Mass spectrometry in high-throughput clinical biomarker assays: multiple reaction monitoring.

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 micro-flow, high-pH, reversed-phase peptide fractionation and collection system for targeted and in-depth proteomics of low-abundance proteins in limiting samples.

We present a method and a simple system for high-pH RP-LC peptide fractionation of small sample amounts (30-60 µg), at micro-flow rates with micro-liter fraction collection using ammonium bicarbonate as an optimized buffer for system stability and robustness. The method is applicable to targeted mass spectrometry approaches and to in-depth proteomic studies where the amount of sample is limited. Using targeted proteomics with peptide standards, we present the method's analytical parameters, and potential in increasing the detection of low-abundance proteins that are difficult to quantify with direct targeted or global LC-MS analyses. This fractionation system increased peptide signals by up to 18-fold, while maintaining high quantitative precision, with high fractionation reproducibility across varied sample sets. In real applications, it increased the detection of targeted endogenous peptides by two-fold in a 25 cell-cycle-control protein panel, and in-depth MS analyses of nuclear extracts, it allowed the detection of up to 8,896 proteins with 138,417 peptides in 24-concatenated fractions compared to 3,344 proteins with 23,093 peptides without fractionation. In a relevant biological problem of CDK4/6-inhibitors and breast cancer, the method reproduced known information and revealed novel insights, highlighting that it can be successfully applied in studies involving low-abundance proteins and limited samples. •Tested nine high-pH buffer/solvent systems to obtain a robust, effective, and reproducible micro-flow fractionation method which was devoid of commonly encountered LC clogging/pressure issues after months of use.•Peptide enrichment method to improve detection and quantitation of low-abundance proteins in targeted and in-depth proteomic studies.•Can be applied to diverse protein samples where the available amount is limited.

A systematic review and meta-analysis of thigmotactic behaviour in the open field test in rodent models associated with persistent pain.

Thigmotaxis is an innate predator avoidance behaviour of rodents. To gain insight into how injury and disease models, and analgesic drug treatments affect thigmotaxis, we performed a systematic review and meta-analysis of studies that assessed thigmotaxis in the open field test. Systematic searches were conducted of 3 databases in October 2020, March and August 2022. Study design characteristics and experimental data were extracted and analysed using a random-effects meta-analysis. We also assessed the correlation between thigmotaxis and stimulus-evoked limb withdrawal. This review included the meta-analysis of 165 studies We report thigmotaxis was increased in injury and disease models associated with persistent pain and this increase was attenuated by analgesic drug treatments in both rat and mouse experiments. Its usefulness, however, may be limited in certain injury and disease models because our analysis suggested that thigmotaxis may be associated with the locomotor function. We also conducted subgroup analyses and meta-regression, but our findings on sources of heterogeneity are inconclusive because analyses were limited by insufficient available data. It was difficult to assess internal validity because reporting of methodological quality measures was poor, therefore, the studies have an unclear risk of bias. The correlation between time in the centre (type of a thigmotactic metric) and types of stimulus-evoked limb withdrawal was inconsistent. Therefore, stimulus-evoked and ethologically relevant behavioural paradigms should be viewed as two separate entities as they are conceptually and methodologically different from each other.

MRM-based multiplexed quantitation of 67 putative cardiovascular disease biomarkers in human plasma.

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 (r >0.98) 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.

An integrated genomic, proteomic and biochemical analysis of (+)-3-carene biosynthesis in Sitka spruce (Picea sitchensis) genotypes that are resistant or susceptible to white pine weevil.

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.

Comparison of standard- and nano-flow liquid chromatography platforms for MRM-based quantitation of putative plasma biomarker proteins.

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.

Diagnosing pleural effusions using mass spectrometry-based multiplexed targeted proteomics quantitating mid- to high-abundance markers of cancer, infection/inflammation and tuberculosis.

