Kinase-impaired BTK mutations are susceptible to clinical-stage BTK and IKZF1/3 degrader NX-2127.

Increasing use of covalent and noncovalent inhibitors of Bruton's tyrosine kinase (BTK) has elucidated a series of acquired drug-resistant BTK mutations in patients with B cell malignancies. Here we identify inhibitor resistance mutations in BTK with distinct enzymatic activities, including some that impair BTK enzymatic activity while imparting novel protein-protein interactions that sustain B cell receptor (BCR) signaling. Furthermore, we describe a clinical-stage BTK and IKZF1/3 degrader, NX-2127, that can bind and proteasomally degrade each mutant BTK proteoform, resulting in potent blockade of BCR signaling. Treatment of chronic lymphocytic leukemia with NX-2127 achieves >80% degradation of BTK in patients and demonstrates proof-of-concept therapeutic benefit. These data reveal an oncogenic scaffold function of mutant BTK that confers resistance across clinically approved BTK inhibitors but is overcome by BTK degradation in patients.

Borrelia PeptideAtlas: A proteome resource of common Borrelia burgdorferi isolates for Lyme research.

Lyme disease, caused by an infection with the spirochete Borrelia burgdorferi, is the most common vector-borne disease in North America. B. burgdorferi strains harbor extensive genomic and proteomic variability and further comparison is key to understanding the spirochetes infectivity and biological impacts of identified sequence variants. To achieve this goal, both transcript and mass spectrometry (MS)-based proteomics was applied to assemble peptide datasets of laboratory strains B31, MM1, B31-ML23, infective isolates B31-5A4, B31-A3, and 297, and other public datasets, to provide a publicly available Borrelia PeptideAtlas http://www.peptideatlas.org/builds/borrelia/. Included is information on total proteome, secretome, and membrane proteome of these B. burgdorferi strains. Proteomic data collected from 35 different experiment datasets, with a total of 855 mass spectrometry runs, identified 76,936 distinct peptides at a 0.1% peptide false-discovery-rate, which map to 1,221 canonical proteins (924 core canonical and 297 noncore canonical) and covers 86% of the total base B31 proteome. The diverse proteomic information from multiple isolates with credible data presented by the Borrelia PeptideAtlas can be useful to pinpoint potential protein targets which are common to infective isolates and may be key in the infection process.

The PeptideAtlas of a widely cultivated fish Labeo rohita: A resource for the Aquaculture Community.

Labeo rohita (Rohu) is one of the most important fish species produced in world aquaculture. Integrative omics research provides a strong platform to understand the basic biology and translate this knowledge into sustainable solutions in tackling disease outbreak, increasing productivity and ensuring food security. Mass spectrometry-based proteomics has provided insights to understand the biology in a new direction. Very little proteomics work has been done on 'Rohu' limiting such resources for the aquaculture community. Here, we utilised an extensive mass spectrometry based proteomic profiling data of 17 histologically normal tissues, plasma and embryo of Rohu to develop an open source PeptideAtlas. The current build of "Rohu PeptideAtlas" has mass-spectrometric evidence for 6015 high confidence canonical proteins at 1% false discovery rate, 2.9 million PSMs and ~150 thousand peptides. This is the first open-source proteomics repository for an aquaculture species. The 'Rohu PeptideAtlas' would promote basic and applied aquaculture research to address the most critical challenge of ensuring nutritional security for a growing population.

Label-free plasma proteomics for the identification of the putative biomarkers of oral squamous cell carcinoma.

