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Background Breast cancer is the most common malignancy in woman and hormone receptor positive breast cancer represents approximately 70% of all cases. These patients are treated with endocrine therapies aimed at disrupting hormone receptor signaling and estrogen production which improves survival and allows a cure in early stages. However, recurrent disease, metastatic dissemination and drug resistance limit the survival of patients. The limitations regarding endocrine therapy have prompted the search for new therapeutic targets, such as CDK4/6 Inhibitors. Despite the improved disease control that CDK4/6 Inhibitors offer to patients, not all patients respond to these drugs and some patients whose tumors respond to CDK4/6 Inhibitors eventually develop acquired resistance. No proven biomarkers of CDK4/6 Inhibitors efficacy exist to date, and there is a need for diagnostic tools that could stratify patients to save costs and the burden of unnecessary therapy. Our aim is to perform a quantitative evaluation of marker proteins with a developed multiplexed panel using targeted mass spectrometry (MS)-based proteomics for 25 proteins from the CDK/RB/E2F-pathway which have been shown in the literature to be central to CDK4/6I resistance. Material and Methods We developed Multiple Reaction Monitoring (MRM) MS methods for the 25 target proteins from the CDK/RB/E2F-pathway using synthetic heavy-isotope-labeled standards with the aim of creating MRM assays to enable specific, sensitive and precise quantitation of these proteins in small amounts of cell line and tissue samples. Moreover, we developed a high resolution peptide fractionation system using high-pH micro-flow liquid chromatography (LC) which is required to overcome the problem of small samples amounts while improving analytical assay sensitivity in the analysis of complex biological matrices such as breast cancer biopsies. The MCF-7 human breast cancer cell line was used as model during method development. Proteins from cell lysates were isolated, reduced, alkylated and digested with trypsin. The resulting tryptic peptides were micro-flow fractionated into 70 fractions and the developed nano-LC-MS MRM assays were used for peptide detection and quantification. Data were analyzed using Skyline. Results Our developed micro-flow fractionation method allowed us to work on limited amounts of samples (60ug), and increased the possibility to detecting low abundance proteins such as cell cycle components. Using the MCF-7 cell model, we are able to identify and quantify 17 proteins out of the 25 from our panel: CDK1, CDK2, CDK4, Cyclin B1, Cyclin D1, Cyclin D3, Cyclin E1, RB1, E2F-3, E2F-4, E2F-5, ESR1, TOP2A, TYMS, EZH2, MKI67, BIRC5. Conclusion We have developed a highly specific MS-based multiplexed assay with peptide standards targeting 25 proteins relevant to CDK4/6 Inhibitors breast cancer treatment. Our micro-flow fractionation method increased assay sensitivity and allows for the analysis of small sample amounts. In the future we will apply this workflow to samples such as Patient Derived Xenografts models, breast cancer tissues and FFPE samples in order to identify the predictive value of these potential biomarkers for responsiveness to CDK4/6 Inhibitors. Citation Format: Marta Zurawska, Adriana Aguilar-Mahecha, Mark Basik, Michal Dadlez, Dominik Domanski. Development of a multiplexed protein panel using a targeted proteomics approach for the study of CDK4/6 inhibitors resistance in hormone receptor positive breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-48.

Simple Summary Our study aimed to advance the understanding of vulvar squamous cell carcinoma (VSCC) biology by recognizing biological pathways that drive the progression of this disease. We applied the experimental path from global proteomic analysis of vulvar tumors to the targeted and quantitative assessment of specific proteins both in the tumors and blood of VSCC patients. The proteomic analysis has advanced the knowledge on VSCC biology by pointing at inflammation as a driver of progression and by providing grounds for the hypothesis of vulvovaginal microflora disturbances as a trigger for the inflammatory response. The study results indicate prognostic protein markers and potential therapeutic targets for improved and personalized management of VSCC. Abstract Current knowledge on the biology of squamous cell vulvar carcinoma (VSCC) is limited. We aimed to identify protein markers of VSCC tumors that would permit to stratify patients by progression risk. Early-stage tumors from patients who progressed (progVSCC) and from those who were disease-free (d-fVSCC) during follow-up, along with normal vulvar tissues were examined by mass spectrometry-based proteomics. Differentially expressed proteins (DEPs) were then verified in solid tissues and blood samples of patients with VSCC tumors and vulvar premalignant lesions. In progVSCC vs. d-fVSCC tumors, the immune response was the most over-represented Gene Ontology category for the identified DEPs. Pathway profiling suggested bacterial infections to be linked to aggressive VSCC phenotypes. High Mobility Group AT-Hook 2 (HMGA2) and Proteinase 3 (PRTN3) were revealed as proteins predicting VSCC progression. HMGA2 and PRTN3 abundances are associated with an aggressive phenotype, and hold promise as markers for VSCC patient stratification. It appears that vulvovaginal microflora disturbances trigger an inflammatory response contributing to cancer progression, suggesting that bacterial rather than viral infection status should be considered in the development of targeted therapies in VSCC.

Background A knowledge on the biology of squamous cell vulvar carcinoma (VSCC) is limited. We aimed to identify protein markers of VSCC tumors stratifying patients by progression risk. Early stage VSCC tumors from patients who progressed (progVSCC) and from those who were disease-free (d-fVSCC) during follow-up, along with normal vulvar tissues were examined by mass spectrometry-based proteomics.Results In progVSCC vs d-fVSCC tumors, the immune response was the most over-represented Gene Ontology category for the identified DEPs. Interestingly, pathway profiling suggested bacterial infections to be linked to aggressive VSCC phenotypes. Differentially expressed proteins (DEPs) were verified in solid tissues and blood samples of patients with VSCC tumors and vulvar premalignant lesions. High Mobility Group AT-Hook 2 (HMGA2) and Proteinase 3 (PRTN3) were revealed as protein markers predicting VSCC progression.Conclusions HMGA2 and PRTN3 abundances are associated with aggressive VSCC phenotype, and hold promise as markers for patient stratification. It appears that vulvovaginal microflora disturbances trigger inflammatory response contributing to cancer progression.

