Proteome alteration induced by hTERT transfection of human fibroblast cells

Background: Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT) gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (W138). Cytosolic and nuclear fractions of W138 cells, empty vector transfected W138 (W138-HPV) and hTERT W138 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis) analysis. Only spots that had a similar abundance in W138 and W138-HPV, but were differentially expressed in W138 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control W138-HPV cells. The proteome alteration induced by hTERT W138 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. Results: 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. Conclusion: We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest that telomerase expression enhances natural cell repair mechanisms and stress resistance probably required for long term resistance of immortalized cells. Thus, hTERT transfected cells can not be only consider as an immortal equivalent to parental cells but also as cells which are over-resistant to stresses. These findings are the prerequisite for any larger proteomics aiming to evaluate anti-telomerase drugs proteome alteration and thus therapeutics induced cell reactions

Sweat Proteomics in Cystic Fibrosis: Discovering Companion Biomarkers for Precision Medicine and Therapeutic Development

peer reviewedIn clinical routine, the diagnosis of cystic fibrosis (CF) is still challenging regardless of international consensus on diagnosis guidelines and tests. For decades, the classical Gibson and Cooke test measuring sweat chloride concentration has been a keystone, yet, it may provide normal or equivocal results. As of now, despite the combination of sweat testing, CFTR genotyping, and CFTR functional testing, a small fraction (1–2%) of inconclusive diagnoses are reported and justifies the search for new CF biomarkers. More importantly, in the context of precision medicine, with a view to early diagnosis, better prognosis, appropriate clinical follow-up, and new therapeutic development, discovering companion biomarkers of CF severity and phenotypic rescue are of utmost interest. To date, previous sweat proteomic studies have already documented disease-specific variations of sweat proteins (e.g., in schizophrenia and tuberculosis). In the current study, sweat samples from 28 healthy control subjects and 14 patients with CF were analyzed by nanoUHPLC-Q-Orbitrap-based shotgun proteomics, to look for CF-associated changes in sweat protein composition and abundance. A total of 1057 proteins were identified and quantified at an individual level, by a shotgun label-free approach. Notwithstanding similar proteome composition, enrichment, and functional annotations, control and CF samples featured distinct quantitative proteome profiles significantly correlated with CF, accounting for the respective inter-individual variabilities of control and CF sweat. All in all: (i) 402 sweat proteins were differentially abundant between controls and patients with CF, (ii) 68 proteins varied in abundance between F508del homozygous patients and patients with another genotype, (iii) 71 proteins were differentially abundant according to the pancreatic function, and iv) 54 proteins changed in abundance depending on the lung function. The functional annotation of pathophysiological biomarkers highlighted eccrine gland cell perturbations in: (i) protein biosynthesis and trafficking, (ii) CFTR proteostasis and membrane stability, and (iii) cell-cell adherence, membrane integrity, and cytoskeleton crosstalk. Cytoskeleton-related biomarkers were of utmost interest because of the consistency between variations observed here in CF sweat and variations previously documented in other CF tissues. From a clinical stance, nine candidate biomarkers of CF diagnosis (CUTA, ARG1, EZR, AGA, FLNA, MAN1A1, MIA3, LFNG, SIAE) and seven candidate biomarkers of CF severity (ARG1, GPT, MDH2, EML4 (F508del homozygous), MGAT1 (pancreatic insufficiency), IGJ, TOLLIP (lung function impairment)) were deemed suitable for further verification.MucoSweatOmic

From Downscaling to Single-Cell Proteomic: Understanding and minimizing the downscaling effect

peer reviewedINTRODUCTION: Cellular systems consist of a variety of cells with distinct molecular and functional properties based on their location and timing. The characterization of proteome heterogeneity in these systems is a key to enhancing medical research and precision medicine, which requires quantification of proteins at the single-cell level. Liquid chromatography mass spectrometry (LC-MS) is a well-suited technique for proteomics analysis. However, the drop in performance is an inherent effect of decreasing the starting sample material amount. This downscaling effect is strongly related to the sensibility of MS instrument and the peptide loss during sample preparation. A comprehensive study of all contributions to the sample downscaling effect is essential to properly optimize single-cell proteomic methods and thus for minimizing the performance drop. METHODS: Here we present results from downscaled proteomic analysis together with a software-assisted strategy to evaluate and furthering the understanding of the sample downscaling effect on the performance drop for bottom-up proteomic analysis. In this approach, a sample condition (e.g., sample preparation protocol, LC-MS method, MS instruments) is evaluated by monitoring performance when the injected quantity of peptides is reduced. Each peptide signal intensity is monitored in function of the injected quantity in LC-MS. The signal drop for each peptides can then be study independently in function of their intrinsic properties (e.g., mass, charge, hydrophobicity factor, acidic/basis residue ratio) to highlight the causes of peptide loss. PRELIMINARY DATA: This approach was first used to evaluate and compare the performances of LC-MS methods (e.g. sensitivity, feature detection) in a high throughput context on QExactive (Thermo) and timsTOF (Bruker) instruments. This study led to a comprehensive comparison of the performances of these two MS for proteomics of low amount to single-cell level samples. The same workflow was then applied to evaluate specific peptide interactions (binding) with the vial surface. Peptides from a HeLa tryptic digest standard were chosen as peptide mix model for this study to avoid the contribution of sample preparation on the analysis performance. This study was first conducted with Total Recovery glass vials from Waters. The loss of peptide signal was assessed by a downscaling experiment on a set of vials containing peptides at different concentration levels (from 180ng/µL to 10ng/µL). As expected, the total peptide signal decreases as the total peptide concentration is reduced. However, the drop in peptide signal was not homogenous in regards to peptide hydrophobicity factors. This observation has been related to preferential and significant peptide binding on the vial surface. These interactions becoming non-negligible when peptide concentration is downscaled. Based on these results, vials molded in different polymeric materials (e.g., glass-filled polymers, Cyclic olefin polymers, polypropylene, poly(methyl methacrylate, polyether ether ketone) were tested with our downscaling approach. The results helped determine the best candidate polymeric material for vials or other laboratory consumables, minimizing peptide-surface interactions, for single cell proteomic analysis or low starting material experiments LC-MS analyses. As preliminary results, poly(methyl methacrylate), PMMA, vials showed promising behaviors for downscaled proteomics increasing the number of hydrophobic peptides detected by LC-MS compared to glass vial. This improvement leads to a 15% increase in identified proteins. NOVEL ASPECT: Influence of microtube material for single-cell sample preparation and related bioinformatic tools for performance check and protocol optimization.ChipOmics (Win2WAL

