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CE-MS-based urinary biomarkers to distinguish non-significant from significant prostate cancer.
Frantzi, M, Gomez Gomez, E, Blanca Pedregosa, A, Valero Rosa, J, Latosinska, A, Culig, Z, Merseburger, AS, Luque, RM, Requena Tapia, MJ, Mischak, H, et al
British journal of cancer. 2019;(12):1120-1128
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Abstract
BACKGROUND Prostate cancer progresses slowly when present in low risk forms but can be lethal when it progresses to metastatic disease. A non-invasive test that can detect significant prostate cancer is needed to guide patient management. METHODS Capillary electrophoresis/mass spectrometry has been employed to identify urinary peptides that may accurately detect significant prostate cancer. Urine samples from 823 patients with PSA (<15 ng/ml) were collected prior to biopsy. A case-control comparison was performed in a training set of 543 patients (nSig = 98; nnon-Sig = 445) and a validation set of 280 patients (nSig = 48, nnon-Sig = 232). Totally, 19 significant peptides were subsequently combined by a support vector machine algorithm. RESULTS Independent validation of the 19-biomarker model in 280 patients resulted in a 90% sensitivity and 59% specificity, with an AUC of 0.81, outperforming PSA (AUC = 0.58) and the ERSPC-3/4 risk calculator (AUC = 0.69) in the validation set. CONCLUSIONS This multi-parametric model holds promise to improve the current diagnosis of significant prostate cancer. This test as a guide to biopsy could help to decrease the number of biopsies and guide intervention. Nevertheless, further prospective validation in an external clinical cohort is required to assess the exact performance characteristics.
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A Mass Spectrometry-Based Structural Assay for Activation-Dependent Conformational Changes in β-Arrestins.
Zhao, Y, Xiao, K
Methods in molecular biology (Clifton, N.J.). 2019;:293-308
Abstract
β-Arrestins are key regulation proteins for G protein-coupled receptors (GPCRs) signaling. Experimental evidence suggests that β-arrestins undergo conformational changes concomitant with binding to activated, phosphorylated GPCRs. We developed a mass spectrometry-based structural proteomic assay to monitor conformational changes associated with the activation of β-arrestins. This assay utilizes synthesized phosphopeptides mimicking phosphorylated C-terminal tails of GPCRs to activate β-arrestins. The activation-dependent conformational changes of β-arrestins are revealed using limited proteolysis coupled with both SDS-PAGE and mass spectrometry analysis. As an in vitro β-arrestin activation assay, this mass spectrometry-based structural method can be adapted as a simple but useful tool to study the nature and extent of conformational changes of β-arrestins downstream of different receptors as well as β-arrestin conformations associated with different functions, such as desensitization, internalization, and signaling.
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Using X-ray Footprinting and Mass Spectrometry to Study the Structure and Function of Membrane Proteins.
Gupta, S
Protein and peptide letters. 2019;(1):44-54
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Abstract
BACKGROUND Membrane proteins are crucial for cellular sensory cascades and metabolite transport, and hence are key pharmacological targets. Structural studies by traditional highresolution techniques are limited by the requirements for high purity and stability when handled in high concentration and nonnative buffers. Hence, there is a growing requirement for the use of alternate methods in a complementary but orthogonal approach to study the dynamic and functional aspects of membrane proteins in physiologically relevant conditions. In recent years, significant progress has been made in the field of X-ray radiolytic labeling in combination with mass spectroscopy, commonly known as X-ray Footprinting and Mass Spectrometry (XFMS), which provide residue-specific information on the solvent accessibility of proteins. In combination with both lowresolution biophysical methods and high-resolution structural data, XFMS is capable of providing valuable insights into structure and dynamics of membrane proteins, which have been difficult to obtain by standalone high-resolution structural techniques. The XFMS method has also demonstrated a unique capability for identification of structural waters and their dynamics in protein cavities at both a high degree of spatial and temporal resolution, and thus capable of identifying conformational hot-spots in transmembrane proteins. CONCLUSION We provide a perspective on the place of XFMS amongst other structural biology methods and showcase some of the latest developments in its usage for studying conformational changes in membrane proteins.
