An "on-matrix" digestion procedure for AP-MS experiments dissects the interplay between complex-conserved and serotype-specific reactivities in Dengue virus-human plasma interactome.

The interactions between the four Dengue virus (DENV) serotypes and plasma proteins are crucial in the initial steps of viral infection to humans. Affinity purification combined with quantitative mass spectrometry analysis, has become one of the most powerful tools for the investigation on novel protein-protein interactions. Using this approach, we report here that a significant number of bait-interacting proteins do not dissociate under standard elution conditions, i.e. acid pH and chaotropic agents, and that this problem can be circumvented by using the "on-matrix" digestion procedure described here. This procedure enabled the identification of 16 human plasma proteins interacting with domain III from the envelope protein of DENV serotypes 1, 3 and 4 that would have not been detected otherwise and increased the known DIIIE interactors in human plasma to 59 proteins. Selected Reaction Monitoring analysis evidenced DENV interactome in human plasma is rather conserved although significant differences on the reactivity of viral serotypes with specific proteins do exist. A comparison between the serotype-dependent profile of reactivity and the conservation pattern of amino acid residues suggests an evolutionary selection of highly conserved interactions with the host and other interactions mediated for surface regions of higher variability. SIGNIFICANCE: False negative results on the identification of interacting proteins in pull-down experiments compromise the subsequent interpretation of results and the formulation of a working hypothesis for the derived future work. In this study we demonstrate the presence of bait-interacting proteins reluctant to dissociate under elution conditions of acid pH and presence of chaotropics. We propose the direct proteolytic digestion of proteins while still bound to the affinity matrix ("on-matrix" digestion) and evaluate the impact of this methodology in the comparative study of the interactome of the four serotypes of Dengue virus mediated by the domain III of the viral envelope glycoprotein. Fifty nine proteins were identified as putative interaction partners of Dengue virus (IPs) either due to direct binding or by co-isolation with interacting proteins. Collectively the IPs identified from the pull-down with the recombinant domain III proteins representing the four viral serotypes, 29% were identified only after "on-matrix" digestion which demonstrate the usefulness of this method of recovering bait-bound proteins. Results highlight a particular importance of "on-matrix" digestion procedure for comparative studies where a stronger interaction with one of the interest baits could prevent a bound protein to elute under standard conditions thus leading to misinterpretation as absent in the interactome of this particular bait. The analysis of the Interaction Network indicates that Dengue virus interactome mediated by the domain III of the envelope protein is rather conserved in the viral complex suggesting a key role of these interactions for viral infection thus making candidates to explore for potential biomarkers of clinical outcome in DENV-caused disease. Interestingly, some particular IPs exhibit significant differences in the strength of the interaction with the viral serotypes representing interactions that involve more variable regions in the surface of the domain III. Since such variable regions are the consequence of the interaction with antibodies generated by human immune response; this result relates the interaction with proteins from human plasma with the interplay of the virus and the human immune system.

Targeted Proteomics Identifies Proteomic Signatures in Liquid Biopsies of the Endometrium to Diagnose Endometrial Cancer and Assist in the Prediction of the Optimal Surgical Treatment.

Purpose: Endometrial cancer (EC) diagnosis relies on the observation of tumor cells in endometrial biopsies obtained by aspiration (i.e., uterine aspirates), but it is associated with 22% undiagnosed patients and up to 50% of incorrectly assigned EC histotype and grade. We aimed to identify biomarker signatures in the fluid fraction of these biopsies to overcome these limitations.Experimental Design: The levels of 52 proteins were measured in the fluid fraction of uterine aspirates from 116 patients by LC-PRM, the latest generation of targeted mass-spectrometry acquisition. A logistic regression model was used to assess the power of protein panels to differentiate between EC and non-EC patients and between EC histologic subtypes. The robustness of the panels was assessed by the "leave-one-out" cross-validation procedure performed within the same cohort of patients and an independent cohort of 38 patients.Results: The levels of 28 proteins were significantly higher in patients with EC (n = 69) compared with controls (n = 47). The combination of MMP9 and KPYM exhibited 94% sensitivity and 87% specificity for detecting EC cases. This panel perfectly complemented the standard diagnosis, achieving 100% of correct diagnosis in this dataset. Nine proteins were significantly increased in endometrioid EC (n = 49) compared with serous EC (n = 20). The combination of CTNB1, XPO2, and CAPG achieved 95% sensitivity and 96% specificity for the discrimination of these subtypes.Conclusions: We developed two uterine aspirate-based signatures to diagnose EC and classify tumors in the most prevalent histologic subtypes. This will improve diagnosis and assist in the prediction of the optimal surgical treatment. Clin Cancer Res; 23(21); 6458-67. ©2017 AACR.

Large-Scale SRM Screen of Urothelial Bladder Cancer Candidate Biomarkers in Urine.

