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Stable isotope labeling by amino acids in cell culture (SILAC) is an established technique used in quantitative mass spectrometry (MS)-based proteomics. SILAC is also used to generate stable isotope labeled (SIL) antibodies for internal standards (IS) used in LC-MS/MS bioassays to improve quantitative robustness.Total antibody (TAb) is measured to evaluate pharmacokinetic (PK) of antibody drug conjugate (ADC) candidates measured by either ligand binding (LBA) or LC-MS/MS. Herein, we describe an application of SILAC, where multiple SIL combinations of an antibody are used for cassette dosing and PK evaluation.Our preclinical studies demonstrate SILAC-labeled ADC therapeutics did not alter antibody PK. Furthermore, with cassette dosing SIL antibodies exhibited comparable exposure to discretely administered unlabeled test articles in rats.In addition, SIL antibodies were conjugated to cytotoxic payloads to create SIL ADCs and cassette dosed in a cynomolgus monkey PK study and SIL ADCs yielded comparable PK results to discrete dosed unlabeled ADCs.In conclusion, SIL antibodies used with a cassette dosing strategy increases PK screening throughput of ADC candidates in preclinical species. Additionally, cassette dosing strategy further facilitates the responsible use of laboratory animals to achieve the three-Rs (Replacement, Reduction, and Refinement).

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INTRODUCTION: Host-microbe interactions are important to human health and ecosystems globally, so elucidating the complex host-microbe interactions and associated protein expressions drives the need to develop sensitive and accurate biochemical techniques. Current proteomics techniques reveal information from the point of view of either the host or microbe, but do not provide data on the corresponding partner. Moreover, it remains challenging to simultaneously study host-microbe proteomes that reflect the direct competition between host and microbe. This raises the need to develop a dual-species proteomics method for host-microbe interactions. OBJECTIVES: We aim to establish a forward + reverse Stable Isotope Labeling with Amino acids in Cell culture (SILAC) proteomics approach to simultaneously label and quantify newly-expressed proteins of host and microbe without physical isolation, for investigating mechanisms in direct host-microbe interactions. METHODS: Using Caenorhabditis elegans-Pseudomonas aeruginosa infection model as proof-of-concept, we employed SILAC proteomics and molecular pathway analysis to characterize the differentially-expressed microbial and host proteins. We then used molecular docking and chemical characterization to identify chemical inhibitors that intercept host-microbe interactions and eliminate microbial infection. RESULTS: Based on our proteomics results, we studied the iron competition between pathogen iron scavenger and host iron uptake protein, where P. aeruginosa upregulated pyoverdine synthesis protein (PvdA) (fold-change of 5.2313) and secreted pyoverdine, and C. elegans expressed ferritin (FTN-2) (fold-change of 3.4057). Targeted intervention of iron competition was achieved using Galangin, a ginger-derived phytochemical that inhibited pyoverdine production and biofilm formation in P. aeruginosa. The Galangin-ciprofloxacin combinatorial therapy could eliminate P. aeruginosa biofilms in a fish wound infection model, and enabled animal survival. CONCLUSION: Our work provides a novel SILAC-based proteomics method that can simultaneously evaluate host and microbe proteomes, with future applications in higher host organisms and other microbial species. It also provides insights into the mechanisms dictating host-microbe interactions, offering novel strategies for anti-infective therapy.

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Co-fractionation mass spectrometry (CF-MS) uses biochemical fractionation to isolate and characterize macromolecular complexes from cellular lysates without the need for affinity tagging or capture. In recent years, this has emerged as a powerful technique for elucidating global protein-protein interaction networks in a wide variety of biospecimens. This review highlights the latest advancements in CF-MS experimental workflows including machine learning-guided analyses, for uncovering dynamic and high-resolution protein interaction landscapes with enhanced sensitivity, accuracy and throughput, enabling better biophysical characterization of endogenous protein complexes. By addressing challenges and emergent opportunities in the field, this review underscores the transformative potential of CF-MS in advancing our understanding of functional protein interaction networks in health and disease.

