Biomarkers of chronic airflow limitation and COPD identified by mass spectrometry.

Rationale: COPD affects 300 million people worldwide and is the third leading cause of death according to World Health Organization global health estimates. Early symptoms are subtle, and so COPD is often diagnosed at an advanced stage. Thus, there is an unmet need for biomarkers that can identify individuals at early stages of the disease before clinical symptoms have manifested. To date, few biomarkers are available for clinical diagnostic use in COPD. Methods: We evaluated a panel of serum biomarkers related to inflammation and infection for their ability to discriminate between 77 subjects with chronic airflow limitation (CAL) and 142 subjects with COPD, versus 150 healthy subjects (divided into two control groups that were matched with regards to age, gender and smoking to CAL and COPD). Healthy subjects and CAL were from Burden of Obstructive Lung Disease (BOLD), a population-based study. CAL was defined by post-bronchodilatory forced expiratory volume in 1 s/forced vital capacity ratio <0.7 in the BOLD population. COPD subjects were from Tools for Identifying Exacerbations (TIE), a COPD patient cohort. Quantification of 100 biomarker candidates was done by liquid chromatography-tandem mass spectrometry. Results: Several protein-derived peptides were upregulated in CAL, compared to controls; most notably peptides representing histidine-rich glycoprotein (HRG), α1-acid glycoprotein (AGP1), α1-antitrypsin (α1AT) and fibronectin. Out of these, HRG-, AGP1- and α1AT-specific peptides were also elevated in the COPD cohort. Conclusion: HRG, AGP1 and α1AT biomarkers distinguish subjects with CAL and COPD from healthy controls. HRG and AGP1 represent novel findings.

Triplexed CEA-NSE-PSA Immunoassay Using Time-Gated Terbium-to-Quantum Dot FRET.

Time-gated Förster resonance energy transfer (TG-FRET) between Tb complexes and luminescent semiconductor quantum dots (QDs) provides highly advantageous photophysical properties for multiplexed biosensing. Multiplexed Tb-to-QD FRET immunoassays possess a large potential for in vitro diagnostics, but their performance is often insufficient for their application under clinical conditions. Here, we developed a homogeneous TG-FRET immunoassay for the quantification of carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), and prostate-specific antigen (PSA) from a single serum sample by multiplexed Tb-to-QD FRET. Tb-IgG antibody donor conjugates were combined with compact QD-F(ab')2 antibody acceptor conjugates with three different QDs emitting at 605, 650, and 705 nm. Upon antibody-antigen-antibody sandwich complex formation, the QD acceptors were sensitized via FRET from Tb, and the FRET ratios of QD and Tb TG luminescence intensities increased specifically with increasing antigen concentrations. Although limits of detection (LoDs: 3.6 ng/mL CEA, 3.5 ng/mL NSE, and 0.3 ng/mL PSA) for the triplexed assay were slightly higher compared to the single-antigen assays, they were still in a clinically relevant concentration range and could be quantified in 50 µL serum samples on a B·R·A·H·M·S KRYPTOR Compact PLUS clinical immunoassay plate reader. The simultaneous quantification of CEA, NSE, and PSA at different concentrations from the same serum sample demonstrated actual multiplexing Tb-to-QD FRET immunoassays and the potential of this technology for translation into clinical diagnostics.

Soluble intercellular adhesion molecule (ICAM)-1 detects invasive fungal infections in patients following liver transplantation.

PURPOSE: Despite antifungal prophylaxis, liver transplanted patients are endangered by invasive fungal infections (IFI). Routinely used microbiological procedures are hallmarked by significant weaknesses, which may lead to a delay in antifungal treatment. METHODS: Culture-based fungal findings, routinely used biomarkers of infection/inflammation (e.g., procalcitonin or C-reactive protein), as well as corresponding plasma concentrations of soluble Intercellular Adhesion Molecule (ICAM)-1 were analysed in 93 patients during a period of 28 days following liver transplantation (LTX). RESULTS: Plasmatic sICAM-1 was significantly elevated in patients affected by an IFI within the first 28 days in comparison to fungally colonised or unobtrusive LTX patients. sICAM-1 might therefore be helpful for the identification of IFI patients after LTX (e.g., Receiver Operating Characteristic (ROC)-Area Under the Curve (AUC): 0.714 at 14d after LTX). The diagnostic performance of sICAM-1 was further improved by its combined use with different other IFI biomarkers (e.g., midregional proadrenomedullin). CONCLUSION: The diagnostic deficiencies of routinely used microbiological procedures for IFI detection in patients after LTX may be reduced by plasmatic sICAM-1 measurements. Clinical Trial Notation. German Clinical Trials Register: DRKS00005480.