Offline high-pH reversed-phase fractionation is widely used to reduce sample complexity in proteomic workflows. This is due to the semi-orthogonality and high peak resolution of the two separations. Offline 2D fractionation, however, is low throughput and requires several manual manipulations and is prone to sample losses. To address these issues, we developed an online two dimensional high-pH - low-pH reversed-phase-reversed-phase (2D RPRP) LC-MRM method whereby hundreds of peptides can be quantified in a single LC-MS/MS injection. The method allowed the reproducible and sensitive quantitation of a test panel of 367 peptides (168 proteins) from undepleted and non-enriched human plasma. Of these, we were able to detect and quantify 95 peptides (29 proteins) by 2D-RPRP that were not detectable by 1D LC-MRM-MS. Online 2D RPRP resulted in an average increase of roughly 10-fold in sensitivity compared to traditional 1D low-pH separations, while improving reproducibility and sample throughput relative to offline 2D RPRP by factors of 1.7 and 5, respectively, compared to offline 2D RPRP. This paper serves as proof-of-concept of the feasibility and efficacy of online 2D RPRP at analytical flow rates for highly multiplexed targeted proteomic analyses.

Pleural effusion (PE), excess fluid in the pleural space, is often observed in lung cancer patients and also forms due to many benign ailments. Classifying it quickly is critical, but this remains an analytical challenge often lengthening the diagnosis process or exposing patients to unnecessary risky invasive procedures. We tested the analysis of PE using a multiplexed cytokeratin (CK) panel with targeted mass spectrometry–based quantitation for its rapid classification. CK markers are often assessed in pathological examinations for cancer diagnosis and guiding treatment course. We developed methods to simultaneously quantify 33 CKs in PE using peptide standards for increased analytical specificity and a simple CK enrichment method to detect their low amounts. Analyzing 121 PEs associated with a variety of lung cancers and noncancerous causes, we show that abundance levels of 10 CKs can be related to PE etiology. CK-6, CK-7, CK-8, CK-18, and CK-19 were found at significantly higher levels in cancer-related PEs. Additionally, elevated levels of vimentin and actin differentiated PEs associated with bacterial infections. A classifier algorithm effectively grouped PEs into cancer-related or benign PEs with 81% sensitivity and 79% specificity. A set of undiagnosed PEs showed that our method has potential to shorten PE diagnosis time. For the first time, we show that a cancer-relevant panel of simple-epithelial CK markers currently used in clinical assessment can also be quantitated in PEs. Additionally, while requiring less invasive sampling, our methodology demonstrated a significant ability to identify cancer-related PEs in clinical samples and thus could improve patient care in the future.

Seed germination provides an excellent model to study the process of mitochondrial biogenesis. It is a complex and strictly regulated process which requires a proper biogenesis of fully active organelles from existing promitochondrial structures. We have previously reported that the lack of the inner mitochondrial membrane protease FTSH4 delayed Arabidopsis seed germination. Here, we implemented a targeted mass spectrometry-based approach, Multiple Reaction Monitoring (MRM), with stable-isotope-labeled standard peptides for increased sensitivity, to quantify mitochondrial proteins in dry and germinating wild-type and ftsh4 mutant seeds, lacking the FTSH4 protease. Using total seed protein extracts we measured the abundance of the peptide targets belonging to the OXPHOS complexes, AOX1A, transport, and inner membrane scaffold as well as mitochondrial proteins that are highly specific to dry and germinating seeds. The MRM assay showed that the abundance of these proteins in ftsh4 did not differ substantially from that observed in wild-type at the level of dry seed and after stratification, but we observed a reduction in protein abundance in most of the examined OXPHOS subunits in the later stages of germination. These changes in OXPHOS protein levels in ftsh4 mutants were accompanied by a lower cytochrome pathway activity as well as an increased AOX1A amount at the transcript and protein level and alternative pathway activity. The analyses of the steady-state transcript levels of mitochondrial and nuclear genes encoding OXPHOS subunits did not show significant difference in their amount, indicating that the observed changes in the OXPHOS occurred at the post-transcriptional level. At the time when ftsh4 seeds were fully germinated, the abundance of the OXPHOS proteins in the mutant was either slightly lowered or comparable to these amounts in wild-type seeds at the similar developmental stage. By the implementation of an integrative approach combining targeted proteomics, quantitative transcriptomics, and physiological studies we have shown that the FTSH4 protease has an important role in the biogenesis of OXPHOS and thus biogenesis of mitochondria during germination of Arabidopsis seeds.

We present a novel way to select for highly polygenic traits. For millennia, humans have used observable phenotypes to selectively breed stronger or more productive livestock and crops. Selection on genotype, using single-nucleotide polymorphisms (SNPs) and genome profiling, is also now applied broadly in livestock breeding programs; however, selection on protein/peptide or mRNA expression markers has not yet been proven useful. Here we demonstrate the utility of protein markers to select for disease-resistant hygienic behavior in the European honey bee (Apis mellifera L.). Robust, mechanistically-linked protein expression markers, by integrating cis- and trans- effects from many genomic loci, may overcome limitations of genomic markers to allow for selection. After three generations of selection, the resulting marker-selected stock outperformed an unselected benchmark stock in terms of hygienic behavior, and had improved survival when challenged with a bacterial disease or a parasitic mite, similar to bees selected using a phenotype–based assessment for this trait. This is the first demonstration of the efficacy of protein markers for industrial selective breeding in any agricultural species, plant or animal.

Purpose The goal of this work was to develop an LC-MRM assay for the quantitative analysis of a set of established and diagnostically important cytokeratin (CK) markers used in cancer diagnosis, prognosis, and therapy monitoring. Second, the potential of this assay in lung cancer diagnosis through pleural effusion (PE) analysis was examined. Experimental design A multiplexed MRM assay was developed for 17 CKs and their select caspase-cleaved fragments. Isotope-labeled standard peptides were used for high assay specificity and absolute peptide quantitation; with robust standard-flow LC coupled to a latest-generation triple-quadrupole instrument for high sensitivity. The potential clinical applicability was demonstrated by the analysis of 118 PE samples. Results The MRM assay was evaluated for endogenous detection, linearity, precision, upper and lower limits of quantification, selectivity, reproducibility and peptide stability, and is generally applicable to any epithelial cancer study. A set of 118 patients with known pathologies allowed us to define the range of CK levels in clinical PE samples. Specific CKs were able to differentiate cancer-related PEs from those caused by benign ailments. In addition, they allowed to differentiate between PEs from subjects with small cell lung cancer versus non-small cell lung carcinoma, and to further differentiate the latter into its two subtypes, adenocarcinoma and squamous cell carcinoma. Conclusion and clinical relevance An MRM-based CK assay for carcinoma studies can differentiate between the three lung cancer histological types using less-invasive PE sampling providing potential therapy-guiding information on patients that are inoperable.