Characterization of the Human Eccrine Sweat Proteome—A Focus on the Biological Variability of Individual Sweat Protein Profiles

The potential of eccrine sweat as a bio-fluid of interest for diagnosis and personalized therapy has not yet been fully evaluated, due to the lack of in-depth sweat characterization studies. Thanks to recent developments in omics, together with the availability of accredited sweat collection methods, the analysis of human sweat may now be envisioned as a standardized, non-invasive test for individualized monitoring and personalized medicine. Here, we characterized individual sweat samples, collected from 28 healthy adult volunteers under the most standardized sampling methodology, by applying optimized shotgun proteomics. The thorough characterization of the sweat proteome allowed the identification of 983 unique proteins from which 344 were identified across all samples. Annotation-wise, the study of the sweat proteome unveiled the over-representation of newly addressed actin dynamics, oxidative stress and proteasome-related functions, in addition to well-described proteolysis and anti-microbial immunity. The sweat proteome composition correlated with the inter-individual variability of sweat secretion parameters. In addition, both gender-exclusive proteins and gender-specific protein abundances were highlighted, despite the high similarity between human female and male sweat proteomes. In conclusion, standardized sample collection coupled with optimized shotgun proteomics significantly improved the depth of sweat proteome coverage, far beyond previous similar studies. The identified proteins were involved in many diverse biological processes and molecular functions, indicating the potential of this bio-fluid as a valuable biological matrix for further studies. Addressing sweat variability, our results prove the proteomic profiling of sweat to be a promising bio-fluid analysis for individualized, non-invasive monitoring and personalized medicine.MucoSweatOmic

Development of a new integrated biosensor system for an accurate diagnosis of prostate cancer using optoacoustic detection

The prostate cancer is the most common male-specific cancer observed in the European Union and is the second leading cause of cancer death in men in our industrialized countries. The choice of treatment and its efficiency is largely dependent on the stage and on the degree of advancement of the cancer when it is diagnosed. Screening procedures like digital rectal examination (DRE) and free prostate specific antigen (PSA) level testing are well established but lack accuracy, yielding only 80% of prostate cancers diagnosed in an early stage. By providing a more accurate and precise tool for diagnosing prostate cancer in its early stages, the percentage of curable cancer patients would increase radically. Current imaging techniques have limited value, thus a major challenge in current prostate cancer oncology is to develop more accurate imaging assessments. An efficient imaging technique which significantly improves the sensitivity and specificity of diagnosing, staging and predicting the behaviour of prostate cancer would be extremely valuable. The ADONIS Project intends to prove the concept of using optoacoustic imaging in combination with biologically functionalized nanoparticles as an integrated biosensor based system for the production of specific and sensitive data for accurate diagnosis of prostate cancer. The achievement of this objective requires excellent know-how on a variety of scientific and technologic fields, brought by the partners of ADONIS, coming from five European countries, such as laser and ultrasound technologies and image reconstruction techniques, the bio-functionalization of nanoparticles, the system integration and, finally, experiments and competent evaluation of the results for their application potential. The development of the biosensor is firstly performed to target the Prostate Specific Membrane Antigen (PSMA), a transmembrane protein considered as a suitable biomarker for prostate cancer and which is under intense investigation for use as an imaging and therapeutic target. To allow the detection optimization of the biosensor, a 3D cellular culture technique (Rotating Cell Culture System) is developed with LNCaP cells (a human prostate carcinoma cell line reported to express PSMA) to be closest to the in vivo aspect for which a three-dimensional aspect of tumor for the biosensor detection is needed. Detection and localisation of PSMA on LNCaP cell surface was performed by immunostainning on monolayer culture and on spheroid slices. Then, by backscattered electron (BSE) microscopy analysis, detection of nanoparticles on cells surface shows the successful binding of the biosensor to the cells expressing PSMA. In prospect, the detection of the biosensor will be tested on large spheroids and finally tested on in vivo model.FP6 STREP ADONIS Projec