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Bio-guided Purification and Mass Spectrometry Characterisation Exploring the Lysozyme-like Protein from Enterococcus lactis Q1, an Unusual Marine Bacterial Strain.
Ben Braïek, O, Smaoui, S, Fleury, Y, Morandi, S, Hani, K, Ghrairi, T
Applied biochemistry and biotechnology. 2019;(1):43-53
Abstract
Lactic acid bacteria produce various antibacterial peptides such as bacteriocins that are active against pathogenic and spoilage microorganisms. Very little attention has been paid to the production of lysozyme as an antimicrobial enzyme. The present work represents one of the few studies reporting lysozyme production by enterococci. Indeed, this study was first conducted to evaluate the antimicrobial activity of Enterococcus lactis Q1, an enterocin P-producing strain previously isolated from fresh shrimp (Penaeus vannamei), against multidrug-resistant clinical isolates. Results showed significant inhibitory activity (P < 0.05) towards diverse pathogens. The purification of the antimicrobial substances produced by Q1 strain leads to the isolation of two active fractions. The SDS-PAGE and mass spectrometry analyses of fraction number 2 (fraction 2) revealed the presence of a protein with molecular mass of 14.3 kDa. Additionally, the experimental results are consistent with mass spectra of industrial lysozyme (Fluka ref. 62970). The lysozyme produced by Enterococcus lactis Q1 strain was confirmed by a plate method against Micrococcus luteus ATCC 4698. Also, sensitivity of the Q1 strain to different concentrations of lysozyme was investigated. For the first time, this study shows that E. lactis Q1 produces lysozyme which could be an excellent candidate in food biopreservation or production of functional foods to promote health benefits.
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A concept to make low-abundance endogenous renin accessible to mass spectrometry: A multistep experimental design approach.
Burdman, I, Burckhardt, BB
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences. 2019;:121856
Abstract
Renin is the rate-limiting step within the renin-angiotensin-aldosterone system, but the reliable quantification of human endogenous renin levels by liquid chromatography coupled with mass spectrometry remains challenging. The complex sample matrix triggering ion suppression and the detection of the low-abundance as well as the proteolytical-resistant renin make a hybrid approach using immunocapture coupled with LC-HRMS a promising method for investigation. Therefore, in-silico digestion and BLAST® experiments were conducted in order to identify the unique amino acid sequence for mass spectrometric detection. To enhance mass spectrometric response, impacting parameters within the denaturation, alkylation, and digestion experiments were identified and optimized by a multistep Design of Experiments process. The optimal denaturation buffer consisted of RapiGest® and urea, leading to a signature peptide intensity increase of 56% at 20 °C, whereas the optimal reducing agent improved intensity by 27%. The most effective generation of signature peptide I was achieved using a high trypsin concentration and a low incubation temperature enhancing digestion by 75%. The applicability of this hybrid approach was confirmed in human matrix and allowed for a fivefold reduction in total assay procedure time without limiting the reliable quantification if compared to a conventional digestion approach.
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Fast Photochemical Oxidation of Proteins Coupled with Mass Spectrometry.
Shi, L, Gross, ML
Protein and peptide letters. 2019;(1):27-34
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Abstract
BACKGROUND Determination of the composition and some structural features of macromolecules can be achieved by using structural proteomics approaches coupled with mass spectrometry (MS). One approach is hydroxyl radical protein footprinting whereby amino-acid side chains are modified with reactive reagents to modify irreversibly a protein side chain. The outcomes, when deciphered with mass-spectrometry-based proteomics, can increase our knowledge of structure, assembly, and conformational dynamics of macromolecules in solution. Generating the hydroxyl radicals by laser irradiation, Hambly and Gross developed the approach of Fast Photochemical Oxidation of Proteins (FPOP), which labels proteins on the sub millisecond time scale and provides, with MS analysis, deeper understanding of protein structure and protein-ligand and protein- protein interactions. This review highlights the fundamentals of FPOP and provides descriptions of hydroxyl-radical and other radical and carbene generation, of the hydroxyl labeling of proteins, and of determination of protein modification sites. We also summarize some recent applications of FPOP coupled with MS in protein footprinting. CONCLUSION We survey results that show the capability of FPOP for qualitatively measuring protein solvent accessibility on the residue level. To make these approaches more valuable, we describe recent method developments that increase FPOP's quantitative capacity and increase the spatial protein sequence coverage. To improve FPOP further, several new labeling reagents including carbenes and other radicals have been developed. These growing improvements will allow oxidative- footprinting methods coupled with MS to play an increasingly significant role in determining the structure and dynamics of macromolecules and their assemblies.