There exists a growing demand in the proteomics community to standardize experimental methods and liquid chromatography–mass spectrometry (LC/MS) platforms in order to enable the acquisition of more precise and accurate quantitative data. This necessity is heightened by the evolving trend of verifying and validating candidate disease biomarkers in complex biofluids, such as blood plasma, through targeted multiple reaction monitoring (MRM)-based approaches with stable isotope-labeled standards (SIS). Considering the lack of performance standards for quantitative plasma proteomics, we previously developed two reference kits to evaluate the MRM with SIS peptide approach using undepleted and non-enriched human plasma. The first kit tests the effectiveness of the LC/MRM-MS platform (kit #1), while the second evaluates the performance of an entire analytical workflow (kit #2). Here, these kits have been refined for practical use and then evaluated through intra- and inter-laboratory testing on 6 common LC/MS platforms. For an identical panel of 22 plasma proteins, similar concentrations were determined, regardless of the kit, instrument platform, and laboratory of analysis. These results demonstrate the value of the kit and reinforce the utility of standardized methods and protocols. Biological significance The proteomics community needs standardized experimental protocols and quality control methods in order to improve the reproducibility of MS-based quantitative data. This need is heightened by the evolving trend for MRM-based validation of proposed disease biomarkers in complex biofluids such as blood plasma. We have developed two kits to assist in the inter- and intra-laboratory quality control of MRM experiments: the first kit tests the effectiveness of the LC/MRM-MS platform (kit #1), while the second evaluates the performance of an entire analytical workflow (kit #2). In this paper, we report the use of these kits in intra- and inter-laboratory testing on 6 common LC/MS platforms. This article is part of a Special Issue entitled: Standardization and Quality Control in Proteomics.

We present a novel way to select for highly polygenic traits. For millennia, humans have used observable phenotypes to selectively breed stronger or more productive livestock and crops. Selection on genotype, using single-nucleotide polymorphisms (SNPs) and quantitative trait loci (QTLs), is also now applied broadly in livestock breeding programs; however, selection on protein or mRNA expression markers have not been proved useful yet. Here we demonstrate the utility of protein markers to select for disease-resistant behaviour in the European honey bees (Apis melliferaL.). Robust, mechanistically-linked protein expression markers, by integrating cis and trans effects from many genomic loci, may overcome limitations of genomic markers to allow for selection. After three generations of selection, the resulting stock performed as well or better than bees selected using phenotype–based assessment of this trait, when challenged with disease. This is the first demonstration of the efficacy of protein markers for selective breeding in any agricultural species, plant or animal.Significance statementThe honey bee has been in the news a lot recently, largely because of world-wide die-offs due to the parasitic Varroa mite, which is becoming resistant to the chemical controls the bee industry uses. In this study, we show that robust expression biomarkers of a disease-resistance trait can be used, in an out-bred population, to select for that trait. After three generations of selection, the resulting stock performed as well or better than bees selected using the phenotypic best method for assessing this trait when challenged with disease. This is the first demonstration of an expression marker for selective breeding in any agricultural species, plant or animal. This also represents a completely novel way to select for highly polygenic traits.

Three common urological diseases are bladder cancer, urinary tract infection, and hematuria. Seventeen bladder cancer biomarkers were previously discovered using iTRAQ - these findings were verified by MRM-MS in this current study. Urine samples from 156 patients with hernia (n=57, control), bladder cancer (n=76), or urinary tract infection/hematuria (n=23) were collected and subjected to multiplexed LC-MRM/MS to determine the concentrations of 63 proteins that are normally considered to be plasma proteins, but which include proteins found in our earlier iTRAQ study. Sixty-five stable isotope-labeled standard proteotypic peptides were used as internal standards for 63 targeted proteins. Twelve proteins showed higher concentrations in the bladder cancer group than in the hernia and the urinary tract infection/hematuria groups, and thus represent potential urinary biomarkers for detection of bladder cancer. Prothrombin had the highest AUC (0.796), with 71.1% sensitivity and 75.0% specificity for differentiating bladder cancer (n=76) from non-cancerous (n=80) patients. The multiplexed MRM-MS data was used to generate a six-peptide marker panel. This six-peptide panel (afamin, adiponectin, complement C4 gamma chain, apolipoprotein A-II precursor, ceruloplasmin, and prothrombin) can discriminate bladder cancer subjects from non-cancerous subjects with an AUC of 0.814, with a 76.3% positive predictive value, and a 77.5% negative predictive value. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.

In this study we demonstrate the use of a multiplexed MRM-based assay to distinguish among normal (NL) and iron-metabolism disorder mouse models, particularly, iron-deficiency anemia (IDA), inflammation (INFL), and inflammation and anemia (INFL + IDA). Our initial panel of potential biomarkers was based on the analysis of 14 proteins expressed by candidate genes involved in iron transport and metabolism. Based on this study, we were able to identify a panel of 8 biomarker proteins: apolipoprotein A4 (APO4), transferrin, transferrin receptor 1, ceruloplasmin, haptoglobin, lactoferrin, hemopexin, and matrix metalloproteinase-8 (MMP8) that clearly distinguish among the normal and disease models. Within this set of proteins, transferrin showed the best individual classification accuracy over all samples (72%) and within the NL group (94%). Compared to the best single-protein biomarker, transferrin, the use of the composite 8-protein biomarker panel improved the classification accuracy from 94% to 100% in the NL group, from 50% to 72% in the INFL group, from 66% to 96% in the IDA group, and from 79% to 83% in the INFL + IDA group. Based on these findings, validation of the utility of this potentially important biomarker panel in human samples in an effort to differentiate IDA, inflammation, and combinations thereof, is now warranted. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.