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CHCHD4 (MIA40) is central to the functions of the mitochondrial disulfide relay system (DRS). CHCHD4 is essential and evolutionarily conserved. Previously, we have shown CHCHD4 to be a critical regulator of tumour cell growth. Here, we use integrated analysis of our genome-wide CRISPR/Cas9 and SILAC proteomic screening data to delineate mechanisms of CHCHD4 essentiality in cancer. We identify a shortlist of common essential genes/proteins regulated by CHCHD4, including subunits of complex I that are known DRS substrates, and genes/proteins involved in key metabolic pathways. Our study highlights a range of CHCHD4-regulated nuclear encoded mitochondrial genes/proteins essential for tumour cell growth.

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Over the last few decades, the study of congenital heart disease (CHD) has benefited from various model systems and the development of molecular biological techniques enabling the analysis of single gene as well as global effects. In this chapter, we first describe different models including CHD patients and their families, animal models ranging from invertebrates to mammals, and various cell culture systems. Moreover, techniques to experimentally manipulate these models are discussed. Second, we introduce cardiac phenotyping technologies comprising the analysis of mouse and cell culture models, live imaging of cardiogenesis, and histological methods for fixed hearts. Finally, the most important and latest molecular biotechniques are described. These include genotyping technologies, different applications of next-generation sequencing, and the analysis of transcriptome, epigenome, proteome, and metabolome. In summary, the models and technologies presented in this chapter are essential to study the function and development of the heart and to understand the molecular pathways underlying CHD.

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Although costly to maintain, protein homeostasis is indispensable for normal cellular function and long-term health. In mammalian cells and tissues, daily variation in global protein synthesis has been observed, but its utility and consequences for proteome integrity are not fully understood. Using several different pulse-labelling strategies, here we gain direct insight into the relationship between protein synthesis and abundance proteome-wide. We show that protein degradation varies in-phase with protein synthesis, facilitating rhythms in turnover rather than abundance. This results in daily consolidation of proteome renewal whilst minimising changes in composition. Coupled rhythms in synthesis and turnover are especially salient to the assembly of macromolecular protein complexes, particularly the ribosome, the most abundant species of complex in the cell. Daily turnover and proteasomal degradation rhythms render cells and mice more sensitive to proteotoxic stress at specific times of day, potentially contributing to daily rhythms in the efficacy of proteasomal inhibitors against cancer. Our findings suggest that circadian rhythms function to minimise the bioenergetic cost of protein homeostasis through temporal consolidation of protein turnover.

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The expression level of individual proteins varies markedly during the progression of the growth phase in bacteria. A set of proteins was quantified in Escherichia coli total proteome during 14 days of batch cultivation using pulse stable isotope labeled amino acids in cell culture (SILAC)-based quantitative mass spectrometry.

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The role of protein turnover in pancreatic ductal adenocarcinoma (PDA) metastasis has not been previously investigated. We introduce dynamic stable-isotope labeling of organoids (dSILO): a dynamic SILAC derivative that combines a pulse of isotopically labeled amino acids with isobaric tandem mass-tag (TMT) labeling to measure proteome-wide protein turnover rates in organoids. We applied it to a PDA model and discovered that metastatic organoids exhibit an accelerated global proteome turnover compared to primary tumor organoids. Globally, most turnover changes are not reflected at the level of protein abundance. Interestingly, the group of proteins that show the highest turnover increase in metastatic PDA compared to tumor is involved in mitochondrial respiration. This indicates that metastatic PDA may adopt alternative respiratory chain functionality that is controlled by the rate at which proteins are turned over. Collectively, our analysis of proteome turnover in PDA organoids offers insights into the mechanisms underlying PDA metastasis.

