Evaluation of Protein Identification and Quantification by the diaPASEF Method on timsTOF SCP.

Accurate and precise quantification is crucial in modern proteomics, particularly in the context of exploring low-amount samples. While the innovative 4D-data-independent acquisition (DIA) quantitative proteomics facilitated by timsTOF mass spectrometers gives enhanced sensitivity and selectivity for protein identification, the diaPASEF (parallel accumulation-serial fragmentation combined with data-independent acquisition) parameters have not been systematically optimized, and a comprehensive evaluation of the quantification is currently lacking. In this study, we conducted a thorough optimization of key parameters on a timsTOF SCP instrument, including sample loading amount (50 ng), ramp/accumulation time (140 ms), isolation window width (20 m/z), and gradient time (60 min). To further improve the identification of proteins in low-amount samples, we utilized different column settings and introduced 0.02% n-dodecyl-ß-d-maltoside (DDM) in the sample reconstitution solution, resulting in a remarkable 19-fold increase in protein identification at the single-cell-equivalent level. Moreover, a comprehensive comparison of protein quantification using a tandem mass tag reporter (TMT-reporter), complement TMT ions (TMTc), and diaPASEF revealed a strong correlation between these methods. Both diaPASEF and TMTc have effectively addressed the issue of ratio compression, highlighting the diaPASEF method's effectiveness in achieving accurate quantification data compared to TMT reporter quantification. Additionally, an in-depth analysis of in-group variation positioned diaPASEF between the TMT-reporter and TMTc methods. Therefore, diaPASEF quantification on the timsTOF SCP instrument emerges as a precise and accurate methodology for quantitative proteomics, especially for samples with small amounts.

Selective CK1α degraders exert antiproliferative activity against a broad range of human cancer cell lines.

Molecular-glue degraders are small molecules that induce a specific interaction between an E3 ligase and a target protein, resulting in the target proteolysis. The discovery of molecular glue degraders currently relies mostly on screening approaches. Here, we describe screening of a library of cereblon (CRBN) ligands against a panel of patient-derived cancer cell lines, leading to the discovery of SJ7095, a potent degrader of CK1α, IKZF1 and IKZF3 proteins. Through a structure-informed exploration of structure activity relationship (SAR) around this small molecule we develop SJ3149, a selective and potent degrader of CK1α protein in vitro and in vivo. The structure of SJ3149 co-crystalized in complex with CK1α + CRBN + DDB1 provides a rationale for the improved degradation properties of this compound. In a panel of 115 cancer cell lines SJ3149 displays a broad antiproliferative activity profile, which shows statistically significant correlation with MDM2 inhibitor Nutlin-3a. These findings suggest potential utility of selective CK1α degraders for treatment of hematological cancers and solid tumors.

The orally bioavailable GSPT1/2 degrader SJ6986 exhibits in vivo efficacy in acute lymphoblastic leukemia.

Advancing cure rates for high-risk acute lymphoblastic leukemia (ALL) has been limited by the lack of agents that effectively kill leukemic cells, sparing normal hematopoietic tissue. Molecular glues direct the ubiquitin ligase cellular machinery to target neosubstrates for protein degradation. We developed a novel cereblon modulator, SJ6986, that exhibits potent and selective degradation of GSPT1 and GSPT2 and cytotoxic activity against childhood cancer cell lines. Here, we report in vitro and in vivo testing of the activity of this agent in a panel of ALL cell lines and xenografts. SJ6986 exhibited similar cytotoxicity to the previously described GSPT1 degrader CC-90009 in a panel of leukemia cell lines in vitro, resulting in apoptosis and perturbation of cell cycle progression. SJ6986 was more effective than CC-90009 in suppressing leukemic cell growth in vivo, partly attributable to favorable pharmacokinetic properties, and did not significantly impair differentiation of human CD34+ cells ex vivo. Genome-wide CRISPR/Cas9 screening of ALL cell lines treated with SJ6986 confirmed that components of the CRL4CRBN complex, associated adaptors, regulators, and effectors were integral in mediating the action of SJ6986. SJ6986 is a potent, selective, orally bioavailable GSPT1/2 degrader that shows broad antileukemic activity and has potential for clinical development.

DRAK2 contributes to type 1 diabetes by negatively regulating IL-2 sensitivity to alter regulatory T cell development.