The analytical performance of a standard-flow ultra-high-performance liquid chromatography (UHPLC) and a nano-flow high-performance liquid chromatography (HPLC) system, interfaced to the same state-of-the-art triple-quadrupole mass spectrometer, were compared for the multiple reaction monitoring (MRM)-mass spectrometry (MS)-based quantitation of a panel of 48 high-to-moderate-abundance cardiovascular disease-related plasma proteins. After optimization of the MRM transitions for sensitivity and testing for chemical interference, the optimum sensitivity, loading capacity, gradient, and retention-time reproducibilities were determined. We previously demonstrated the increased robustness of the standard-flow platform, but we expected that the standard-flow platform would have an overall lower sensitivity. This study was designed to determine if this decreased sensitivity could be compensated for by increased sample loading. Significantly fewer interferences with the MRM transitions were found for the standard-flow platform than for the nano-flow platform (2 out of 103 transitions compared with 42 out of 103 transitions, respectively), which demonstrates the importance of interference-testing when nano-flow systems are used. Using only interference-free transitions, 36 replicate LC/MRM-MS analyses resulted in equal signal reproducibilities between the two platforms (9.3 % coefficient of variation (CV) for 88 peptide targets), with superior retention-time precision for the standard-flow platform (0.13 vs. 6.1 % CV). Surprisingly, for 41 of the 81 proteotypic peptides in the final assay, the standard-flow platform was more sensitive while for 9 of 81 the nano-flow platform was more sensitive. For these 81 peptides, there was a good correlation between the two sets of results (R 2 = 0.98, slope = 0.97). Overall, the standard-flow platform had superior performance metrics for most peptides, and is a good choice if sufficient sample is available.

Conifers are extremely long-lived plants that have evolved complex chemical defenses in the form of oleoresin terpenoids to resist attack from pathogens and herbivores. In these species, terpenoid diversity is determined by the size and composition of the terpene synthase (TPS) gene family and the single- and multi-product profiles of these enzymes. The monoterpene (+)-3-carene is associated with resistance of Sitka spruce (Picea sitchensis) to white pine weevil (Pissodes strobi). We used a combined genomic, proteomic and biochemical approach to analyze the (+)-3-carene phenotype in two contrasting Sitka spruce genotypes. Resistant trees produced significantly higher levels of (+)-3-carene than susceptible trees, in which only trace amounts were detected. Biosynthesis of (+)-3-carene is controlled, at the genome level, by a small family of closely related (+)-3-carene synthase (PsTPS-3car) genes (82-95% amino acid sequence identity). Transcript profiling identified one PsTPS-3car gene (PsTPS-3car1) that is expressed in both genotypes, one gene (PsTPS-3car2) that is expressed only in resistant trees, and one gene (PsTPS-3car3) that is expressed only in susceptible trees. The PsTPS-3car2 gene was not detected in genomic DNA of susceptible trees. Target-specific selected reaction monitoring confirmed this pattern of differential expression of members of the PsTPS-3car family at the proteome level. Kinetic characterization of the recombinant PsTPS-3car enzymes identified differences in the activities of PsTPS-3car2 and PsTPS-3car3 as a factor contributing to the different (+)-3-carene profiles of resistant and susceptible trees. In conclusion, variation of the (+)-3-carene phenotype is controlled by copy number variation of PsTPS-3car genes, variation of gene and protein expression, and variation in catalytic efficiencies.

Introduction The application of the concept of tumour-specific markers to assess a possible disease outcome and to choose appropriate treatment options, is still obstructed by the limited knowledge on vulvar carcinoma (VC) biology. We aimed to identify protein markers of VC that would be indicative of a tumour that is more likely to progress. Material and methods Primary tumour samples from 28 patients with early stage squamous cell VC and 14 samples of normal vulvar tissue were studied using iTRAQ analysis. The results obtained for tumour samples of VC patients that progressed during 8–12 years of follow-up period (‘progVC’, n=14) were be compared to those obtained for samples of patients who were disease-free at the time of last observation (‘d-fVC’, n=14). The differentially expressed proteins were subsequently validated using targeted proteomics methods (PRM) and immunohistochemistry using a larger sample set. Results and discussions 5510 proteins were identified in the analysed samples. Gene Ontology (GO) analysis of the proteins differentially expressed in progVC and d-fVC tumours proteins indicated an immune response as the most over-represented GO category in this sample comparison. The top 47 candidate markers were chosen for further validation. Correlation of the validation results with clinical parameters of the enrolled VC patients indicated that HMGA2, ANO1, PRTN3, UBE2C, ABI3BP and PRELP should be considered as potential protein markers for the prediction of progression in VC patients. Conclusion Our findings provide evidence showing that deregulation of eight proteins’ abundance is significantly associated with aggressive phenotype of VC. Their immunohistochemical assessment hold promise as a patient stratification tool. Acknowledgement This work was supported by the Polish National Science Centre grant No. 2013/10/E/NZ5/00663.

Induction of terpene synthase (TPS) gene expression and enzyme activity is known to occur in response to various chemical and biological stimuli in several species of spruce (genus Picea). However, high sequence identity between TPS family members has made it difficult to determine the induction patterns of individual TPS at the protein and transcript levels and whether specific TPS enzymes respond differentially to treatment. In the present study we used a multi-level approach to measure the induction and activity of TPS enzymes in protein extracts of Norway spruce (Picea abies) bark tissue following treatment with methyl jasmonate (MeJA). Measurements were made on the transcript, protein, enzyme activity and metabolite levels. Using a relatively new proteomics application, selective reaction monitoring (SRM), it was possible to differentiate and quantitatively measure the abundance of several known TPS proteins and three 1-deoxy-D-xylulose 5-phosphate synthase (DXS) isoforms in Norway spruce. Protein levels of individual TPS and DXS enzymes were differentially induced upon MeJA treatment and good correlation was generally observed between induction of transcripts, proteins, and enzyme activities. Most of the mono- and diterpenoid metabolites accumulated with similar temporal patterns of induction as part of the coordinated multi-compound chemical defense response. Protein and enzyme activity levels of the monoTPS (+)-3-carene synthase and the corresponding accumulation of (+)-3-carene was induced to a higher fold change than any other TPS or metabolite measured, indicating an important role in the induced terpenoid defense response in Norway spruce.