Overview on the ADONIS Project: Accurate Diagnosis of prostate cancer using Optoacoustic detection of biologically functionalized gold Nanoparticles - A new Integrated Biosensor System

Overview on the ADONIS Project: Accurate Diagnosis of prostate cancer using Optoacoustic detection of biologically functionalized gold Nanoparticles - A new Integrated Biosensor SystemFP6 STREP ADONIS Projec

Minimizing the peptide/protein loss due to interactions with custom consumable materials for proteomic study of protein samples at very low concentrations

peer reviewedLiquid chromatography-mass spectrometry (LC-MS) proteomics is challenged by the detection limit of current mass analyzers for low concentration – low quantity samples (e.g., single cell proteomics and tissue microdissection proteomics) but also especially by material losses during sample preparation steps. The binding of peptides and proteins to the surfaces of consumables significantly reduces the effective concentration in the solution, which further drastically decreases proteomic performance when the concentration of the starting material decreases. In addition to the optimization of the experimental protocols for the preparation of the samples and the technical evolution of the mass analyzers, the nature of the polymeric surface of consumables is crucial. We have therefore designed microvials suitable for proteomic low concentration sample preparation that can be molded in different polymeric materials. A comparative study has shown that the nature of the polymer material has a strong influence on proteomic performances for low concentration samples ranging from 1.5 to 10 ng/µl of proteins (injected quantity is less than 10ng). The nature of the vial has less impact on protein concentrations above 10ng/µl. Peptide loss due to surface interaction is not homogeneous with respect to peptide hydrophobicity, hydrophobic peptides being more impacted on commonly used vials. A polar polymer surface should therefore reduce hydrophobic interactions with peptides. Some polymer materials show promising behaviors for low concentration to single cell proteomic samples, leading to a 20% increase in identification compared to commercially available vials commonly used for proteomics.ChipOmics (Win2WAL

Optoacoustic specific detection of prostate cancer using functionalized gold nanorods

A major challenge in prostate cancer oncology is to develop more accurate imaging assessments than those currently available. Indeed, an efficient imaging technique which significantly improves the sensitivity and specificity of the diagnostic and predicting the cancer behaviour would be extremely valuable. This project intends to prove the concept of using optoacoustic imaging in combination with biologically functionalized nanoparticles as an integrated biosensor based system for the production of specific and sensitive data for accurate diagnosis of prostate cancer. This concept results on the use of contrast agents which transform an incident luminous energy into local heating inducing a pressure wave detectable by acoustic (echography). For the optoacoustic detection, the nanoparticles used must present a maximum of absorption in the optical transparency window of the human tissues in order to allow their and subsequently the tissue specific excitations while avoiding unwanted destructive energy transfers. According to these characteristics (energy transfer by thermoelastic reaction), rod-like gold nanoparticles (stick form) with a maximum of absorption towards 760 nm were produced by using a “bottom-up” approach with dynamic templates (surfactant). These nanoparticles are then coupled with an antibody directed against the cancerous cells to guarantee the specific detection of the particles. The development of the biosensor is firstly performed to target the Prostate Specific Membrane Antigen (PSMA), a transmembrane protein considered as a suitable biomarker for prostate cancer. Detection and localisation of PSMA on LNCaP fixed cell surface was performed by immunostainning on monolayer cell culture and on spheroid slices. Then, by backscattered electron (BSE) microscopy analysis and two-photon luminescence imaging, detection of nanoparticles on fixed and living cell surface shows the successful binding of the biosensor to the cells expressing PSMA. In prospect, the detection of the biosensor will be tested on spheroids, on human biopsies and finally on in vivo models (mouse xenograft models).FP6 STREP ADONIS Projec

Minimize peptide/protein loss due to interactions with custom consumable materials for very low concentration protein samples

Liquid chromatography-mass spectrometry (LC-MS) proteomics is challenged by the detection limit of current mass analyzers for low concentration samples but also especially by material losses during sample preparation steps. The binding of peptides and proteins to the surfaces of consumables significantly reduces the effective concentration in the solution, which further drastically decreases proteomic performance when the concentration of the starting material decreases. In addition to the optimization of the experimental protocols for the preparation of the samples, the nature of the polymeric surface of consumables is crucial. We have therefore designed microvials suitable for low protein concentration sample preparation that can be molded in different polymeric materials. A comparative study has shown that the nature of the polymer material has a strong influence on proteomic performances for low concentration samples ranging from 1.5 to 10ng/µl of proteins (injected quantity less than 10ng). Peptide loss due to surface interaction is not homogeneous with respect to peptide hydrophobicity, hydrophobic peptides being more impacted on commonly used vials. A polar polymer surface should therefore reduce hydrophobic interactions with peptides. Some polymer materials show promising behaviors for low concentration samples, leading to a 20% increase in identification compared to vials commonly used for proteomics.ChipOmics (Win2Wal