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Allele-specific epigenetic events regulate the expression of specific genes such as tumor suppressor genes. Methods to biochemically identify epigenetic regulators remain limited. Here, we used insertional chromatin immunoprecipitation (iChIP) to address this issue. iChIP combined with quantitative mass spectrometry identified DNA methyltransferase 1 (DNMT1) and epigenetic regulators as proteins that potentially interact with a region of the p16INK4A gene that is CpG-methylated in one allele in HCT116 cells. Some of the identified proteins are involved in the CpG methylation of this region, and of these, DEAD-box helicase 24 (DDX24) contributes to CpG methylation by regulating the protein levels of DNMT1. Thus, iChIP is a useful method to identify proteins which bind to a target locus of interest.

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This study delves into the biomolecular mechanisms underlying the antitumoral efficacy of a hybrid nanosystem, comprised of a silver core@shell (Ag@MSNs) functionalized with transferrin (Tf). Employing a SILAC proteomics strategy, we identified over 150 de-regulated proteins following exposure to the nanosystem. These proteins play pivotal roles in diverse cellular processes, including mitochondrial fission, calcium homeostasis, endoplasmic reticulum (ER) stress, oxidative stress response, migration, invasion, protein synthesis, RNA maturation, chemoresistance, and cellular proliferation. Rigorous validation of key findings substantiates that the nanosystem elicits its antitumoral effects by activating mitochondrial fission, leading to disruptions in calcium homeostasis, as corroborated by RT-qPCR and flow cytometry analyses. Additionally, induction of ER stress was validated through western blotting of ER stress markers. The cytotoxic action of the nanosystem was further affirmed through the generation of cytosolic and mitochondrial reactive oxygen species (ROS). Finally, in vivo experiments using a chicken embryo model not only confirmed the antitumoral capacity of the nanosystem, but also demonstrated its efficacy in reducing cellular proliferation. These comprehensive findings endorse the potential of the designed Ag@MSNs-Tf nanosystem as a groundbreaking chemotherapeutic agent, shedding light on its multifaceted mechanisms and in vivo applicability.

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One of the main obstacles to therapeutic success in colorectal cancer (CRC) is the development of acquired resistance to treatment with drugs such as 5-fluorouracil (5-FU). Whilst some resistance mechanisms are well known, it is clear from the stasis in therapy success rate that much is still unknown. Here, a proteomics approach is taken towards identification of candidate proteins using 5-FU-resistant sublines of human CRC cell lines generated in house. Using a multiplexed stable isotope labelling with amino acids in cell culture (SILAC) strategy, 5-FU-resistant and equivalently passaged sensitive cell lines were compared to parent cell lines by growing in Heavy medium with 2D liquid chromatography and Orbitrap Fusion™ Tribrid™ Mass Spectrometry analysis. Among 3003 commonly quantified proteins, six (CD44, APP, NAGLU, CORO7, AGR2, PLSCR1) were found up-regulated, and six (VPS45, RBMS2, RIOK1, RAP1GDS1, POLR3D, CD55) down-regulated. A total of 11 of the 12 proteins have a known association with drug resistance mechanisms or role in CRC oncogenesis. Validation through immunodetection techniques confirmed high expression of CD44 and CD63, two known drug resistance mediators with elevated proteomics expression results. The information revealed by the sensitivity of this method warrants it as an important tool for elaborating the complexity of acquired drug resistance in CRC.