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An investigation on the metabolic pathways of synthetic isoflavones by gas chromatography coupled to high accuracy mass spectrometry.
Iannone, M, Botrè, F, Parenti, S, Jardines, D, de la Torre, X
Rapid communications in mass spectrometry : RCM. 2019;(19):1485-1493
Abstract
RATIONALE Isoflavones are a group of flavonoids that may be of interest in sport doping because they can be used by athletes in the recovery periods after the administration of anabolic steroids, with the aim of increasing the natural production of luteinizing hormone (LH) and, consequently, the biosynthesis of endogenous androgens. METHODS The in vivo metabolism of methoxyisoflavone (5-methyl-7-methoxyisoflavone) and ipriflavone (7-isopropoxyisoflavone), respectively present in a dietary supplement and in a pharmaceutical preparation, was investigated. The study was carried out by the analysis of urinary samples collected from male Caucasian subjects before, during and after the oral administration of methoxyisoflavone or ipriflavone. After enzymatic hydrolysis and liquid-liquid extraction, all urinary samples were analyzed by gas chromatography/quadrupole time-of-flight (qTOF MS system/qTOF) electron ionization mass spectrometry (EI-MS). RESULTS Eight metabolites of methoxyisoflavone and six metabolites of ipriflavone were isolated. The corresponding accurate mass spectra are specific for isoflavone structures and revealed also a retro-Diels-Alder fragmentation. CONCLUSIONS When excreted in large amounts, the urinary metabolites of methoxyisoflavone and ipriflavone can be traced to potential confounding factors in doping analysis. As methoxyisoflavone and ipriflavone have been shown to inhibit the enzyme aromatase, thus interfering with the normal metabolic pathways of testosterone, the detection of their intake, by screening for the presence of their main metabolites in urine, might be helpful in routine doping control analysis.
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Effects of anagliptin on plasma glucagon levels and gastric emptying in patients with type 2 diabetes: An exploratory randomized controlled trial versus metformin.
Nakagawa, T, Nagai, Y, Yamamoto, Y, Miyachi, A, Hamajima, H, Mieno, E, Takahashi, M, Inoue, E, Tanaka, Y
Diabetes research and clinical practice. 2019;:107892
Abstract
AIMS: Glucagon has an important role in glucose homeostasis. Recently, a new plasma glucagon assay based on liquid chromatography-high resolution mass spectrometry was developed. We evaluated the influence of a dipeptidyl peptidase-4 inhibitor (anagliptin) on plasma glucagon levels in Japanese patients with type 2 diabetes by using this new assay. METHODS Twenty-four patients with type 2 diabetes were enrolled in a prospective, single-center, randomized, open-label study and were randomly allocated to 4 weeks of treatment with metformin (1000 mg/day) or anagliptin (200 mg/day). A liquid test meal labeled with sodium [13C] acetate was ingested before and after the treatment period. Samples of blood and expired air were collected over 3 h. Plasma levels of glucose, glucagon, C-peptide, glucagon-like peptide-1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP) were measured, and gastric emptying was also evaluated. RESULTS Twenty-two patients completed the study (metformin group: n = 10; anagliptin group: n = 12). Glycemic control showed similar improvement in both groups. In the anagliptin group, there was a slight decrease of the incremental area under the plasma concentration versus time curve for glucagon after the test meal (P = 0.048). In addition, the plasma level of active GLP-1 and GIP was increased, and plasma C-peptide was also increased versus baseline. Neither anagliptin nor metformin delayed gastric emptying. CONCLUSIONS In patients with type 2 diabetes maintained endogenous insulin secretion, anagliptin increased the plasma level of active GLP-1 and GIP in association with a slight stimulation of insulin secretion and slight inhibition of glucagon secretion, but did not delay gastric emptying. Clinical Trial Registry: University hospital Medical Information Network UMIN000028293.