Mass spectrometry-based label-free proteomics is becoming the analytical tool of interest to identify and quantitate the biomarkers for cancer. The oral squamous cell carcinoma (OSCC) which is one of the leading cancers worldwide, lacks biomarkers for the early prognosis and diagnosis. The present study profiled plasma proteome of the Indian OSCC human patients using a label-free mass spectrometry proteomics approach. The study first time utilized the three most widely used data analysis software MaxQuant (MQ), Proteome discoverer (PD), and Trans proteomic pipeline (TPP) together for label-free quantitation of the proteins. The study identified 16 proteins which can be used as a signature protein panel for OSCC. The pathway analysis showed predominant involvement of the immune system, hemostasis as the major pathways that are indicative of the disease progression. The network analysis showed maximum interaction for the Fibronectin and C-reactive protein. The study demonstrates that plasma proteins contain signatures that can be used as putative biomarkers for OSCC. Based on the label-free quantitation and the mechanistic analysis C-reactive protein, Carbonic anhydrase-1, and Fibronectin are identified as putative biomarkers of OSCC. Once these findings are validated in the large cohorts these protein signatures can be used as a biomarker for OSCC. SIGNIFICANCE: The oral squamous cell carcinoma (OSCC) is the eighteenth most prevalent malignancy in the world and ranks second in India. There are no biomarkers that could be indicative of the diseased state. Various studies have been carried out on saliva and tumors of OSCC patients in India, but none of the studies have profiled the plasma. We utilized the label-free approach for the first time on the Indian population in generating the panel of plasma proteins which could be indicative of the diseased state. The study identified Carbonic anhydrase 1, C-reactive protein, and Fibronectin proteins as putative biomarkers for the OSCC. The study obtained the signature panel by utilizing the 3 most widely used software for the label-free quanatitation (LFQ) namely MaxQuant, Proteome Discoverer, and Trans proteomic pipeline. The utilization of 3 software for the LFQ reduced the bias and provided a comprehensive list of proteins. All the differential proteins were mechanistically studied for their biological relevance and the pathway and network analysis were carried out. The study helps us in increasing the understanding of the proteins which are involved in the progression of the diseases. Studying the panel of proteins from all biofluids along with plasma in large cohorts of the population will help in understanding the disease in greater depth and help in identifying the relevant biomarkers for the OSCC.

Organ-Based Proteome and Post-Translational Modification Profiling of a Widely Cultivated Tropical Water Fish, Labeo rohita.

Proteomics has enormous applications in human and animal research. However, proteomic studies in fisheries science are quite scanty particularly for economically important species. Few proteomic studies have been carried out in model fish species, but comprehensive proteomics of aquaculture species are still scarce. This study aimed to perform a comprehensive organ-based protein profiling of important tissue samples for one of the most important aquaculture species,Labeo rohita.Deep proteomic profiling of 17 histologically normal tissues, blood plasma, and embryo provided mass-spectrometric evidence for 8498 proteins at 1% false discovery rate that make up about 26% of the total annotated protein-coding sequences in Rohu. Tissue-wise expression analysis was performed, and the presence of several biologically important proteins was also verified using a targeted proteomic approach. We identified the global post-translational modifications (PTMs) in terms of acetylation (N-terminus and lysine), methylation (N-terminus, lysine, and arginine), and phosphorylation (serine, threonine, and tyrosine) to present a comprehensive proteome resource. An interactive web-based portal has been developed for an overall landscape of protein expression across the studied tissues of Labeo rohita (www.fishprot.org). This draft proteome map of Labeo rohita would advance basic and applied research in aquaculture to meet the most critical challenge of providing food and nutritional security to an increasing world population.

Recent Progress in Lyme Disease and Remaining Challenges.

Lyme disease (also known as Lyme borreliosis) is the most common vector-borne disease in the United States with an estimated 476,000 cases per year. While historically, the long-term impact of Lyme disease on patients has been controversial, mounting evidence supports the idea that a substantial number of patients experience persistent symptoms following treatment. The research community has largely lacked the necessary funding to properly advance the scientific and clinical understanding of the disease, or to develop and evaluate innovative approaches for prevention, diagnosis, and treatment. Given the many outstanding questions raised into the diagnosis, clinical presentation and treatment of Lyme disease, and the underlying molecular mechanisms that trigger persistent disease, there is an urgent need for more support. This review article summarizes progress over the past 5 years in our understanding of Lyme and tick-borne diseases in the United States and highlights remaining challenges.

A comprehensive spectral assay library to quantify the Escherichia coli proteome by DIA/SWATH-MS.

Data-Independent Acquisition (DIA) is a method to improve consistent identification and precise quantitation of peptides and proteins by mass spectrometry (MS). The targeted data analysis strategy in DIA relies on spectral assay libraries that are generally derived from a priori measurements of peptides for each species. Although Escherichia coli (E. coli) is among the best studied model organisms, so far there is no spectral assay library for the bacterium publicly available. Here, we generated a spectral assay library for 4,014 of the 4,389 annotated E. coli proteins using one- and two-dimensional fractionated samples, and ion mobility separation enabling deep proteome coverage. We demonstrate the utility of this high-quality library with robustness in quantitation of the E. coli proteome and with rapid-chromatography to enhance throughput by targeted DIA-MS. The spectral assay library supports the detection and quantification of 91.5% of all E. coli proteins at high-confidence with 56,182 proteotypic peptides, making it a valuable resource for the scientific community. Data and spectral libraries are available via ProteomeXchange (PXD020761, PXD020785) and SWATHAtlas (SAL00222-28).