Urothelial bladder cancer is a condition associated with high recurrence and substantial morbidity and mortality. Noninvasive urinary tests that would detect bladder cancer and tumor recurrence are required to significantly improve patient care. Over the past decade, numerous bladder cancer candidate biomarkers have been identified in the context of extensive proteomics or transcriptomics studies. To translate these findings in clinically useful biomarkers, the systematic evaluation of these candidates remains the bottleneck. Such evaluation involves large-scale quantitative LC-SRM (liquid chromatography-selected reaction monitoring) measurements, targeting hundreds of signature peptides by monitoring thousands of transitions in a single analysis. The design of highly multiplexed SRM analyses is driven by several factors: throughput, robustness, selectivity and sensitivity. Because of the complexity of the samples to be analyzed, some measurements (transitions) can be interfered by coeluting isobaric species resulting in biased or inconsistent estimated peptide/protein levels. Thus the assessment of the quality of SRM data is critical to allow flagging these inconsistent data. We describe an efficient and robust method to process large SRM data sets, including the processing of the raw data, the detection of low-quality measurements, the normalization of the signals for each protein, and the estimation of protein levels. Using this methodology, a variety of proteins previously associated with bladder cancer have been assessed through the analysis of urine samples from a large cohort of cancer patients and corresponding controls in an effort to establish a priority list of most promising candidates to guide subsequent clinical validation studies.

Development of a sequential workflow based on LC-PRM for the verification of endometrial cancer protein biomarkers in uterine aspirate samples.

About 30% of endometrial cancer (EC) patients are diagnosed at an advanced stage of the disease, which is associated with a drastic decrease in the 5-year survival rate. The identification of biomarkers in uterine aspirate samples, which are collected by a minimally invasive procedure, would improve early diagnosis of EC. We present a sequential workflow to select from a list of potential EC biomarkers, those which are the most promising to enter a validation study. After the elimination of confounding contributions by residual blood proteins, 52 potential biomarkers were analyzed in uterine aspirates from 20 EC patients and 18 non-EC controls by a high-resolution accurate mass spectrometer operated in parallel reaction monitoring mode. The differential abundance of 26 biomarkers was observed, and among them ten proteins showed a high sensitivity and specificity (AUC > 0.9). The study demonstrates that uterine aspirates are valuable samples for EC protein biomarkers screening. It also illustrates the importance of a biomarker verification phase to fill the gap between discovery and validation studies and highlights the benefits of high resolution mass spectrometry for this purpose. The proteins verified in this study have an increased likelihood to become a clinical assay after a subsequent validation phase.

Parallel reaction monitoring using quadrupole-Orbitrap mass spectrometer: Principle and applications.

Targeted mass spectrometry-based approaches are nowadays widely used for quantitative proteomics studies and more recently have been implemented on high resolution/accurate mass (HRAM) instruments resulting in a considerable performance improvement. More specifically, the parallel reaction monitoring technique (PRM) performed on quadrupole-Orbitrap mass spectrometers, leveraging the high resolution and trapping capabilities of the instrument, offers a clear advantage over the conventional selected reaction monitoring (SRM) measurements executed on triple quadrupole instruments. Analyses performed in HRAM mode allow for an improved discrimination between signals derived from analytes and those resulting from matrix interferences translating in the reliable quantification of low abundance components. The purpose of the study defines various implementation schemes of PRM, namely: (i) exploratory experiments assessing the detectability of very large sets of peptides (100-1000), (ii) wide-screen analyses using (crude) internal standards to obtain statistically meaningful (relative) quantitative analyses, and (iii) precise/accurate quantification of a limited number of analytes using calibrated internal standards. Each of the three implementation schemes requires specific acquisition methods with defined parameters to appropriately control the acquisition during the actual peptide elution. This tutorial describes the different PRM approaches and discusses their benefits and limitations in terms of quantification performance and confidence in analyte identification.

Dataset on protein composition of a human plasma sub-proteome able to modulate the Dengue 2 virus infection in Huh 7.5 cells.

The four serotypes of dengue virus (DENV1-4) are the causal agents of the emerging disease Dengue Fever and its severe forms. DENV is inoculated into human blood through a mosquito bite. Thus, plasma is an important media for DENV dissemination in infected persons and several important interactions should take place for the virus with human plasma proteins that strongly influence or may determine the course of the infection. This dataset contains 239 proteins identified in the elution fractions of human plasma subjected to DE-52 anion exchange chromatography. Data on DENV2 infection of Huh 7.5 cells in presence of the human plasma fraction is also presented.

Targeted Proteomics to Assess the Response to Anti-Angiogenic Treatment in Human Glioblastoma (GBM).