Drak2-deficient (Drak2-/-) mice are resistant to multiple models of autoimmunity yet effectively eliminate pathogens and tumors. Thus, DRAK2 represents a potential target to treat autoimmune diseases. However, the mechanisms by which DRAK2 contributes to autoimmunity, particularly type 1 diabetes (T1D), remain unresolved. Here, we demonstrate that resistance to T1D in non-obese diabetic (NOD) mice is due to the absence of Drak2 in T cells and requires the presence of regulatory T cells (Tregs). Contrary to previous hypotheses, we show that DRAK2 does not limit TCR signaling. Rather, DRAK2 regulates IL-2 signaling by inhibiting STAT5A phosphorylation. We further demonstrate that enhanced sensitivity to IL-2 in the absence of Drak2 augments thymic Treg development. Overall, our data indicate that DRAK2 contributes to autoimmunity in multiple ways by regulating thymic Treg development and by impacting the sensitivity of conventional T cells to Treg-mediated suppression.

Mutual antagonism between glucocorticoid and canonical Wnt signaling pathways in B-cell acute lymphoblastic leukemia.

Glucocorticoids (GCs; i.e., steroids) are important chemotherapeutic agents in the treatment of B-cell precursor acute lymphoblastic leukemia (B-ALL) and de novo GC resistance predicts relapse and poor clinical outcome in patients. Glucocorticoids induce B-ALL cell apoptosis through activation of glucocorticoid receptor (GR), a ligand-induced nuclear receptor transcription factor (TF). We previously identified disruptions to glucocorticoid receptor (GR)-bound cis -regulatory elements controlling TLE1 expression in GC-resistant primary B-ALL cells from patients. TLE1 is a GC-response gene up-regulated by steroids and functions as a canonical Wnt signaling repressor. To better understand the mechanistic relationship between GC signaling and canonical Wnt signaling, we performed diverse functional analyses that identified extensive crosstalk and mutual antagonism between these two signaling pathways in B-ALL. We determined that crosstalk and antagonism was driven by the binding of GR and the canonical Wnt signaling TFs LEF1 and TCF7L2 to overlapping sets of cis -regulatory elements associated with genes impacting cell death and cell proliferation, and was further accompanied by overlapping and opposing transcriptional programs. Our data additionally suggest that cis -regulatory disruptions at TLE1 are linked to GC resistance through a dampening of the GC response and GC-mediated apoptosis via enhanced canonical Wnt signaling. As a result of the extensive genomic and gene regulatory connectivity between these two signaling pathways, our data supports the importance of canonical Wnt signaling in mediating GC resistance in B-ALL.

A phospho-tyrosine-based signaling module using SPOP, CSK, and LYN controls TLR-induced IRF activity.

Toll-like receptors (TLRs) recognize pathogen- and host-derived factors and control immune responses via the adaptor protein MyD88 and members of the interferon regulatory transcription factor (IRF) family. IRFs orchestrate key effector functions, including cytokine release, cell differentiation, and, under certain circumstances, inflammation pathology. Here, we show that IRF activity is generically controlled by the Src kinase family member LYN, which phosphorylates all TLR-induced IRFs at a conserved tyrosine residue, resulting in K48-linked polyubiquitination and proteasomal degradation of IRFs. We further show that LYN activity is controlled by the upstream kinase C-terminal Src kinase (CSK), whose activity, in turn, is controlled by the adaptor protein SPOP, which serves as molecular bridge to recruit CSK into the TLR signaling complex and to activate CSK catalytic activity. Consistently, deletion of SPOP or CSK results in increased LYN activity, LYN-directed IRF degradation, and inhibition of IRF transcriptional activity. Together, the data reveal a key regulatory mechanism for IRF family members controlling TLR biology.

JUMPn: A Streamlined Application for Protein Co-Expression Clustering and Network Analysis in Proteomics.

With recent advances in mass spectrometry-based proteomics technologies, deep profiling of hundreds of proteomes has become increasingly feasible. However, deriving biological insights from such valuable datasets is challenging. Here we introduce a systems biology-based software JUMPn, and its associated protocol to organize the proteome into protein co-expression clusters across samples and protein-protein interaction (PPI) networks connected by modules (e.g., protein complexes). Using the R/Shiny platform, the JUMPn software streamlines the analysis of co-expression clustering, pathway enrichment, and PPI module detection, with integrated data visualization and a user-friendly interface. The main steps of the protocol include installation of the JUMPn software, the definition of differentially expressed proteins or the (dys)regulated proteome, determination of meaningful co-expression clusters and PPI modules, and result visualization. While the protocol is demonstrated using an isobaric labeling-based proteome profile, JUMPn is generally applicable to a wide range of quantitative datasets (e.g., label-free proteomics). The JUMPn software and protocol thus provide a powerful tool to facilitate biological interpretation in quantitative proteomics.