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In response to genotoxic stress, cells evolved with a complex signaling network referred to as the DNA damage response (DDR). It is now well established that the DDR depends upon various posttranslational modifications; among them, ubiquitylation plays a key regulatory role. Here, we profiled ubiquitylation in response to the DNA alkylating agent methyl methanesulfonate (MMS) in the budding yeast Saccharomyces cerevisiae using quantitative proteomics. To discover new proteins ubiquitylated upon DNA replication stress, we used stable isotope labeling by amino acids in cell culture, followed by an enrichment of ubiquitylated peptides and LC-MS/MS. In total, we identified 1853 ubiquitylated proteins, including 473 proteins that appeared upregulated more than 2-fold in response to MMS treatment. This enabled us to localize 519 ubiquitylation sites potentially regulated upon MMS in 435 proteins. We demonstrated that the overexpression of some of these proteins renders the cells sensitive to MMS. We also assayed the abundance change upon MMS treatment of a selection of yeast nuclear proteins. Several of them were differentially regulated upon MMS treatment. These findings corroborate the important role of ubiquitin-proteasome-mediated degradation in regulating the DDR.

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The carryover of trace allergens in complex food matrices poses challenges for detection techniques. Here, we demonstrate an accurate UPLC-MS/MS quantification assay for the shrimp allergen tropomyosin with a full-length isotope-labelled recombinant tropomyosin (TM-I) internal standard in complex food matrices. The TM-I, expressed based on the SILAC technique, exhibited a high isotope labelling ratio (>99%), purity, and alignment with the natural sequence. This method determined the tropomyosin ranging from 0.2 to 100 ng/mL. Mean recoveries ranged from 89 to 116%, with intra- and inter-day RSDs below 12%, for three signature peptides across three types of commercially processed food matrices. The limits of quantitation were 1 µg/g in pop food and sauce, and 10 µg/g in surimi product, respectively. This study supports the use of recombinant full-length isotope-labelled proteins rather than stable-isotope labelling peptides as internal standards to achieve more accurate quantitation of food allergens as the digestion error is corrected.

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Understanding the molecular functions of less-studied proteins is an important task of life science research. Despite reports of basic leucine zipper and W2 domain-containing protein 2 (BZW2) promoting cancer progression first emerging in 2017, little is known about its molecular function. Using a quantitative proteomic approach to identify its interacting proteins, we found that BZW2 interacts with both endoplasmic reticulum (ER) and mitochondrial proteins. We thus hypothesized that BZW2 localizes to and promotes the formation of ER-mitochondria contact sites and that such localization would promote calcium transport from ER to the mitochondria and promote ATP production. Indeed, we found that BZW2 localized to ER-mitochondria contact sites and that BZW2 knockdown decreased ER-mitochondria contact, mitochondrial calcium levels, and ATP production. These findings provide key insights into molecular functions of BZW2, the potential role of BZW2 in cancer progression, and highlight the utility of interactome data in understanding the function of less-studied proteins.

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Cullin-RING finger ligases represent the largest family of ubiquitin ligases. They are responsible for the ubiquitination of ∼20% of cellular proteins degraded through the proteasome, by catalyzing the transfer of E2-loaded ubiquitin to a substrate. Seven cullins are described in vertebrates. Among them, cullin 4 (CUL4) associates with DNA damage-binding protein 1 (DDB1) to form the CUL4-DDB1 ubiquitin ligase complex, which is involved in protein ubiquitination and in the regulation of many cellular processes. Substrate recognition adaptors named DDB1/CUL4-associated factors (DCAFs) mediate the specificity of CUL4-DDB1 and have a short structural motif of approximately forty amino acids terminating in tryptophan (W)-aspartic acid (D) dipeptide, called the WD40 domain. Using different approaches (bioinformatics/structural analyses), independent studies suggested that at least sixty WD40-containing proteins could act as adaptors for the DDB1/CUL4 complex. To better define this association and classification, the interaction of each DCAFs with DDB1 was determined, and new partners and potential substrates were identified. Using BioID and affinity purification-mass spectrometry approaches, we demonstrated that seven WD40 proteins can be considered DCAFs with a high confidence level. Identifying protein interactions does not always lead to identifying protein substrates for E3-ubiquitin ligases, so we measured changes in protein stability or degradation by pulse-stable isotope labeling with amino acids in cell culture to identify changes in protein degradation, following the expression of each DCAF. In conclusion, these results provide new insights into the roles of DCAFs in regulating the activity of the DDB1-CUL4 complex, in protein targeting, and characterized the cellular processes involved.

