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Frank Klont, Linda Bras, Justina Clarinda Wolters, Sara Ongay, Rainer Bischoff, Gyorgy B Halmos, Péter Horvatovich
For mass spectrometry-based proteomics, the selected sample preparation strategy is a key determinant for information that will be obtained. However, the corresponding selection is often not based on a fit-for-purpose evaluation. Here we report a comparison of in-gel (IGD), in-solution (ISD), on-filter (OFD), and on-pellet digestion (OPD) workflows on the basis of targeted (QconCAT-multiple reaction monitoring (MRM) method for mitochondrial proteins) and discovery proteomics (data dependent acquisition, DDA) analyses using three different human head and neck tissues (i...
April 2, 2018: Analytical Chemistry
Brahim Achour, Alyssa Dantonio, Mark Niosi, Jonathan J Novak, Zubida M Al-Majdoub, Theunis C Goosen, Amin Rostami-Hodjegan, Jill Barber
Quantitative proteomic methods require optimization at several stages, including sample preparation, LC-MS/MS and data analysis, with the final analysis stage being less widely appreciated by end-users. Achour et al. (2017b) previously reported measurement of eight uridine-5'-diphospho-glucuronosyltransferases (UGT) generated by two laboratories [using stable isotope-labeled (SIL) peptides or quantitative concatemer (QconCAT)], which reflected significant disparity between proteomic methods. Initial analysis of QconCAT data showed lack of correlation with catalytic activity for several UGTs (1A4, 1A6, 1A9, 2B15) and moderate correlations for UGTs 1A1, 1A3 and 2B7 (Rs=0...
March 26, 2018: Drug Metabolism and Disposition: the Biological Fate of Chemicals
Sacha K Stelder, Celia Benito de Moya, Huub C J Hoefsloot, Leo J de Koning, Stanley Brul, Chris G de Koster
Spores of Bacillus cereus pose a threat to food safety due to their high resistance to the heat or acid treatments commonly used to make food microbiologically safe. Spores may survive these treatments and later resume growth either on foodstuffs or, after ingestion, upon entering the gut they are capable of producing toxins, which cause either vomiting or diarrhea. The outer layers of the spore, the spore coat and exosporium, consist primarily of proteins that may serve as potential biomarkers for detection...
February 2, 2018: Journal of Proteome Research
Mukesh Kumar, Shai R Joseph, Martina Augsburg, Aliona Bogdanova, David Drechsel, Nadine L Vastenhouw, Frank Buchholz, Marc Gentzel, Andrej Shevchenko
Absolute quantification of proteins elucidates the molecular composition, regulation and dynamics of multiprotein assemblies and networks. Here we report on a method termed MS Western that accurately determines the molar abundance of dozens of user-selected proteins at the subfemtomole level in whole cell or tissue lysates without metabolic or chemical labeling and without using specific antibodies. MS Western relies on GeLC-MS/MS and quantifies proteins by in-gel codigestion with an isotopically labeled QconCAT protein chimera composed of concatenated proteotypic peptides...
February 2018: Molecular & Cellular Proteomics: MCP
Nobuaki Takemori, Ayako Takemori, Yuki Tanaka, Yaeta Endo, Jane L Hurst, Guadalupe Gómez-Baena, Victoria M Harman, Robert J Beynon
A major challenge in proteomics is the absolute accurate quantification of large numbers of proteins. QconCATs, artificial proteins that are concatenations of multiple standard peptides, are well established as an efficient means to generate standards for proteome quantification. Previously, QconCATs have been expressed in bacteria, but we now describe QconCAT expression in a robust, cell-free system. The new expression approach rescues QconCATs that previously were unable to be expressed in bacteria and can reduce the incidence of proteolytic damage to QconCATs...
December 2017: Molecular & Cellular Proteomics: MCP
Brahim Achour, Alyssa Dantonio, Mark Niosi, Jonathan J Novak, John K Fallon, Jill Barber, Philip C Smith, Amin Rostami-Hodjegan, Theunis C Goosen
Quantitative characterization of UDP-glucuronosyltransferase (UGT) enzymes is valuable in glucuronidation reaction phenotyping, predicting metabolic clearance and drug-drug interactions using extrapolation exercises based on pharmacokinetic modeling. Different quantitative proteomic workflows have been employed to quantify UGT enzymes in various systems, with reports indicating large variability in expression, which cannot be explained by inter-individual variability alone. To evaluate the effect of methodological differences on end-point UGT abundance quantification, eight UGT enzymes were quantified in 24 matched liver microsomal samples by two laboratories using stable isotope-labeled (SIL) peptides or quantitative concatemer (QconCAT) standard, and measurements were assessed against catalytic activity in seven enzymes (n=59)...