Impact of biological matrix on inflammatory protein biomarker quantification based on targeted mass spectrometry.

BACKGROUND: Serum and plasma are widely used matrices in biological and clinical studies. To improve reliability and consistency of markers quantification, the influence of these matrices on proteins was evaluated by targeted mass spectrometry. RESULTS: 65 proteins were quantified in matched blood samples collected in serum, ethylenediaminetetraacetic acid and heparin plasma tubes from 40 healthy and 10 pathological individuals. Only 52% of the proteins were not impacted by any of the biological matrices tested, and the effects on quantification of proteins affected was matrix and protein dependent. CONCLUSION: Matrix comparisons using mass spectrometry is therefore recommended to assess the relevance of using surrogate matrix, performing biomarker discovery study or evaluating the clinical use of biomarkers in large clinical cohorts.

Towards libraries of luminescent lanthanide complexes and labels from generic synthons.

A synthetic approach is developed to obtain families of luminescent lanthanide complexes and markers from a generic family of precursors built from nonadentate coordination sites. The syntheses of the precursors, based on a directed regioselective nucleophilic aromatic substitution on polyfluoropyridines, are described. Functionalisation of the synthons on the aromatic moieties allowed the introduction of labelling functions and/or the extension of the electronic delocalisation, with concomitant changes in the spectroscopic properties. The synthesis of two such families of ligands and of some of their complexes of Eu(III) and Tb(III) are described, and the photo-physical properties of the complexes were measured, revealing excellent luminescence quantum yields reaching unity in some cases. For some of these complexes, the emphasis was further put on the preparation of an N-hydroxylsuccinimide (NHS) ester as activated function for labelling. The Tb and La complexes in the NHS activated form were synthesized and fully characterized. The labelling was first demonstrated on amino functionalized polymer beads and characterized by time-resolved luminescence microscopy. In a second step, the activated Tb complex was used for the labelling of GFR44 monoclonal antibody, and was applied to the detection of carcinoembryonic antigene (CEA) within the frame of a time-resolved fluoroimmunoassay. Comparison with a commercially available kit based on a europium cryptate as energy donor confirms the efficiency of Tb to act as an energy donor with an unoptimised 35% increase of the detection efficiency.

POSVP(21), a major secretory androgen-dependent protein from sand rat seminal vesicles, identified as a transgelin.

The seminal vesicles of adult sand rat contain a major secretory protein band (MW 21 kDa) designated as Psammomys obesus seminal vesicles protein of 21 kDa (POSVP(21)). This protein is abundant in secretions, regulated by androgens and also present in the vaginal plug. POSVP(21) accounts for over 22.3% of soluble proteins from homogenate during the breeding season, 13.3% during the middle season and 5.3% during the hormonal regression season. It is absent during the non-breeding season. POSVP(21) is localized in the cytoplasm of epithelial cells and in secretory products in the lumen. It presents an immunological homology with two epididymal proteins with the same molecular weight and a high degree of homology with transgelin from rat (Rattus norvegicus).

Industrial process proteomics: alfalfa protein patterns during wet fractionation processing.

A proteome reference map of major soluble proteins from Medicago sativa (alfalfa) leaves and stems has been established for the first time. Among 195 spots analyzed by mass spectrometry and N-terminal Edman sequencing, 117 spots were unambiguously identified, representing 87 different proteins. Of these 87 proteins, 13 proteins were directly identified from the partial genome of Medicago sativa, 30 from expressed sequenced tags (ESTs) of the model legume Medicago truncatula and 44 from closely relative species by a cross-species protein identification method. The proteome map of Medicago sativa was then set as a reference to study the major high protein content products that are generated during the wet fractionation process of alfalfa green biomass. Using two-dimensional electrophoresis, we studied the variation of the protein patterns at different steps of the industrial-scale process. We clearly show that the process induces significant changes including chemical modifications, proteolytic events, and heat-shock protein responses. Strikingly, a certain level of cellular regulation is conserved during biomass processing, as exemplified by the induction of some heat shock proteins. Finally, all the results obtained in this proteomic study may help to identify novel products and to improve process designs in alfalfa biomass plants.