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Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS) through gain-of-function effects, yet the mechanisms by which misfolded mutant SOD1 (mutSOD1) protein impairs human motor neurons (MNs) remain unclear. Here, we use induced-pluripotent-stem-cell-derived MNs coupled to metabolic stable isotope labeling and mass spectrometry to investigate proteome-wide degradation dynamics. We find several proteins, including the ALS-causal valosin-containing protein (VCP), which predominantly acts in proteasome degradation and autophagy, that degrade slower in mutSOD1 relative to isogenic control MNs. The interactome of VCP is altered in mutSOD1 MNs in vitro, while VCP selectively accumulates in the affected motor cortex of ALS-SOD1 patients. Overexpression of VCP rescues mutSOD1 toxicity in MNs in vitro and in a C. elegans model in vivo, in part due to its ability to modulate the degradation of insoluble mutSOD1. Our results demonstrate that VCP contributes to mutSOD1-dependent degeneration, link two distinct ALS-causal genes, and highlight selective protein degradation impairment in ALS pathophysiology.

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Self-renewal and differentiation of mouse embryonic stem cells (mESCs) are greatly affected by the extracellular matrix (ECM) environment; the composition and stiffness of which are sensed by the cells via integrin-associated focal adhesions (FAs) which link the cells to the ECM. Although FAs have been studied extensively in differentiated cells, their composition and function in mESCs are not as well elucidated. To gain more detailed knowledge of the molecular compositions of FAs in mESCs, we adopted the proximity-dependent biotinylation (BioID) proteomics approach. Paxillin, a known FA protein (FAP), is fused to the promiscuous biotin ligase TurboID as bait. We employed both SILAC- and label-free (LF)-based quantitative proteomics to strengthen as well as complement individual approach. The mass spectrometry data derived from SILAC and LF identified 38 and 443 proteins, respectively, with 35 overlapping candidates. Fifteen of these shared proteins are known FAPs based on literature-curated adhesome and 7 others are among the reported "meta-adhesome", suggesting the components of FAs are largely conserved between mESCs and differentiated cells. Furthermore, the LF data set contained an additional 18 literature-curated FAPs. Notably, the overlapped proteomics data failed to detect LIM-domain proteins such as zyxin family proteins, which suggests that FAs in mESCs are less mature than differentiated cells. Using the LF approach, we are able to identify PDLIM7, a LIM-domain protein, as a FAP in mESCs. This study illustrates the effectiveness of TurboID in mESCs. Importantly, we found that application of both SILAC and LF methods in combination allowed us to analyze the TurboID proteomics data in an unbiased, stringent and yet comprehensive manner.

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The p38 members of the mitogen-activated protein kinases (MAPKs) family mediate various cellular responses to stress conditions, inflammatory signals, and differentiation factors. They are constitutively active in chronic inflammatory diseases and some cancers. The differences between their transient effects in response to signals and the chronic effect in diseases are not known. The family is composed of four isoforms, of which p38α seems to be abnormally activated in diseases. p38α and p38ß are almost identical in sequence, structure, and biochemical and pharmacological properties, and the specific unique effects of each of them, if any, have not yet been revealed. This study aimed to reveal the specific effects induced by p38α and p38ß, both when transiently activated in response to stress and when chronically active. This was achieved via large-scale proteomics and phosphoproteomics analyses using stable isotope labeling of two experimental systems: one, mouse embryonic fibroblasts (MEFs) deficient in each of these p38 kinases and harboring either an empty vector or vectors expressing p38αWT, p38ßWT, or intrinsically active variants of these MAPKs; second, induction of transient stress by exposure of MEFs, p38α-/-, and p38ß-/- MEFs to anisomycin. Significant differences in the repertoire of the proteome and phosphoproteome between cells expressing active p38α and p38ß suggest distinct roles for each kinase. Interestingly, in both cases, the constitutive activation induced adaptations of the cells to the chronic activity so that known substrates of p38 were downregulated. Within the dramatic effect of p38s on the proteome and phosphoproteome, some interesting affected phosphorylation sites were those found in cancer-associated p53 and Hspb1 (HSP27) proteins and in cytoskeleton-associated proteins. Among these, was the stronger direct phosphorylation by p38α of p53-Ser309, which was validated on the Ser315 in human p53. In summary, this study sheds new light on the differences between chronic and transient p38α and p38ß signaling and on the specific targets of these two kinases.