Whole genome sequence and comparative analysis of Borrelia burgdorferi MM1.

Lyme disease is caused by spirochaetes of the Borrelia burgdorferi sensu lato genospecies. Complete genome assemblies are available for fewer than ten strains of Borrelia burgdorferi sensu stricto, the primary cause of Lyme disease in North America. MM1 is a sensu stricto strain originally isolated in the midwestern United States. Aside from a small number of genes, the complete genome sequence of this strain has not been reported. Here we present the complete genome sequence of MM1 in relation to other sensu stricto strains and in terms of its Multi Locus Sequence Typing. Our results indicate that MM1 is a new sequence type which contains a conserved main chromosome and 15 plasmids. Our results include the first contiguous 28.5 kb assembly of lp28-8, a linear plasmid carrying the vls antigenic variation system, from a Borrelia burgdorferi sensu stricto strain.

mRNA transcript distribution bias between Borrelia burgdorferi bacteria and their outer membrane vesicles.

Borrelia burgdorferi, a bacterium in the spirochete phylum, is the causative agent of Lyme disease. Borrelia burgdorferi has a linear chromosome with a number of circular and linear plasmids. Bacteria, including B. burgdorferi, release spherical outer membrane vesicles (OMVs) that are known to carry secretory products including metabolites, nucleic acids and proteins. Herein, we provide the first comparative transcriptomic analysis of the vesicles released from B. burgdorferi. We identified a total of ∼1200 unique transcripts with at least one mapped read from the bacterial cell and its OMVs. We compared the spectrum of transcripts between bacterial cell and its OMVs, and found a biased distribution based on the source of transcripts, i.e. plasmid-encoded transcripts are more likely to be enriched in the OMVs. We validated the distribution for some of the transcripts by qPCR. This analysis provides the first evidence that some of the B. burgdorferi transcripts are preferentially packaged in OMV, which further suggest that the bacteria might use its OMVs for bacteria-bacteria or bacteria-host communications. This report also suggests a possible involvement of Borrelia-derived OMVs in the development of Lyme disease in both early and post disease syndromes.

Quantitative mass spectrometry analysis reveals a panel of nine proteins as diagnostic markers for colon adenocarcinomas.

Adenocarcinomas are cancers originating from the gland forming cells of the colon and rectal lining, and are known to be the most common type of colorectal cancers. The current diagnosis strategies for colorectal cancers include biopsy, laboratory tests, and colonoscopy which are time consuming. Identification of protein biomarkers could aid in the detection of colon adenocarcinomas (CACs). In this study, tissue proteome of colon adenocarcinomas (n = 11) was compared with the matched control specimens (n = 11) using isobaric tags for relative and absolute quantitation (iTRAQ) based liquid chromatography-mass spectrometry (LC-MS/MS) approach. A list of 285 significantly altered proteins was identified in colon adenocarcinomas as compared to its matched controls, which are associated with growth and malignancy of the tumors. Protein interaction analysis revealed the association of altered proteins in colon adenocarcinomas with various transcription factors and their targets. A panel of nine proteins was validated using multiple reaction monitoring (MRM). Additionally, S100A9 was also validated using immunoblotting. The identified panel of proteins may serve as potential biomarkers and thereby aid in the detection of colon adenocarcinomas.

Identification of Highly Expressed Plasmodium Vivax Proteins from Clinical Isolates Using Proteomics.