Glioblastoma (GBM) is a highly aggressive primary brain tumor with dismal outcome for affected patients. Because of the significant neo-angiogenesis exhibited by GBMs, anti-angiogenic therapies have been intensively evaluated during the past years. Recent clinical studies were however disappointing, although a subpopulation of patients may benefit from such treatment. We have previously shown that anti-angiogenic targeting in GBM increases hypoxia and leads to a metabolic adaptation toward glycolysis, suggesting that combination treatments also targeting the glycolytic phenotype may be effective in GBM patients. The aim of this study was to identify marker proteins that are altered by treatment and may serve as a short term readout of anti-angiogenic therapy. Ultimately such proteins could be tested as markers of efficacy able to identify patient subpopulations responsive to the treatment. We applied a proteomics approach based on selected reaction monitoring (SRM) to precisely quantify targeted protein candidates, selected from pathways related to metabolism, apoptosis and angiogenesis. The workflow was developed in the context of patient-derived intracranial GBM xenografts developed in rodents and ensured the specific identification of human tumor versus rodent stroma-derived proteins. Quality control experiments were applied to assess sample heterogeneity and reproducibility of SRM assays at different levels. The data demonstrate that tumor specific proteins can be precisely quantified within complex biological samples, reliably identifying small concentration differences induced by the treatment. In line with previous work, we identified decreased levels of TCA cycle enzymes, including isocitrate dehydrogenase, whereas malectin, calnexin, and lactate dehydrogenase A were augmented after treatment. We propose the most responsive proteins of our subset as potential novel biomarkers to assess treatment response after anti-angiogenic therapy that warrant future analysis in clinical GBM samples.

Novel interactions of domain III from the envelope glycoprotein of dengue 2 virus with human plasma proteins.

Blood cells and plasma are important media for the four serotypes of dengue virus (DENV1-4) spreading into an infected person. Thus, interactions with human plasma proteins are expected to be decisive in the course of the viral infection. Affinity purification followed by MS analysis (AP/MS) was used to isolate and identify plasma-derived proteins capable to interact with a recombinant protein comprising the domain III of the envelope protein of DENV2 (DIIIE2). The elution of the AP potently inhibits DENV2 infection. Twenty-nine proteins were identified using a label-free approach as specifically captured by DIIIE2. Of these, a direct interaction with C reactive protein, thrombin and Inter-alpha-inhibitor complexes was confirmed by ELISA. Results provide further evidence of a significant representation of proteins from complement and coagulation cascades on DENV2 interactome in human plasma and stand out the domain III of the viral envelope protein as participant on these interactions. A functional clustering analysis highlights the presence of three structural motifs among putative DIIIE2-binding proteins: hydroxylation and EGF-like calcium-binding- and Gla domains. BIOLOGICAL SIGNIFICANCE: Early cycles of dengue virus replication take place in human blood cells. Thus, the characterization of the interactome of dengue virus proteins in human plasma can lead to the identification of pivotal interactions for the infection that can eventually constitute the target for the development of methods to control dengue virus-caused disease. In this work we identified 29 proteins from human plasma that potentially interact with the envelope protein of dengue 2 virus either directly or through co-complex formation. C reactive protein, thrombin and Inter-alpha-inhibitor complexes were validated as interactors of the domain III of the envelope protein of dengue 2. Results highlight the presence of three structural motifs among putative DIIIE2-binding proteins: hydroxylation and EGF-like calcium-binding- and Gla domains. This finding together with the participation of domain III of the envelope protein on the interactions with human plasma proteins should contribute to a better understanding of dengue virus interactome in human plasma. Such knowledge can contribute to the development of more effective treatments to infected persons.

Structural characterization and biological implications of sulfated N-glycans in a serine protease from the neotropical moth Hylesia metabus (Cramer [1775]) (Lepidoptera: Saturniidae).

Contact with the urticating setae from the abdomen of adult females of the neo-tropical moth Hylesia metabus gives rise to an urticating dermatitis, characterized by intense pruritus, generalized malaise and occasionally ocular lesions (lepidopterism). The setae contain a pro-inflammatory glycosylated protease homologous to other S1A serine proteases of insects. Deglycosylation with PNGase F in the presence of a buffer prepared with 40% H2 (18)O allowed the assignment of an N-glycosylation site. Five main paucimannosidic N-glycans were identified, three of which were exclusively α(1-6)-fucosylated at the proximal GlcNAc. A considerable portion of these N-glycans are anionic species sulfated on either the 4- or the 6-position of the α(1-6)-mannose residue of the core. The application of chemically and enzymatically modified variants of the toxin in an animal model in guinea pigs showed that the pro-inflammatory and immunological reactions, e.g. disseminated fibrin deposition and activation of neutrophils, are due to the presence of sulfate-linked groups and not on disulfide bonds, as demonstrated by the reduction and S-alkylation of the toxin. On the other hand, the hemorrhagic vascular lesions observed are attributed to the proteolytic activity of the toxin. Thus, N-glycan sulfation may constitute a defense mechanism against predators.