There is a need for the development of a rapid method for identifying chemicals that disrupt thyroid hormone (TH) action while maintaining complex tissue structure and biological variation. Moreover, no assay to date allows a simultaneous screen of an individual's response to multiple chemicals. A cultured tail fin biopsy or C-fin assay was developed using Rana catesbeiana tadpoles. Multiple tail fin biopsies were taken per tadpole, cultured in serum-free medium, and then each biopsy was exposed to a different treatment condition. The effects of known disruptors of TH action were evaluated in the C-fin assay. Chemical exposure was performed +/- 10 nM 3,3',5-triiodothyronine and real-time quantitative polymerase chain reaction (QPCR) of two TH-responsive transcripts, TH receptor beta (TRbeta) and the Rana larval keratin type I (RLKI), was performed. Within 48 h of exposure to Triac (1-100 nM), roscovitine (0.6-60 microM), or genistein (1-100 microM), perturbations in TH signaling were detected. Tetrabromobisphenol A (TBBPA) (10-1,000 nM) showed no effect. Acetochlor (1-100 nM) elicited a modest effect on the TH-dependent induction of TRbeta transcript. These data reveal that a direct tissue effect may not be critical for TBBPA and acetochlor to disrupt TH action previously observed in intact tadpoles.

The involvement of phosphorylation signaling pathways in postembryonic development of the frog is poorly understood. The thyroid hormone, 3, 5, 3′-triiodothyronine (T3), is essential for inducing tadpole metamorphosis and we show that the cyclin-dependent kinase (Cdk) inhibitor, roscovitine, prevents T3-dependent regression of cultured tail tips from Rana catesbeiana tadpoles. Using tail tips from precociously induced and naturally metamorphosing tadpoles, our data suggest that protein phosphorylation is important in the establishment of the T3-dependent proapoptotic gene expression program. Our evidence indicates that Cdk8 is the most likely candidate for this proapoptotic activity with the expression of its regulatory subunit, cyclin C, identified as a novel T3-responsive gene. We suggest that this activity is crucial for the genetic reprogramming required for tail regression and demonstrate that protein phosphorylation is important in T3-induced apoptosis in normal cells during development. Developmental Dynamics 237:3787–3797, 2008. © 2008 Wiley-Liss, Inc.

Thyroid hormone (TH)-regulated gene expression is mainly mediated by TH binding to nuclear thyroid hormone receptors (TRs). Despite extensive studies in mammalian cell lines that show that phosphorylation signaling pathways are important in TH action, little is known about their roles on TH signaling in vivo during development. Anuran metamorphosis is a postembryonic process that is absolutely dependent upon TH and tadpole tail resorption can be precociously induced by exogenous administration of 3,5,3′-triiodothyronine (T3). We demonstrate that genistein (a major isoflavone in soy products and tyrosine kinase inhibitor) and the PKC inhibitor (H7) prevent T3-induced regression of the Rana catesbeiana tadpole tail. T3-induced protein kinase C tyrosine phosphorylation and kinase activity are inhibited by genistein while T3-induced up-regulation of TRβ mRNA, but not TRα mRNA, is significantly attenuated, most likely through inhibition of T3-dependent phosphorylation of the TRα protein. This phosphorylation may be modulated through PKC. These data demonstrate that T3 signaling in the context of normal cells in vivo includes phosphorylation as an important factor in establishing T3-dependent tail regression during development. Developmental Dynamics 236:777–790, 2007. © 2007 Wiley-Liss, Inc.

Thyroid hormones (THs) are critical for the growth, development, and homeostasis of many organisms and are necessary for metamorphosis of Xenopus laevis tadpoles. TH-induced metamorphosis requires alterations in the transcriptome and the proteome. However, only a few of the molecular components of this developmental program have been identified and their interrelationship remains unclear. Using a cDNA array comprised of 420 known anuran genes and quantitative PCR, we have identified 93 TH-responsive genes in the tail of premetamorphic tadpoles after exogenous administration of T3. Fifty-three of these mRNA transcripts have not previously been characterized as TH responsive in any species. The gene expression profiles show distinctive temporal patterns with most transcript steady-state levels increasing after induction of metamorphosis. Two-dimensional gel electrophoresis of total protein extracts from the tail shows changes in steady-state levels of many proteins after T3 treatment. Of the up-regulated proteins, 10 were identified by peptide mass mapping. These data identify potential components involved in the regulation of Xenopus tail regression by T3 and begin to address a critical question regarding the interrelationship between the transcriptome and the proteome in TH-dependent developmental processes.

One challenge in Multiple Reaction Monitoring (MRM)-based proteomics is to select the most appropriate surrogate peptides to represent a target protein. We present here a software package to automatically generate these most appropriate surrogate peptides for an LC/MRM–MS analysis. Our method integrates information about the proteins, their tryptic peptides, and the suitability of these peptides for MRM which is available online in UniProtKB, NCBI's dbSNP, ExPASy, PeptideAtlas, PRIDE, and GPMDB. The scoring algorithm reflects our knowledge in choosing the best candidate peptides for MRM, based on the uniqueness of the peptide in the targeted proteome, its physiochemical properties, and whether it previously has been observed. The modularity of the workflow allows further extension and additional selection criteria to be incorporated. We have developed a simple Web interface where the researcher provides the protein accession number, the subject organism, and peptide-specific options. Currently, the software is designed for human and mouse proteomes, but additional species can be easily be added. Our software improved the peptide selection by eliminating human error, considering multiple data sources and all of the isoforms of the protein, and resulted in faster peptide selection — approximately 50 proteins per hour compared to 8 per day. Biological significance Compiling a list of optimal surrogate peptides for target proteins to be analyzed by LC/MRM–MS has been a cumbersome process, in which expert researchers retrieved information from different online repositories and used their own reasoning to find the most appropriate peptides. Our scientific workflow automates this process by integrating information from different data sources including UniProt, Global Proteome Machine, NCBI's dbSNP, and PeptideAtlas, simulating the researchers' reasoning, and incorporating their knowledge of how to select the best proteotypic peptides for an MRM analysis. The developed software can help to standardize the selection of peptides, eliminate human error, and increase productivity.