Plasmodium vivax is the most geographically widespread species responsible for malaria in humans. Our study focused on identifying highly expressed parasite proteins using a shotgun proteomics approach. Parasites (P. vivax) are isolated from seven patient samples using saponin lysis. Protein extracts from these parasites are processed and subjected to LC-MS/MS analysis. An overall proteome coverage of 605 P. vivax proteins along with 1670 human host proteins are obtained upon combining the data from LC-MS/MS runs. While a major proportion of the P. vivax proteins are either hypothetical or involved in basic cellular activities, few proteins such as tryptophan-rich antigen (Pv-fam-a; PVX_090265), Pv-fam-d protein (PVX_101520), Plasmodium exported protein (PVX_003545), Pvstp1 (PVX_094303) and hypothetical protein (PVX_083555) are detected in more than 80% of the clinical isolates and found to be unique to P. vivax without orthologs in P. falciparum. Our proteomics study on individual parasite isolates reveals highly expressed P. vivax proteins, few of which may be good candidates for vivax malaria diagnosis due to their abundance and absence in P. falciparum. This study represents the first step towards the identification of biomarkers for P. vivax malaria. In future, their clinical diagnostic values must be explored and validated on large patient cohorts.

Global proteomic profiling identifies etoposide chemoresistance markers in non-small cell lung carcinoma.

Chemoresistance is one of the leading health concerns in cancer treatment. Understanding the mechanism of chemoresistance is the best way to improve the survival of the patient. Etoposide and its analogues are widely used as antitumor drugs in lung cancer but many etoposide resistant lung cancer cases has been identified in recent years. The present study aims to explore the cellular response of lung cancer cell lines to etoposide and finding the potential chemoresistant marker proteins. Multiple proteomic platforms like 2-DE, DIGE and iTRAQ have been used to study the global proteome profile of NCI-H460 and etoposide resistant NCI-H460R cell lines. Our study revealed that etoposide treatment leads to alteration of 83 proteins in NCI-H460R cell lines. The functional analysis highlighted the role of the differential expressed proteins in cellular signaling, apoptosis, and cytoskeleton reorganization. Our study has identified several new proteins like RHOC, DLG5, UGDH, TMOD3 in addition to known chemoresistance associated proteins. In silico prediction of the important selected candidates are further validated at protein and mRNA level. Further, functional studies of newly identified candidate genes RHOC and DLG5 revealed that chemotherapeutic resistance is associated with their elevated level and may serve as novel targets for therapeutic intervention. BIOLOGICAL SIGNIFICANCE: Etoposide and its analogues have been used for lung cancer treatment for a while and it was reported that many non small cell lung carcinoma patients are resistant to etoposide. Although etoposide show drug resistance, the exact mechanism was not well understood. The present study focused on the global proteome analysis of NCI-H460 and NCI-H460R cell lines using multiple proteomic platforms to understand the potential chemoresistant markers for etoposide. Our multi-proteomic analysis has showed differential expression of 83 proteins involved in oxidative phosphorylation, metabolic, protein folding, cytoskeleton associated protein along with apoptotic pathway has been identified. In addition, quite a few interesting proteins such as RHOC, DLG5, HSP90, citrate synthase, UDP-glucose-6-dehydrogenase, Tropomodulin-3 are involved in chemoresistance has been observed. Overall, this is the first comprehensive proteomic study on etoposide resistant cell line NCI-H460 to explore the mechanism of chemoresistance in lung cancer.

Meeting Report: "Proteomics from Discovery to Function:" 6th Annual Meeting of Proteomics Society, India and International Conference-A Milestone for the Indian Proteomics Community.

Proteomics is at the epicenter of post-genomics biotechnologies that are currently driving the next generation system science. Moreover, proteomics is a truly global science. The 6(th) Annual Meeting of Proteomics Society, India (PSI) and International Conference on "Proteomics from Discovery to Function" held from December 7-9, 2014, was a transformative endeavor for global proteomics, bringing together the luminaries in the field of proteomics for the very first time in India. This meeting report presents the lessons learned and the highlights of this international scientific conference that was comprised of nine thematic sessions, pre- and post-conference workshops, and an opportunity to cultivate enduring collaborations for proteomics science to benefit both India and global society. The conference had an unforgettable impression on the participants: for the first time, India hosted past and present President and Council members from the Human Proteome Organization (HUPO), along with eminent scientists and young scholars from India and abroad in the field of proteomics at such a large scale, a major highlight of this international event. In all, the PSI 2014 was a milestone conference that has firmly poised the Indian life sciences community as a leading contributor to post-genomics life sciences, thus cultivating crucial trans-generational capacity and inspiration by recognizing the emerging scholars and omics systems scientists who can think and conduct science from cell to society.