Deep Profiling of Microgram-Scale Proteome by Tandem Mass Tag Mass Spectrometry.

Tandem mass tag (TMT)-based mass spectrometry (MS) enables deep proteomic profiling of more than 10,000 proteins in complex biological samples but requires up to 100 µg protein in starting materials during a standard analysis. Here, we present a streamlined protocol to quantify more than 9000 proteins with 0.5 µg protein per sample by 16-plex TMT coupled with two-dimensional liquid chromatography and tandem mass spectrometry (LC/LC-MS/MS). In this protocol, we optimized multiple conditions to reduce sample loss, including processing each sample in a single tube to minimize surface adsorption, increasing digestion enzymes to shorten proteolysis and function as carriers, eliminating a desalting step between digestion and TMT labeling, and developing miniaturized basic pH LC for prefractionation. By profiling 16 identical human brain tissue samples of Alzheimer's disease (AD), vascular dementia (VaD), and non-dementia controls, we directly compared this new microgram-scale protocol to the standard-scale protocol, quantifying 9116 and 10,869 proteins, respectively. Importantly, bioinformatics analysis indicated that the microgram-scale protocol had adequate sensitivity and reproducibility to detect differentially expressed proteins in disease-related pathways. Thus, this newly developed protocol is of general application for deep proteomics analysis of biological and clinical samples at sub-microgram levels.

High-throughput and Deep-proteome Profiling by 16-plex Tandem Mass Tag Labeling Coupled with Two-dimensional Chromatography and Mass Spectrometry.

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an expanded 16-plex TMT method has been introduced, which further increases the throughput of proteomic studies. In this manuscript, we present an optimized protocol for 16-plex TMT-based deep-proteome profiling, including protein sample preparation, enzymatic digestion, TMT labeling reaction, two-dimensional reverse-phase liquid chromatography (LC/LC) fractionation, tandem mass spectrometry (MS/MS), and computational data processing. The crucial quality control steps and improvements in the process specific for the 16-plex TMT analysis are highlighted. This multiplexed process offers a powerful tool for profiling a variety of complex samples such as cells, tissues, and clinical specimens. More than 10,000 proteins and posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination in highly complex biological samples from up to 16 different samples can be quantified in a single experiment, providing a potent tool for basic and clinical research.

Deep Multilayer Brain Proteomics Identifies Molecular Networks in Alzheimer's Disease Progression.

Alzheimer's disease (AD) displays a long asymptomatic stage before dementia. We characterize AD stage-associated molecular networks by profiling 14,513 proteins and 34,173 phosphosites in the human brain with mass spectrometry, highlighting 173 protein changes in 17 pathways. The altered proteins are validated in two independent cohorts, showing partial RNA dependency. Comparisons of brain tissue and cerebrospinal fluid proteomes reveal biomarker candidates. Combining with 5xFAD mouse analysis, we determine 15 Aß-correlated proteins (e.g., MDK, NTN1, SMOC1, SLIT2, and HTRA1). 5xFAD shows a proteomic signature similar to symptomatic AD but exhibits activation of autophagy and interferon response and lacks human-specific deleterious events, such as downregulation of neurotrophic factors and synaptic proteins. Multi-omics integration prioritizes AD-related molecules and pathways, including amyloid cascade, inflammation, complement, WNT signaling, TGF-ß and BMP signaling, lipid metabolism, iron homeostasis, and membrane transport. Some Aß-correlated proteins are colocalized with amyloid plaques. Thus, the multilayer omics approach identifies protein networks during AD progression.

Deep multiomics profiling of brain tumors identifies signaling networks downstream of cancer driver genes.

High throughput omics approaches provide an unprecedented opportunity for dissecting molecular mechanisms in cancer biology. Here we present deep profiling of whole proteome, phosphoproteome and transcriptome in two high-grade glioma (HGG) mouse models driven by mutated RTK oncogenes, PDGFRA and NTRK1, analyzing 13,860 proteins and 30,431 phosphosites by mass spectrometry. Systems biology approaches identify numerous master regulators, including 41 kinases and 23 transcription factors. Pathway activity computation and mouse survival indicate the NTRK1 mutation induces a higher activation of AKT downstream targets including MYC and JUN, drives a positive feedback loop to up-regulate multiple other RTKs, and confers higher oncogenic potency than the PDGFRA mutation. A mini-gRNA library CRISPR-Cas9 validation screening shows 56% of tested master regulators are important for the viability of NTRK-driven HGG cells, including TFs (Myc and Jun) and metabolic kinases (AMPKa1 and AMPKa2), confirming the validity of the multiomics integrative approaches, and providing novel tumor vulnerabilities.