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Efficient ligand discovery from natural herbs by integrating virtual screening, affinity mass spectrometry and targeted metabolomics.
Wang, Z, Liang, H, Cao, H, Zhang, B, Li, J, Wang, W, Qin, S, Wang, Y, Xuan, L, Lai, L, et al
The Analyst. 2019;(9):2881-2890
Abstract
Although natural herbs have been a rich source of compounds for drug discovery, identification of bioactive components from natural herbs suffers from low efficiency and prohibitive cost of the conventional bioassay-based screening platforms. Here we develop a new strategy that integrates virtual screening, affinity mass spectrometry (MS) and targeted metabolomics for efficient discovery of herb-derived ligands towards a specific protein target site. Herb-based virtual screening conveniently selects herbs of potential bioactivity whereas affinity MS combined with targeted metabolomics readily screens candidate compounds in a high-throughput manner. This new integrated approach was benchmarked on screening chemical ligands that target the hydrophobic pocket of the nucleoprotein (NP) of Ebola viruses for which no small molecule ligands have been reported. Seven compounds identified by this approach from the crude extracts of three natural herbs were all validated to bind to the NP target in pure ligand binding assays. Among them, three compounds isolated from Piper nigrum (HJ-1, HJ-4 and HJ-6) strongly promoted the formation of large NP oligomers and reduced the protein thermal stability. In addition, cooperative binding between these chemical ligands and an endogenous peptide ligand was observed, and molecular docking was employed to propose a possible mechanism. Taken together, we established a platform integrating in silico and experimental screening approaches for efficient discovery of herb-derived bioactive ligands especially towards non-enzyme protein targets.
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Diagnosis of Hemoglobinopathy and β-Thalassemia by 21 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and Tandem Mass Spectrometry of Hemoglobin from Blood.
He, L, Rockwood, AL, Agarwal, AM, Anderson, LC, Weisbrod, CR, Hendrickson, CL, Marshall, AG
Clinical chemistry. 2019;(8):986-994
Abstract
BACKGROUND Hemoglobinopathies and thalassemias are the most common genetically determined disorders. Current screening methods include cation-exchange HPLC and electrophoresis, the results of which can be ambiguous because of limited resolving power. Subsequently, laborious genetic testing is required for confirmation. METHODS We performed a top-down tandem mass spectrometry (MS/MS) approach with a fast data acquisition (3 min), ultrahigh mass accuracy, and extensive residue cleavage by use of positive electrospray ionization 21 Tesla Fourier transform ion cyclotron resonance-tandem mass spectrometry (21 T FT-ICR MS/MS) for hemoglobin (Hb) variant de novo sequencing and β-thalassemia diagnosis. RESULTS We correctly identified all Hb variants in blind analysis of 18 samples, including the first characterization of homozygous Hb Himeji variant. In addition, an Hb heterozygous variant with isotopologue mass spacing as small as 0.0194 Da (Hb AD) was resolved in both precursor ion mass spectrum (MS1) and product ion mass spectrum (MS2). In blind analysis, we also observed that the abundance ratio between intact δ and β subunits (δ/β) or the abundance ratio between intact δ and α subunits (δ/α) could serve to diagnose β-thalassemia trait caused by a mutation in 1 HBB gene. CONCLUSIONS We found that 21 T FT-ICR MS/MS provides a benchmark for top-down MS/MS analysis of blood Hb. The present method has the potential to be translated to lower resolving power mass spectrometers (lower field FT-ICR mass spectrometry and Orbitrap) for Hb variant analysis (by MS1 and MS2) and β-thalassemia diagnosis (MS1).