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Allergic contact dermatitis (ACD) is the predominant form of immunotoxicity in humans. The sensitizing potential of chemicals can be assessed in vitro. However, a better mechanistic understanding could improve the current OECD-validated test battery. The aim of this study was to get insights into toxicity mechanisms of four contact allergens, p-benzoquinone (BQ), 2,4-dinitrochlorobenzene (DNCB), p-nitrobenzyl bromide (NBB) and NiSO4, by analyzing differential proteome alterations in THP-1 cells using two common proteomics workflows, stable isotope labeling by amino acids in cell culture (SILAC) and label-free quantification (LFQ). Here, SILAC was found to deliver more robust results. Overall, the four allergens induced similar responses in THP-1 cells, which underwent profound metabolic reprogramming, including a striking upregulation of the TCA cycle accompanied by pronounced induction of the Nrf2 oxidative stress response pathway. The magnitude of induction varied between the allergens with DNCB and NBB being most potent. A considerable overlap between transcriptome-based signatures of the GARD assay and the proteins identified in our study was found. When comparing the results of this study to a previous proteomics study in human primary monocyte-derived dendritic cells, we found a rather low share in regulated proteins. However, on pathway level, the overlap was high, indicating that affected pathways rather than single proteins are more eligible to investigate proteomic changes induced by contact allergens. Overall, this study confirms the potential of proteomics to obtain a profound mechanistic understanding, which may help improving existing in vitro assays for skin sensitization.

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Genome-wide association studies along with expression quantitative trait locus (eQTL) mapping have identified hundreds of single-nucleotide polymorphisms (SNPs) and their target genes in prostate cancer (PCa), yet functional characterization of these risk loci remains challenging. To screen for potential regulatory SNPs, we designed a CRISPRi library containing 9,133 guide RNAs (gRNAs) to cover 2,166 candidate SNP loci implicated in PCa and identified 117 SNPs that could regulate 90 genes for PCa cell growth advantage. Among these, rs60464856 was covered by multiple gRNAs significantly depleted in screening (FDR < 0.05). Pooled SNP association analysis in the PRACTICAL and FinnGen cohorts showed significantly higher PCa risk for the rs60464856 G allele (p value = 1.2 × 10-16 and 3.2 × 10-7, respectively). Subsequent eQTL analysis revealed that the G allele is associated with increased RUVBL1 expression in multiple datasets. Further CRISPRi and xCas9 base editing confirmed that the rs60464856 G allele leads to elevated RUVBL1 expression. Furthermore, SILAC-based proteomic analysis demonstrated allelic binding of cohesin subunits at the rs60464856 region, where the HiC dataset showed consistent chromatin interactions in prostate cell lines. RUVBL1 depletion inhibited PCa cell proliferation and tumor growth in a xenograft mouse model. Gene-set enrichment analysis suggested an association of RUVBL1 expression with cell-cycle-related pathways. Increased expression of RUVBL1 and activation of cell-cycle pathways were correlated with poor PCa survival in TCGA datasets. Our CRISPRi screening prioritized about one hundred regulatory SNPs essential for prostate cell proliferation. In combination with proteomics and functional studies, we characterized the mechanistic role of rs60464856 and RUVBL1 in PCa progression.

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