Correction: Comprehensive Analysis of Temporal Alterations in Cellular Proteome of Bacillus subtilis under Curcumin Treatment.

[This corrects the article DOI: 10.1371/journal.pone.0120620.].

The quest of the human proteome and the missing proteins: digging deeper.

Given the diverse range of transcriptional and post-transcriptional mechanisms of gene regulation, the estimates of the human proteome is likely subject to scientific surprises as the field of proteomics has gained momentum worldwide. In this regard, the establishment of the "Human Proteome Draft" using high-resolution mass spectrometry (MS), tissue microarrays, and immunohistochemistry by three independent research groups (laboratories of Pandey, Kuster, and Uhlen) accelerated the pace of proteomics research. The Chromosome Centric Human Proteome Project (C-HPP) has taken initiative towards the completion of the Human Proteome Project (HPP) so as to understand the proteomics correlates of common complex human diseases and biological diversity, not to mention person-to-person and population differences in response to drugs, nutrition, vaccines, and other health interventions and host-environment interactions. Although high-resolution MS-based and antibody microarray approaches have shown enormous promises, we are still unable to map the whole human proteome due to the presence of numerous "missing proteins." In December 2014, at the Indian Institute of Technology Bombay, Mumbai the 6(th) Annual Meeting of the Proteomics Society, India (PSI) and the International Proteomics Conference was held. As part of this interdisciplinary summit, a panel discussion session on "The Quest of the Human Proteome and Missing Proteins" was organized. Eminent scientists in the field of proteomics and systems biology, including Akhilesh Pandey, Gilbert S. Omenn, Mark S. Baker, and Robert L. Mortiz, shed light on different aspects of the human proteome drafts and missing proteins. Importantly, the possible reasons for the "missing proteins" in shotgun MS workflow were identified and debated by experts as low tissue expression, lack of enzymatic digestion site, or protein lost during extraction, among other contributing factors. To capture the missing proteins, the experts' collective view was to study the wider tissue range with multiple digesting enzymes and follow targeted proteomics workflow in particular. On the innovation trajectory from the proteomics laboratory to novel proteomics diagnostics and therapeutics in society, we will also need new conceptual frames for translation science and innovation strategy in proteomics. These will embody both technical as well as rigorous social science and humanities considerations to understand the correlates of the proteome from cell to society.

Proteomics research in India: an update.

After a successful completion of the Human Genome Project, deciphering the mystery surrounding the human proteome posed a major challenge. Despite not being largely involved in the Human Genome Project, the Indian scientific community contributed towards proteomic research along with the global community. Currently, more than 76 research/academic institutes and nearly 145 research labs are involved in core proteomic research across India. The Indian researchers have been major contributors in drafting the "human proteome map" along with international efforts. In addition to this, virtual proteomics labs, proteomics courses and remote triggered proteomics labs have helped to overcome the limitations of proteomics education posed due to expensive lab infrastructure. The establishment of Proteomics Society, India (PSI) has created a platform for the Indian proteomic researchers to share ideas, research collaborations and conduct annual conferences and workshops. Indian proteomic research is really moving forward with the global proteomics community in a quest to solve the mysteries of proteomics. A draft map of the human proteome enhances the enthusiasm among intellectuals to promote proteomic research in India to the world.This article is part of a Special Issue entitled: Proteomics in India.

Comprehensive analysis of temporal alterations in cellular proteome of Bacillus subtilis under curcumin treatment.

Curcumin is a natural dietary compound with antimicrobial activity against various gram positive and negative bacteria. This study aims to investigate the proteome level alterations in Bacillus subtilis due to curcumin treatment and identification of its molecular/cellular targets to understand the mechanism of action. We have performed a comprehensive proteomic analysis of B. subtilis AH75 strain at different time intervals of curcumin treatment (20, 60 and 120 min after the drug exposure, three replicates) to compare the protein expression profiles using two complementary quantitative proteomic techniques, 2D-DIGE and iTRAQ. To the best of our knowledge, this is the first comprehensive longitudinal investigation describing the effect of curcumin treatment on B. subtilis proteome. The proteomics analysis revealed several interesting targets such UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1, putative septation protein SpoVG and ATP-dependent Clp protease proteolytic subunit. Further, in silico pathway analysis using DAVID and KOBAS has revealed modulation of pathways related to the fatty acid metabolism and cell wall synthesis, which are crucial for cell viability. Our findings revealed that curcumin treatment lead to inhibition of the cell wall and fatty acid synthesis in addition to differential expression of many crucial proteins involved in modulation of bacterial metabolism. Findings obtained from proteomics analysis were further validated using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) assay for respiratory activity, resazurin assay for metabolic activity and membrane integrity assay by potassium and inorganic phosphate leakage measurement. The gene expression analysis of selected cell wall biosynthesis enzymes has strengthened the proteomics findings and indicated the major effect of curcumin on cell division.