Deep undepleted human serum proteome profiling toward biomarker discovery for Alzheimer's disease.

BACKGROUND: Blood-based protein measurement is a routine practice for detecting biomarkers in human disease. Comprehensive profiling of blood/plasma/serum proteome is a challenge due to an extremely large dynamic range, as exemplified by a small subset of highly abundant proteins. Antibody-based depletion of these abundant proteins alleviates the problem but introduces experimental variations. We aimed to establish a method for direct profiling of undepleted human serum and apply the method toward biomarker discovery for Alzheimer's disease (AD), as AD is the most common form of dementia without available blood-based biomarkers in clinic. METHODS: We present an ultra-deep analysis of undepleted human serum proteome by combining the latest 11-plex tandem-mass-tag (TMT) labeling, exhaustive two-dimensional liquid chromatography (LC/LC) fractionation (the 1st LC: 3 h for 180 fractions, and the 2nd LC: 3 h gradient per fraction), coupled with high resolution tandem mass spectrometry (MS/MS). AD (n = 6) and control (n = 5) sera were analyzed in this pilot study. In addition, we implemented a multiplexed targeted LC-MS3 method (TOMAHAQ) for the validation of selected target proteins. RESULTS: The TMT-LC/LC-MS/MS platform is capable of analyzing 4826 protein components (4368 genes), covering at least 6 orders of magnitude in dynamic range, representing one of the deepest serum proteome analysis. We defined intra- and inter- group variability in the AD and control groups. Statistical analysis revealed differentially expressed proteins in AD (26 decreased and 4 increased). Notably, these altered proteins are enriched in the known pathways of mitochondria, fatty acid beta oxidation, and AGE/RAGE. Finally, we set up a TOMAHAQ method to confirm the decrease of PCK2 and AK2 in our AD samples. CONCLUSIONS: Our results show an ultra-deep serum discovery study by TMT-LC/LC-MS/MS, and a validation experiment by TOMAHAQ targeted LC-MS3. The MS-based discovery and validation methods are of general use for biomarker discovery from complex biofluids (e.g. serum proteome). This pilot study also identified deregulated proteins, in particular proteins associated with mitochondrial function in the AD serum samples. These proteins may serve as novel AD candidate biomarkers.

Identification of Toll-like receptor signaling inhibitors based on selective activation of hierarchically acting signaling proteins.

Toll-like receptors (TLRs) recognize various pathogen- and host tissue-derived molecules and initiate inflammatory immune responses. Exaggerated or prolonged TLR activation, however, can lead to etiologically diverse diseases, such as bacterial sepsis, metabolic and autoimmune diseases, or stroke. Despite the apparent medical need, no small-molecule drugs against TLR pathways are clinically available. This may be because of the complex signaling mechanisms of TLRs, which are governed by a series of protein-protein interactions initiated by Toll/interleukin-1 receptor homology domains (TIR) found in TLRs and the cytoplasmic adaptor proteins TIRAP and MyD88. Oligomerization of TLRs with MyD88 or TIRAP leads to the recruitment of members of the IRAK family of kinases and the E3 ubiquitin ligase TRAF6. We developed a phenotypic drug screening system based on the inducible homodimerization of either TIRAP, MyD88, or TRAF6, that ranked hits according to their hierarchy of action. From a bioactive compound library, we identified methyl-piperidino-pyrazole (MPP) as a TLR-specific inhibitor. Structure-activity relationship analysis, quantitative proteomics, protein-protein interaction assays, and cellular thermal shift assays suggested that MPP targets the TIR domain of MyD88. Chemical evolution of the original MPP scaffold generated compounds with selectivity for distinct TLRs that interfered with specific TIR interactions. Administration of an MPP analog to mice protected them from TLR4-dependent inflammation. These results validate this phenotypic screening approach and suggest that the MPP scaffold could serve as a starting point for the development of anti-inflammatory drugs.

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification.

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

Extensive Peptide Fractionation and y1 Ion-Based Interference Detection Method for Enabling Accurate Quantification by Isobaric Labeling and Mass Spectrometry.