August 2, 2017: Drug Metabolism and Disposition: the Biological Fate of Chemicals
Richard J Bennett, Deborah M Simpson, Stephen W Holman, Sheila Ryan, Philip Brownridge, Claire E Eyers, John Colyer, Robert J Beynon
The two most common techniques for absolute protein quantification are based on either mass spectrometry (MS) or on immunochemical techniques, such as western blotting (WB). Western blotting is most often used for protein identification or relative quantification, but can also be deployed for absolute quantification if appropriate calibration standards are used. MS based techniques offer superior data quality and reproducibility, but WB offers greater sensitivity and accessibility to most researchers. It would be advantageous to apply both techniques for orthogonal quantification, but workflows rarely overlap...
April 3, 2017: Scientific Reports
Erica T Goddard, Ryan C Hill, Alexander Barrett, Courtney Betts, Qiuchen Guo, Ori Maller, Virginia F Borges, Kirk C Hansen, Pepper Schedin
Normal epithelium exists within a dynamic extracellular matrix (ECM) that is tuned to regulate tissue specific epithelial cell function. As such, ECM contributes to tissue homeostasis, differentiation, and disease, including cancer. Though it is now recognized that the functional unit of normal and transformed epithelium is the epithelial cell and its adjacent ECM, we lack a basic understanding of tissue-specific ECM composition and abundance, as well as how physiologic changes in ECM impact cancer risk and outcomes...
December 2016: International Journal of Biochemistry & Cell Biology
Daryl G S Smith, Geneviève Gingras, Yves Aubin, Terry D Cyr
UNLABELLED: Quantification of the antigens hemagglutinin and neuraminidase in influenza vaccines has been reported using an antibody-free liquid chromatography-mass spectrometry (LC-MS) based method known as MS(E) "Hi3". This approach is based on the average signal intensity of the three most intense tryptic peptides relative to a primary standard. This strategy assumes that the Hi3 signal responses are consistent for all proteins, and therefore comparable to a spiked reference for absolute quantification...
September 2, 2016: Journal of Proteomics
Rebecca J Mackenzie, Craig Lawless, Stephen W Holman, Karin Lanthaler, Robert J Beynon, Chris M Grant, Simon J Hubbard, Claire E Eyers
Chaperones are fundamental to regulating the heat shock response, mediating protein recovery from thermal-induced misfolding and aggregation. Using the QconCAT strategy and selected reaction monitoring (SRM) for absolute protein quantification, we have determined copy per cell values for 49 key chaperones in Saccharomyces cerevisiae under conditions of normal growth and heat shock. This work extends a previous chemostat quantification study by including up to five Q-peptides per protein to improve confidence in protein quantification...
August 2016: Proteomics
Heidi Pertl-Obermeyer, Oliver Trentmann, Kerstin Duscha, H Ekkehard Neuhaus, Waltraud X Schulze
Measurements of protein abundance changes are important for biological conclusions on protein-related processes such as activity or complex formation. Proteomic analyses in general are almost routine tasks in many laboratories, but a precise and quantitative description of (absolute) protein abundance changes require careful experimental design and precise data quality. Today, a vast choice of metabolic labeling and label-free quantitation protocols are available, but the trade-off between quantitative precision and proteome coverage of quantified proteins including missing value problems remain...
2016: Frontiers in Plant Science
Louise Bundgaard, Emøke Bendixen, Mette Aa Sørensen, Victoria M Harman, Robert J Beynon, Lars J Petersen, Stine Jacobsen
In horses, pathological healing with formation of exuberant granulation tissue (EGT) is a particular problem in limb wounds, whereas body wounds tend to heal without complications. Chronic inflammation has been proposed to be central to the pathogenesis of EGT. This study aimed to investigate levels of inflammatory acute phase proteins (APPs) in interstitial fluid from wounds in horses. A novel approach for absolute quantification of proteins, selected reaction monitoring (SRM)-based mass spectrometry in combination with a quantification concatamer (QconCAT), was used for the quantification of five established equine APPs (fibrinogen, serum amyloid A, ceruloplasmin, haptoglobin, and plasminogen) and three proposed equine APPs (prothrombin, α-2-macroglobulin, and α-1-antitrypsin)...
May 2016: Wound Repair and Regeneration
Angelo D'Alessandro, Monika Dzieciatkowska, Ryan C Hill, Kirk C Hansen
BACKGROUND: Laboratory technologies have highlighted the progressive accumulation of the so-called "storage lesion," a wide series of alterations to stored red blood cells (RBCs) that may affect the safety and effectiveness of the transfusion therapy. New improvements in the field are awaited to ameliorate this lesion, such as the introduction of washing technologies in the cell processing pipeline. Laboratory studies that have tested such technologies so far rely on observational qualitative or semiquantitative techniques...