Targeted proteomics using multiple reaction monitoring (MRM) has become the method of choice for the rapid, precise, and reproducible quantitation of proteins in complex biological matrices. MRM-based targeted proteomics allows protein profiling in complex biological samples and accurate comparisons of between samples [1-6], typically using a bottom-up LC-MS/MS approach [7]. While relative quantitation approaches report differences in fold-changes between the samples being compared, absolute methods measure the concentrations [8], ideally based on isotopically labeled internal standard peptides that are analogues of the endogenous proteotypic target peptides [9]. The use of stable-isotope labeled standard (SIS) peptides is a key factor in the precision and reproducibility of absolute quantitation experiments using LC/MRM-MS, but the results may still not be accurate. Experimentally derived concentrations for specific tissues or biofluids can vary as a function of digestion protocol, or even which peptides are selected to represent a given protein [10-12]. This article is protected by copyright. All rights reserved

Multiple reaction monitoring (MRM), sometimes called selected reaction monitoring (SRM), is a directed tandem mass spectrometric technique performed on to triple quadrupole mass spectrometers. MRM assays can be used to sensitively and specifically quantify proteins based on peptides that are specific to the target protein. Stable-isotope-labeled standard peptide analogues (SIS peptides) of target peptides are added to enzymatic digests of samples, and quantified along with the native peptides during MRM analysis. Monitoring of the intact peptide and a collision-induced fragment of this peptide (an ion pair) can be used to provide information on the absolute peptide concentration of the peptide in the sample and, by inference, the concentration of the intact protein. This technique provides high specificity by selecting for biophysical parameters that are unique to the target peptides: (1) the molecular weight of the peptide, (2) the generation of a specific fragment from the peptide, and (3) the HPLC retention time during LC/MRM-MS analysis. MRM is a highly sensitive technique that has been shown to be capable of detecting attomole levels of target peptides in complex samples such as tryptic digests of human plasma. This chapter provides a detailed description of how to develop and use an MRM protein assay. It includes sections on the critical "first step" of selecting the target peptides, as well as optimization of MRM acquisition parameters for maximum sensitivity of the ion pairs that will be used in the final method, and characterization of the final MRM assay.

There are currently no widely accepted biomarkers for non-invasive diagnosis or screening of bladder cancer. There is, therefore, a compelling need to develop more reliable bladder cancer biomarkers, particularly those which can be measured in body fluids. In this book chapter, we describe the proteomic workflow which we used to develop a non-invasive assay for the detection of human bladder tumor in urine specimens. A six-protein biomarker panel was generated by a combination of untargeted mass-spectrometry-based biomarker discovery using an “isobaric tags for relative and absolute quantitation” (iTRAQ) platform, and subsequent biomarker verification using a targeted multiple-reaction-monitoring mass spectrometry (MRM-MS) approach.

Multiple reaction monitoring (MRM), sometimes called selected reaction monitoring (SRM), is a directed tandem mass spectrometric technique performed on to triple quadrupole mass spectrometers. MRM assays can be used to sensitively and specifically quantify proteins based on peptides that are specific to the target protein. Stable-isotope-labeled standard peptide analogues (SIS peptides) of target peptides are added to enzymatic digests of samples, and quantified along with the native peptides during MRM analysis. Monitoring of the intact peptide and a collision-induced fragment of this peptide (an ion pair) can be used to provide information on the absolute peptide concentration of the peptide in the sample and, by inference, the concentration of the intact protein. This technique provides high specificity by selecting for biophysical parameters that are unique to the target peptides: (1) the molecular weight of the peptide, (2) the generation of a specific fragment from the peptide, and (3) the HPLC retention time during LC/MRM-MS analysis. MRM is a highly sensitive technique that has been shown to be capable of detecting attomole levels of target peptides in complex samples such as tryptic digests of human plasma. This chapter provides a detailed description of how to develop and use an MRM protein assay. It includes sections on the critical "first step" of selecting the target peptides, as well as optimization of MRM acquisition parameters for maximum sensitivity of the ion pairs that will be used in the final method, and characterization of the final MRM assay.

β-escin is a mixture of triterpene saponins isolated from the horse chestnut seeds (Aesculus hippocastanum L.). The anti-edematous, anti-inflammatory and venotonic properties of β-escin have been the most extensively clinically investigated effects of this plant-based drug and randomized controlled trials have proved the efficacy of β-escin for the treatment of chronic venous insufficiency. However, despite the clinical recognition of the drug its pharmacological mechanism of action still remains largely elusive. To determine the cellular and molecular basis for the therapeutic effectiveness of β-escin we performed discovery and targeted proteomic analyses and in vitro evaluation of cellular and molecular responses in human endothelial cells under inflammatory conditions. Our results demonstrate that in endothelial cells β-escin potently induces cholesterol synthesis which is rapidly followed with marked fall in actin cytoskeleton integrity. The concomitant changes in cell functioning result in a significantly diminished responses to TNF-α stimulation. These include reduced migration, alleviated endothelial monolayer permeability, and inhibition of NFκB signal transduction leading to down-expression of TNF-α-induced effector proteins. Moreover, the study provides evidence for novel therapeutic potential of β-escin beyond the current vascular indications.

Clinical biomarker discovery, verification, and validation are facilitated by the latest technological advances in mass spectrometry. It is now possible to analyze simultaneously group of tens or hundreds of biomarkers in a blood sample using multiple reaction monitoring (MRM), a tandem mass spectrometric method. However, these newly-developed methods face new challenges, including standardization, calibration, and the determination of analytical and biological variation. Here we illustrate the background, pre-analytical sample preparation, and biomarker assay development using an MRM-mass spectrometric method. In addition, special attention is given to future standardization methods to enable widespread use of the technology.