Pleural effusion (PE) is excess fluid in the pleural cavity that stems from lung cancer, other diseases like extra-pulmonary tuberculosis (TB) and pneumonia, or from a variety of benign conditions. Diagnosing its cause is often a clinical challenge and we have applied targeted proteomic methods with the aim of aiding the determination of PE etiology. We developed a mass spectrometry (MS)-based multiple reaction monitoring (MRM)-protein-panel assay to precisely quantitate 53 established cancer-markers, TB-markers, and infection/inflammation-markers currently assessed individually in the clinic, as well as potential biomarkers suggested in the literature for PE classification. Since MS-based proteomic assays are on the cusp of entering clinical use, we assessed the merits of such an approach and this marker panel based on a single-center 209 patient cohort with established etiology. We observed groups of infection/inflammation markers (ADA2, WARS, CXCL10, S100A9, VIM, APCS, LGALS1, CRP, MMP9, and LDHA) that specifically discriminate TB-PEs and other-infectious-PEs, and a number of cancer markers (CDH1, MUC1/CA-15-3, THBS4, MSLN, HPX, SVEP1, SPINT1, CK-18, and CK-8) that discriminate cancerous-PEs. Some previously suggested potential biomarkers did not show any significant difference. Using a Decision Tree/Multiclass classification method, we show a very good discrimination ability for classifying PEs into one of four types: cancerous-PEs (AUC: 0.863), tuberculous-PEs (AUC of 0.859), other-infectious-PEs (AUC of 0.863), and benign-PEs (AUC: 0.842). This type of approach and the indicated markers have the potential to assist in clinical diagnosis in the future, and help with the difficult decision on therapy guidance.

The therapeutic potential of targeting exchange protein directly activated by cyclic adenosine 3',5'-monophosphate (Epac) for central nervous system trauma.

Millions of people worldwide are affected by traumatic spinal cord injury, which usually results in permanent sensorimotor disability. Damage to the spinal cord leads to a series of detrimental events including ischaemia, haemorrhage and neuroinflammation, which over time result in further neural tissue loss. Eventually, at chronic stages of traumatic spinal cord injury, the formation of a glial scar, cystic cavitation and the presence of numerous inhibitory molecules act as physical and chemical barriers to axonal regrowth. This is further hindered by a lack of intrinsic regrowth ability of adult neurons in the central nervous system. The intracellular signalling molecule, cyclic adenosine 3',5'-monophosphate (cAMP), is known to play many important roles in the central nervous system, and elevating its levels as shown to improve axonal regeneration outcomes following traumatic spinal cord injury in animal models. However, therapies directly targeting cAMP have not found their way into the clinic, as cAMP is ubiquitously present in all cell types and its manipulation may have additional deleterious effects. A downstream effector of cAMP, exchange protein directly activated by cAMP 2 (Epac2), is mainly expressed in the adult central nervous system, and its activation has been shown to mediate the positive effects of cAMP on axonal guidance and regeneration. Recently, using ex vivo modelling of traumatic spinal cord injury, Epac2 activation was found to profoundly modulate the post-lesion environment, such as decreasing the activation of astrocytes and microglia. Pilot data with Epac2 activation also suggested functional improvement assessed by in vivo models of traumatic spinal cord injury. Therefore, targeting Epac2 in traumatic spinal cord injury could represent a novel strategy in traumatic spinal cord injury repair, and future work is needed to fully establish its therapeutic potential.

Targeted proteomics using selected reaction monitoring reveals the induction of specific terpene synthases in a multi-level study of methyl jasmonate-treated Norway spruce (Picea abies).

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.

Assay development for the determination of phosphorylation stoichiometry using multiple reaction monitoring methods with and without phosphatase treatment: application to breast cancer signaling pathways.

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.

C-fin: a cultured frog tadpole tail fin biopsy approach for detection of thyroid hormone-disrupting chemicals.

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.

Inflammatory Proteins HMGA2 and PRTN3 as Drivers of Vulvar Squamous Cell Carcinoma Progression.

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.

Targeted Proteomics Approach Toward Understanding the Role of the Mitochondrial Protease FTSH4 in the Biogenesis of OXPHOS During Arabidopsis Seed Germination.

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.

An MRM-Based Cytokeratin Marker Assay as a Tool for Cancer Studies: Application to Lung Cancer Pleural Effusions.

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.

Analysis of the Rana catesbeiana tadpole tail fin proteome and phosphoproteome during T3-induced apoptosis: identification of a novel type I keratin.

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.

An online 2D-reversed-phase - Reversed-phase chromatographic method for sensitive and robust plasma protein quantitation.

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.

Peptide biomarkers used for the selective breeding of a complex polygenic trait in honey bees.

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.

A Multiplexed Cytokeratin Analysis Using Targeted Mass Spectrometry Reveals Specific Profiles in Cancer-Related Pleural Effusions.

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.

Molecular Mechanism for Cellular Response to ß-Escin and Its Therapeutic Implications.

ß-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 ß-escin have been the most extensively clinically investigated effects of this plant-based drug and randomized controlled trials have proved the efficacy of ß-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 ß-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 ß-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 ß-escin beyond the current vascular indications.