerful, they do not inherently enable direct identification of the cellular targets of promising lead compounds. To overcome this, chemoproteomic platforms like Thermal Proteome Profiling (TPP) and Stability of Proteins from Rates of Oxidation (SPROX) can be implemented to reveal protein targets of biologically active small molecules. Here we utilize both of these chemoproteomic strategies to identify targets of an N-arylbenzimidazole compound, NAB2, which was previously identified for its ability to restore viability in cellular models of PD-associated α-synuclein toxicity. The combined results from our TPP and SPROX analyses of NAB2 and the proteins in a neuroblastoma-derived SHSY5Y cell lysate reveal a previously unrecognized protein target of NAB2. This newly recognized target, Rab1a, is a small GTPase that acts as a molecular switch to regulate ER-to-Golgi trafficking, a process that is disrupted by α-synuclein toxicity and restored by NAB2 treatment. Further validation reveals that NAB2 binds to Rab1a with selectivity for its GDP-bound form and that NAB2 treatment phenocopies Rab1a overexpression in alleviation of α-synuclein toxicity. Finally, we conduct a preliminary investigation into the relationship between Rab1a and the E3 ubiquitin ligase, Nedd4, a previously identified NAB2 target. Together, these efforts expand our understanding of the mechanism of NAB2 in the alleviation of α-synuclein toxicity and reinforce the utility of chemoproteomic identification of the targets of phenotypically active small molecules that regulate complex biological pathways.

Plasmodium chaperonin TRiC/CCT identified as a target of the antihistamine clemastine using parallel chemoproteomic strategy.

The antihistamine clemastine inhibits multiple stages of the Plasmodium parasite that causes malaria, but the molecular targets responsible for its parasite inhibition were unknown. Here, we applied parallel chemoproteomic platforms to discover the mechanism of action of clemastine and identify that clemastine binds to the Plasmodium falciparum TCP-1 ring complex or chaperonin containing TCP-1 (TRiC/CCT), an essential heterooligomeric complex required for de novo cytoskeletal protein folding. Clemastine destabilized all eight P. falciparum TRiC subunits based on thermal proteome profiling (TPP). Further analysis using stability of proteins from rates of oxidation (SPROX) revealed a clemastine-induced thermodynamic stabilization of the Plasmodium TRiC delta subunit, suggesting an interaction with this protein subunit. We demonstrate that clemastine reduces levels of the major TRiC substrate tubulin in P. falciparum parasites. In addition, clemastine treatment leads to disorientation of Plasmodium mitotic spindles during the asexual reproduction and results in aberrant tubulin morphology suggesting protein aggregation. This clemastine-induced disruption of TRiC function is not observed in human host cells, demonstrating a species selectivity required for targeting an intracellular human pathogen. Our findings encourage larger efforts to apply chemoproteomic methods to assist in target identification of antimalarial drugs and highlight the potential to selectively target Plasmodium TRiC-mediated protein folding for malaria intervention.

Comparative Analysis of Mass-Spectrometry-Based Proteomic Methods for Protein Target Discovery Using a One-Pot Approach.

Recently, several mass-spectrometry- and protein-denaturation-based proteomic methods have been developed to facilitate protein target discovery efforts in drug mode-of-action studies. These methods, which include the stability of proteins from rates of oxidation (SPROX), pulse proteolysis (PP), chemical denaturation and protein precipitation (CPP), and thermal proteome profiling (TPP) techniques, have been used in an increasing number of applications in recent years. However, while the advantages and disadvantages to using these different techniques have been reviewed, the analytical characteristics of these methods have not been directly compared. Reported here is such a direct comparison using the well-studied immunosuppressive drug, cyclosporine A (CsA), and the proteins in a yeast cell lysate. Also described is a one-pot strategy that can be utilized with each technique to streamline data acquisition and analysis. We find that there are benefits to utilizing all four strategies for protein target discovery including increased proteomic coverage and reduced false positive rates that approach 0%. Moreover, the one-pot strategy described here makes such an experiment feasible, because of the 10-fold reduction in reagent costs and instrument time it affords.

Quantitative time-resolved chemoproteomics reveals that stable O-GlcNAc regulates box C/D snoRNP biogenesis.

O-linked GlcNAcylation (O-GlcNAcylation), a ubiquitous posttranslational modification on intracellular proteins, is dynamically regulated in cells. To analyze the turnover dynamics of O-GlcNAcylated proteins, we developed a quantitative time-resolved O-linked GlcNAc proteomics (qTOP) strategy based on metabolic pulse-chase labeling with an O-GlcNAc chemical reporter and stable isotope labeling with amino acids in cell culture (SILAC). Applying qTOP, we quantified the turnover rates of 533 O-GlcNAcylated proteins in NIH 3T3 cells and discovered that about 14% exhibited minimal removal of O-GlcNAc or degradation of protein backbones. The stability of those hyperstable O-GlcNAcylated proteins was more sensitive to O-GlcNAcylation inhibition compared with the more dynamic populations. Among the hyperstable population were three core proteins of box C/D small nucleolar ribonucleoprotein complexes (snoRNPs): fibrillarin (FBL), nucleolar protein 5A (NOP56), and nucleolar protein 5 (NOP58). We showed that O-GlcNAcylation stabilized these proteins and was essential for snoRNP assembly. Blocking O-GlcNAcylation on FBL altered the 2'-O-methylation of rRNAs and impaired cancer cell proliferation and tumor formation in vivo.

Chemoproteomic profiling of targets of lipid-derived electrophiles by bioorthogonal aminooxy probe.

Redox imbalance in cells induces lipid peroxidation and generates a class of highly reactive metabolites known as lipid-derived electrophiles (LDEs) that can modify proteins and affects their functions. Identifying targets of LDEs is critical to understand how such modifications are functionally implicated in oxidative-stress associated diseases. Here we report a quantitative chemoproteomic method to globally profile protein targets and sites modified by LDEs. In this strategy, we designed and synthesized an alkyne-functionalized aminooxy probe to react with LDE-modified proteins for imaging and proteomic profiling. Using this probe, we successfully quantified >4000 proteins modified by 4-hydroxy-2-nonenal (HNE) of high confidence in mammalian cell lysate and combined with a tandem-orthogonal proteolysis activity-based protein profiling (TOP-ABPP) strategy, we identified ~400 residue sites targeted by HNE including reactive cysteines in peroxiredoxins, an important family of enzymes with anti-oxidant roles. Our method expands the toolbox to quantitatively profile protein targets of endogenous electrophiles and the enlarged inventory of LDE-modified proteins and sites will contribute to functional elucidation of cellular pathways affected by oxidative stress.