A highly-multiplexed MRM-based assay for determination of cardiovascular disease (CVD) status and disease classification has been developed for clinical research. A high-flow system using ultra-high performance LC and an Agilent 6490 triple quadrupole mass spectrometer, equipped with an ion funnel, provided ease of use and increased the robustness of the assay. The assay uses 135 stable isotope-labeled peptide standards for the quantitation of 67 putative biomarkers of CVD in tryptic digests of whole plasma in a 30-min assay. Eighty-five analyses of the same sample showed no loss of sensitivity (20% CV for 134/135 peptides) and no loss of retention time accuracy (0.5% CV for all peptides). The maximum linear dynamic range of the MRM assays ranged from 10(3) -10(5) for 106 of the assays. Excellent linear responses (r0.98) were obtained for 117 of the 135 peptide targets with attomole level limits of quantitation (20% CV and accuracy 80-120%) for 81 of the 135 peptides. The assay presented in this study is easy to use, robust, sensitive, and has high-throughput capabilities through short analysis time and complete automated sample preparation. It is therefore well suited for CVD biomarker validation and discovery in plasma.

Clinical biomarker discovery, verification, and validation are facilitated by the latest technological advances in mass spectrometry. It is now possible to analyze simultaneously group of tens or hundreds of biomarkers in a blood sample using multiple reaction monitoring (MRM), a tandem mass spectrometric method. However, these newly-developed methods face new challenges, including standardization, calibration, and the determination of analytical and biological variation. Here we illustrate the background, pre-analytical sample preparation, and biomarker assay development using an MRM-mass spectrometric method. In addition, special attention is given to future standardization methods to enable widespread use of the technology.

Background: High-abundance plasma proteins are involved in disease-associated pathways and are useful in the diagnosis of nutritional and disease states. However, little is known about how concentrations of many plasma proteins vary between individuals from different ethnocultural groups with different dietary habits. Objective: We explored the association between plasma proteomic groups, dietary patterns, and ethnicity in the Toronto Nutrigenomics and Health Study, an ethnically diverse population of healthy young adults. Design: Concentrations of 54 high-abundance plasma proteins were measured simultaneously by liquid chromatography/multiple-reaction monitoring–mass spectrometry in 1090 individuals. Principal components analysis was used to identify plasma proteomic groups. Linear regression was used to investigate relations between proteomic groups and previously identified dietary patterns (Western, prudent, Eastern). Differences in individual protein concentrations between ethnocultural groups were tested by using general linear models. Results: Four independent principal components representative of proteomic groups were identified. Principal components 1 and 2 included proteins from multiple pathways. Component 3 was inflammatory, and component 4 included coagulation cascade proteins. East Asians and South Asians had lower component 1 scores, and East Asians had higher component 2 scores. South Asians had higher average scores for component 3. Individual protein concentrations also varied across ethnocultural groups. Principal component 1 was positively associated with the Western dietary pattern and inversely associated with the Eastern pattern. Component 3 was positively associated with the Eastern pattern. Conclusions: Plasma proteomic groups differ between young adults of diverse ethnocultural groups with different dietary habits. These differences may partly account for different rates of cardiometabolic disease later in life. Am J Clin Nutr doi: 10.3945/ajcn.111.022657.

Conifers are extremely long-lived plants that have evolved complex chemical defenses in the form of oleoresin terpenoids to resist attack from pathogens and herbivores. In these species, terpenoid diversity is determined by the size and composition of the terpene synthase (TPS) gene family and the single- and multi-product profiles of these enzymes. The monoterpene (+)-3-carene is associated with resistance of Sitka spruce (Picea sitchensis) to white pine weevil (Pissodes strobi). We used a combined genomic, proteomic and biochemical approach to analyze the (+)-3-carene phenotype in two contrasting Sitka spruce genotypes. Resistant trees produced significantly higher levels of (+)-3-carene than susceptible trees, in which only trace amounts were detected. Biosynthesis of (+)-3-carene is controlled, at the genome level, by a small family of closely related (+)-3-carene synthase (PsTPS-3car) genes (82-95% amino acid sequence identity). Transcript profiling identified one PsTPS-3car gene (PsTPS-3car1) that is expressed in both genotypes, one gene (PsTPS-3car2) that is expressed only in resistant trees, and one gene (PsTPS-3car3) that is expressed only in susceptible trees. The PsTPS-3car2 gene was not detected in genomic DNA of susceptible trees. Target-specific selected reaction monitoring confirmed this pattern of differential expression of members of the PsTPS-3car family at the proteome level. Kinetic characterization of the recombinant PsTPS-3car enzymes identified differences in the activities of PsTPS-3car2 and PsTPS-3car3 as a factor contributing to the different (+)-3-carene profiles of resistant and susceptible trees. In conclusion, variation of the (+)-3-carene phenotype is controlled by copy number variation of PsTPS-3car genes, variation of gene and protein expression, and variation in catalytic efficiencies.

Conifers have evolved complex chemical defenses in the form of oleoresin terpenoids to resist attack from pathogens and herbivores. The large diversity of terpenoid metabolites is determined by the size and composition of the terpene synthase (TPS) gene family, and the single- and multi-product profiles of these enzymes. The monoterpene (+)-3-carene is associated with resistance of Sitka spruce (Picea sitchensis) to white pine weevil (Pissodes strobi). We used a combined genomic, proteomic and biochemical approach to analyze the (+)-3-carene phenotype in two contrasting Sitka spruce genotypes. Resistant trees produced significantly higher levels of (+)-3-carene than susceptible trees, in which only trace amounts were detected. Biosynthesis of (+)-3-carene is controlled, at the genome level, by a small family of closely related (82-95% amino acid sequence identity) (+)-3-carene synthase (PsTPS-3car) genes. Transcript profiling identified one PsTPS-3car gene (PsTPS-3car1) which is expressed in both genotypes, one gene (PsTPS-3car2) expressed only in resistant trees, and one gene (PsTPS-3car3) expressed only in susceptible trees. The PsTPS-3car2 gene was not detected in genomic DNA of susceptible trees. Target-specific selected reaction monitoring substantiated this pattern of differential expression of members of the PsTPS-3car family on the proteome level. Kinetic characterization of the recombinant PsTPS-3car enzymes identified differences in the activities of PsTPS-3car2 and PsTPS-3car3as a factor for the different (+)-3-carene profiles of resistant and susceptible trees. In conclusion, variation of the (+)-3-carene phenotype is controlled by PsTPS-3car gene copy number variation, variation of gene and protein expression, and variation of catalytic efficiencies.