June 2016: Transfusion
Kerry Bauer Scott, Illarion V Turko, Karen W Phinney
Protein quantification based on stable isotope labeling-mass spectrometry involves adding known quantities of stable isotope-labeled internal standards into biological samples. The internal standards are analogous to analyte molecules and quantification is achieved by comparing signals from isotope-labeled and analyte molecules. This methodology is broadly applicable to proteomics research, biomarker discovery and validation, and clinical studies, which require accurate and precise protein abundance measurements...
2016: Methods in Enzymology
Stephen W Holman, Lynn McLean, Claire E Eyers
This study introduces a new reversed-phase liquid chromatography retention time (RT) standard, RePLiCal (Reversed-phase liquid chromatography calibrant), produced using QconCAT technology. The synthetic protein contains 27 lysine-terminating calibrant peptides, meaning that the same complement of standards can be generated using either Lys-C or trypsin-based digestion protocols. RePLiCal was designed such that each constituent peptide is unique with respect to all eukaryotic proteomes, thereby enabling integration into a wide range of proteomic analyses...
March 4, 2016: Journal of Proteome Research
Todd D Johnson, Ryan C Hill, Monika Dzieciatkowska, Vishal Nigam, Atta Behfar, Karen L Christman, Kirk C Hansen
PURPOSE: The purpose of this study was to characterize and quantitatively analyze human cardiac extracellular matrix (ECM) isolated from six different cadaveric donor hearts. EXPERIMENTAL DESIGN: ECM was isolated by decellularization of six human cadaveric donor hearts and characterized by quantifying sulfated glycosaminoglycan content (sGAG) and via PAGE. The protein content was then quantified using ECM-targeted Quantitative conCATamers (QconCAT) by LC-SRM analysis using 83 stable isotope labeled (SIL) peptides representing 48 different proteins...
January 2016: Proteomics. Clinical Applications
Kyle W Anderson, Junjun Chen, Meiyao Wang, Natalia Mast, Irina A Pikuleva, Illarion V Turko
Histone deacetylase (HDAC) inhibition has promise as a therapy for Alzheimer's disease (AD) and other neurodegenerative diseases. Currently, therapeutic HDAC inhibitors target many HDAC isoforms, a particularly detrimental approach when HDAC isoforms are known to have different and specialized functions. We have developed a multiple reaction monitoring (MRM) mass spectrometry assay using stable isotope-labeled QconCATs as internal standards to quantify HDAC isoforms. We further determined a quantitative pattern of specific HDACs expressed in various human and mouse neural tissues...
2015: PloS One
Kerry Bauer Scott, Illarion V Turko, Karen W Phinney
Stable-isotope-labeling mass spectrometry involves the addition of known quantities of stable-isotope labeled standards, which mimic native molecules, to biological samples. We evaluated three conventional internal standard platforms (synthetic peptides, QconCAT constructs, and recombinant proteins) for quantitative accuracy, precision, and inherent advantages and limitations. Internal standards for the absolute quantification of three human cytokine proteins (interferon gamma, interleukin-1 beta, and tumor necrosis factor alpha) were designed and verified...
April 21, 2015: Analytical Chemistry
M D Harwood, B Achour, M R Russell, G L Carlson, G Warhurst, A Rostami-Hodjegan
Transporter proteins expressed in the gastrointestinal tract play a major role in the oral absorption of some drugs, and their involvement may lead to drug-drug interaction (DDI) susceptibility when given in combination with drugs known to inhibit gut wall transporters. Anticipating such liabilities and predicting the magnitude of the impact of transporter proteins on oral drug absorption and DDIs requires quantification of their expression in human intestine, and linking these to data obtained through in vitro experiments...
June 10, 2015: Journal of Pharmaceutical and Biomedical Analysis
Monika Dzieciatkowska, Angelo D'Alessandro, Ryan C Hill, Kirk C Hansen
UNLABELLED: Clinical translation of proteomic technologies is often hampered by technical limitations, including inter-laboratory inconsistencies of label-free derived relative quantification, time-consuming analytical approaches and the subsequent challenge of performing proteomic analyses on large cohorts of subjects. Here we introduce plasma QconCAT-based targeted proteomics, an approach that allows the simultaneous absolute quantitation down to the picogram level of hundreds of proteins in a single liquid chromatography-selected reaction monitoring mass spectrometry run...
April 29, 2015: Journal of Proteomics
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