Proteomics analyses of Bacillus subtilis after treatment with plumbagin, a plant-derived naphthoquinone.

Infectious diseases and increasing antibiotic resistance among diverse classes of microbes are global health concerns and a prime focus of omics systems science applications in novel drug discovery. Plumbagin is a plant-derived naphthoquinone, a natural product that exhibits antibacterial activity against gram-positive bacteria. In the present study, we investigated the antimicrobial effects of plumbagin against Bacillus subtilis using two complementary proteomics techniques: two-dimensional electrophoresis (2-DE) and isobaric tag for relative and absolute quantification (iTRAQ). Comparative quantitative proteomics analysis of plumbagin treated and untreated control samples identified differential expression of 230 proteins (1% FDR, 1.5 fold-change and ≥2 peptides) in B. subtilis after plumbagin treatment. Pathway analysis involving the differentially expressed proteins suggested that plumbagin effectively increases heme and protein biosynthesis, whereas fatty acid synthesis was significantly reduced. Gene expression and metabolic activity assays further corroborated the proteomics findings. We anticipate that plumbagin blocks the cell division by altering the membrane permeability required for energy generation. This is the first report, to the best of our knowledge, offering new insights, at proteome level, for the putative mode(s) of action of plumbagin and attendant cellular targets in B. subtilis. The findings also suggest new ways forward for the modern omics-guided drug target discovery, building on traditional plant medicine.

A comprehensive proteomic analysis of totarol induced alterations in Bacillus subtilis by multipronged quantitative proteomics.

The rapid emergence of microbial drug resistance indicates the urgent need for development of new antimicrobial agents. Bacterial cell division machinery is considered as a promising antimicrobial target. Totarol is a naturally existing diterpenoid, which has the ability to restrain bacterial growth by perturbing the cell division. The present study was conducted to investigate the proteomic alterations in Bacillus subtilis as a consequence of totarol treatment to decipher its mechanism of action and possible molecular targets. Cellular proteome of the totarol treated B. subtilis AH75 strain was analyzed by using multiple complementary proteomic approaches. After the drug treatment, 12, 38 and 139 differentially expressed (1.5 fold change) proteins were identified using 2-DE, DIGE and iTRAQ analyses, respectively. In silico functional analysis of the identified differentially expressed proteins indicated a possible effect of totarol on the central metabolism for energy production, heme biosynthesis and chemotaxis. Interestingly, the primary dehydrogenases, which play a vital role in generating the reducing equivalent, were found to be repressed after totarol treatment indicating an apparent metabolic shutdown. Consequently, multiple cellular assays including resazurin assay and FACS analysis of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) staining confirmed the effect of totarol on respiratory activity and cellular metabolism. BIOLOGICAL SIGNIFICANCE: The exact mechanism of action of totarol is still unclear and further investigations are essential to identify the molecular/cellular targets of this potential antimicrobial agent. The present study demonstrates the application of differential proteome to decipher the mechanism of action and molecular targets of totarol in B. subtilis. Our quantitative proteome analysis revealed that totarol induced alterations in the expression levels of 139 proteins (1.5 fold change and ≥2 peptides) in B. subtilis. Findings obtained from this study indicate that totarol treatment leads to metabolic shutdown by repressing the major central metabolic dehydrogenases in B. subtilis. In addition, expression levels of universal chaperone proteins, heme biosynthesis, and ribosomal proteins were found to be altered, which caused the filamentation of the bacteria. To the best of our knowledge, this is the foremost inclusive investigation describing totarol induced alterations in B. subtilis proteome and diverse physiological processes. We anticipate that this in depth proteomic study may contribute to a better understanding of the mode of action of totarol and its primary molecular and cellular targets.