Isobaric labeling quantification by mass spectrometry (MS) has emerged as a powerful technology for multiplexed large-scale protein profiling, but measurement accuracy in complex mixtures is confounded by the interference from coisolated ions, resulting in ratio compression. Here we report that the ratio compression can be essentially resolved by the combination of pre-MS peptide fractionation, MS2-based interference detection, and post-MS computational interference correction. To recapitulate the complexity of biological samples, we pooled tandem mass tag (TMT)-labeled Escherichia coli peptides at 1:3:10 ratios and added in ∼20-fold more rat peptides as background, followed by the analysis of two-dimensional liquid chromatography (LC)-MS/MS. Systematic investigation shows that quantitative interference was impacted by LC fractionation depth, MS isolation window, and peptide loading amount. Exhaustive fractionation (320 × 4 h) can nearly eliminate the interference and achieve results comparable to the MS3-based method. Importantly, the interference in MS2 scans can be estimated by the intensity of contaminated y1 product ions, and we thus developed an algorithm to correct reporter ion ratios of tryptic peptides. Our data indicate that intermediate fractionation (40 × 2 h) and y1 ion-based correction allow accurate and deep TMT profiling of more than 10 000 proteins, which represents a straightforward and affordable strategy in isobaric labeling proteomics.

Multiple Weak Linear Motifs Enhance Recruitment and Processivity in SPOP-Mediated Substrate Ubiquitination.

Primary sequence motifs, with millimolar affinities for binding partners, are abundant in disordered protein regions. In multivalent interactions, such weak linear motifs can cooperate to recruit binding partners via avidity effects. If linear motifs recruit modifying enzymes, optimal placement of weak motifs may regulate access to modification sites. Weak motifs may thus exert physiological relevance stronger than that suggested by their affinities, but molecular mechanisms of their function are still poorly understood. Herein, we use the N-terminal disordered region of the Hedgehog transcriptional regulator Gli3 (Gli3(1-90)) to determine the role of weak motifs encoded in its primary sequence for the recruitment of its ubiquitin ligase CRL3(SPOP) and the subsequent effect on ubiquitination efficiency. The substrate adaptor SPOP binds linear motifs through its MATH (meprin and TRAF homology) domain and forms higher-order oligomers through its oligomerization domains, rendering SPOP multivalent for its substrates. Gli3 has multiple weak SPOP binding motifs. We map three such motifs in Gli3(1-90), the weakest of which has a millimolar dissociation constant. Multivalency of ligase and substrate for each other facilitates enhanced ligase recruitment and stimulates Gli3(1-90) ubiquitination in in vitro ubiquitination assays. We speculate that the weak motifs enable processivity through avidity effects and by providing steric access to lysine residues that are otherwise not prioritized for polyubiquitination. Weak motifs may generally be employed in multivalent systems to act as gatekeepers regulating post-translational modification.

Quantitative protein analysis by mass spectrometry.

Mass spectrometry is one of the most sensitive methods in analytical chemistry, and its application in proteomics has been rapidly expanded after sequencing the human genome. Mass spectrometry is now the mainstream approach for identification and quantification of proteins and posttranslational modifications, either in small scale or in the entire proteome. Shotgun proteomics can analyze up to 10,000 proteins in a comprehensive study, with detection sensitivity in the picogram range. In this chapter, we describe major experimental steps in a shotgun proteomics platform, including sample preparation in the context of studying protein-protein interaction, mass spectrometric data acquisition, and database search to identify proteins and posttranslational modification analysis. Proteome quantification strategies and bioinformatics analysis are also illustrated. Finally, we discuss the capabilities, limitations, and potential improvements of current platforms.

Integrated approaches for analyzing U1-70K cleavage in Alzheimer's disease.

The accumulation of pathologic protein fragments is common in neurodegenerative disorders. We have recently identified in Alzheimer's disease (AD) the aggregation of the U1-70K splicing factor and abnormal RNA processing. Here, we present that U1-70K can be cleaved into an N-terminal truncation (N40K) in ∼50% of AD cases, and the N40K abundance is inversely proportional to the total level of U1-70K. To map the cleavage site, we compared tryptic peptides of N40K and stable isotope labeled U1-70K by liquid chromatography-tandem mass spectrometry (MS), revealing that the proteolysis site is located in a highly repetitive and hydrophilic domain of U1-70K. We then adapted Western blotting to map the cleavage site in two steps: (i) mass spectrometric analysis revealing that U1-70K and N40K share the same N-termini and contain no major modifications; (ii) matching N40K with a series of six recombinant U1-70K truncations to define the cleavage site within a small region (Arg300 ± 6 residues). Finally, N40K expression led to substantial degeneration of rat primary hippocampal neurons. In summary, we combined multiple approaches to identify the U1-70K proteolytic site and found that the N40K fragment might contribute to neuronal toxicity in Alzheimer's disease.