We have developed a phosphatase-based phosphopeptide quantitation (PPQ) method for determining phosphorylation stoichiometry in complex biological samples. This PPQ method is based on enzymatic dephosphorylation, combined with specific and accurate peptide identification and quantification by multiple reaction monitoring (MRM) with stable-isotope-labeled standard peptides. In contrast with classical MRM methods for the quantitation of phosphorylation stoichiometry, the PPQ-MRM method needs only one nonphosphorylated SIS (stable isotope-coded standard) and two analyses (one for the untreated sample and one for the phosphatase-treated sample), from which the expression and modification levels can accurately be determined. From these analyses, the percent phosphorylation can be determined. In this manuscript, we compare the PPQ-MRM method with an MRM method without phosphatase and demonstrate the application of these methods to the detection and quantitation of phosphorylation of the classic phosphorylated breast cancer biomarkers (ERalpha and HER2), and for phosphorylated RAF and ERK1, which also contain phosphorylation sites of biological importance. Using synthetic peptides spiked into a complex protein digest, we were able to use our PPQ-MRM method to accurately determine the total phosphorylation stoichiometry on specific peptides as well as the absolute amount of the peptide and phosphopeptide present. Analyses of samples containing ERalpha protein revealed that the PPQ-MRM method is capable of determining phosphorylation stoichiometry in proteins from cell lines, and is in good agreement with determinations obtained using the direct MRM approach in terms of phosphorylation and total protein amount.

Induction of terpene synthase (TPS) gene expression and enzyme activity is known to occur in response to various chemical and biological stimuli in several species of spruce (genus Picea). However, high sequence identity between TPS family members has made it difficult to determine the induction patterns of individual TPS at the protein and transcript levels and whether specific TPS enzymes respond differentially to treatment. In the present study we used a multi-level approach to measure the induction and activity of TPS enzymes in protein extracts of Norway spruce (Picea abies) bark tissue following treatment with methyl jasmonate (MeJA). Measurements were made on the transcript, protein, enzyme activity and metabolite levels. Using a relatively new proteomics application, selective reaction monitoring (SRM), it was possible to differentiate and quantitatively measure the abundance of several known TPS proteins and three 1-deoxy-D-xylulose 5-phosphate synthase (DXS) isoforms in Norway spruce. Protein levels of individual TPS and DXS enzymes were differentially induced upon MeJA treatment and good correlation was generally observed between induction of transcripts, proteins, and enzyme activities. Most of the mono- and diterpenoid metabolites accumulated with similar temporal patterns of induction as part of the coordinated multi-compound chemical defense response. Protein and enzyme activity levels of the monoTPS (+)-3-carene synthase and the corresponding accumulation of (+)-3-carene was induced to a higher fold change than any other TPS or metabolite measured, indicating an important role in the induced terpenoid defense response in Norway spruce.

Background Thyroid hormones (THs) are vital in the maintenance of homeostasis and in the control of development. One postembryonic developmental process that is principally regulated by THs is amphibian metamorphosis. This process has been intensively studied at the genomic level yet very little information at the proteomic level exists. In addition, there is increasing evidence that changes in the phosphoproteome influence TH action. Results Here we identify components of the proteome and phosphoproteome in the tail fin that changed within 48 h of exposure of premetamorphic Rana catesbeiana tadpoles to 10 nM 3,5,3'-triiodothyronine (T3). To this end, we developed a cell and protein fractionation method combined with two-dimensional gel electrophoresis and phosphoprotein-specific staining. Altered proteins were identified using mass spectrometry (MS). We identified and cloned a novel Rana larval type I keratin, RLK I, which may be a target for caspase-mediated proteolysis upon exposure to T3. In addition, the RLK I transcript is reduced during T3-induced and natural metamorphosis which is consistent with a larval keratin. Furthermore, GILT, a protein involved in the immune system, is changed in phosphorylation state which is linked to its activation. Using a complementary MS technique for the analysis of differentially-expressed proteins, isobaric tags for relative and absolute quantitation (iTRAQ) revealed 15 additional proteins whose levels were altered upon T3 treatment. The success of identifying proteins whose levels changed upon T3 treatment with iTRAQ was enhanced through de novo sequencing of MS data and homology database searching. These proteins are involved in apoptosis, extracellular matrix structure, immune system, metabolism, mechanical function, and oxygen transport. Conclusion We have demonstrated the ability to derive proteomics-based information from a model species for postembryonic development for which no genome information is currently available. The present study identifies proteins whose levels and/or phosphorylation states are altered within 48 h of the induction of tadpole tail regression prior to overt remodeling of the tail. In particular, we have identified a novel keratin that is a target for T3-mediated changes in the tail that can serve as an indicator of early response to this hormone.

Despite the extensive use of wildlife species in elucidating important biological processes, very few gene expression tools are available. For example, many frog species with different sensitivities and ecological niches are used as sentinel species for environmental contaminants and as developmental models. However, gene expression analyses have been essentially limited to one laboratory species. In an attempt to extend gene expression analyses to relevant indigenous species, we have developed a frog cDNA array with probes designed against conserved protein-encoding sequences. Changes in gene expression profiles were identified in cultured tail tips of Rana catesbeiana tadpoles during induction of tail regression by exogenous thyroid hormone and are associated with a transition from active cell proliferation to increased apoptotic activity. The expression profiles of selected genes representative of different response patterns were further characterized in tails of tadpoles undergoing natural metamorphosis using de novo designed biomarker probes and quantitative real-time polymerase chain reaction analysis. The results support the cross-species application of cDNA arrays that can direct the development of gene expression biomarkers for indigenous wildlife species.