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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
Jiabin Li, Lianqi Zhou, Huanhuan Wang, Hui Yan, Nannan Li, Rui Zhai, Fenglong Jiao, Feiran Hao, Zuyao Jin, Fang Tian, Bo Peng, Yangjun Zhang, Xiaohong Qian
A key step in the workflow of bottom-up proteomics is the proteolysis of proteins into peptides with trypsin. In addition, enzyme-catalytic (18)O labeled peptides as internal standards coupled with multiple reaction monitoring mass spectrometry (MRM MS) for the absolute quantitation of the target proteome is commonly used for its convenient operation and low cost. However, long digestion and labeling times, incomplete digestion and (18)O to (16)O back exchange limit its application, therefore, we developed a rapid and efficient digestion method based on a high ratio of trypsin to protein...
February 21, 2015: Analyst
Crystal S F Cheung, Kyle W Anderson, Meiyao Wang, Illarion V Turko
Quantification by targeted proteomics has largely depended on mass spectrometry and isotope-labeled internal standards. In addition to traditionally used recombinant proteins or synthetic peptides, concatenated peptides (QconCATs) were introduced as a conceptually new source of internal standard. In the present study, we focused on assessing the length of natural flanking sequences, which surround each peptide included in QconCAT and provide for identical rates of analyte and standard digestion by trypsin. We have expressed, purified, and characterized a set of seven (15)N-labeled QconCATs that cover seven tryptic peptides from human clusterin with a length of natural flanking sequences ranging from none (+0) to six amino acid residues (+6) for each tryptic peptide...
January 20, 2015: Analytical Chemistry
Raphael Voges, Stephanie Corsten, Wolfgang Wiechert, Stephan Noack
UNLABELLED: The soil bacterium Corynebacterium glutamicum is one of the best-studied production hosts for industrial biotechnology, and it is primarily used for the large-scale production of essential amino acids, such as l-lysine. For rational strain development, detailed knowledge of intracellular protein concentration is crucial to determine metabolic capacities and limitations. We developed a QconCAT approach for the accurate absolute quantification of key enzymes of C. glutamicum glycolysis and anaplerosis...
January 15, 2015: Journal of Proteomics
Louise Bundgaard, Stine Jacobsen, Thomas F Dyrlund, Mette Aa Sørensen, Victoria M Harman, Robert J Beynon, Philip J Brownridge, Lars J Petersen, Emøke Bendixen
The aim of this study was the development of a quantitative assay that could support future studies of a panel of acute phase proteins (APPs) in the horse. The assay was based on a quantification concatamer (QconCAT) coupled to selected reaction monitoring methodology. Thirty-two peptides, corresponding to 13 putative or confirmed APPs for the Equus caballus (equine) species were selected for the design of a QconCAT construct. The gene encoding the QconCAT was synthesized and expressed as an isotope-labeled chimaeric protein in Escherichia coli...
December 5, 2014: Journal of Proteome Research
Tanveer S Batth, Pragya Singh, Vikram R Ramakrishnan, Mirta M L Sousa, Leanne Jade G Chan, Huu M Tran, Eric G Luning, Eva H Y Pan, Khanh M Vuu, Jay D Keasling, Paul D Adams, Christopher J Petzold
Transformation of engineered Escherichia coli into a robust microbial factory is contingent on precise control of metabolism. Yet, the throughput of omics technologies used to characterize cell components has lagged far behind our ability to engineer novel strains. To expand the utility of quantitative proteomics for metabolic engineering, we validated and optimized targeted proteomics methods for over 400 proteins from more than 20 major pathways in E. coli metabolism. Complementing these methods, we constructed a series of synthetic genes to produce concatenated peptides (QconCAT) for absolute quantification of the proteins and made them available through the Addgene plasmid repository (www...
November 2014: Metabolic Engineering
Robert J Beynon, Stuart D Armstrong, Guadalupe Gómez-Baena, Victoria Lee, Deborah Simpson, Jennifer Unsworth, Jane L Hurst
The high degree of protein sequence similarity in the MUPs (major urinary proteins) poses considerable challenges for their individual differentiation, analysis and quantification. In the present review, we discuss MS approaches for MUP quantification, at either the protein or the peptide level. In particular, we describe an approach to multiplexed quantification based on the design and synthesis of novel proteins (QconCATs) that are concatamers of quantification standards, providing a simple route to the generation of a set of stable-isotope-labelled peptide standards...
August 2014: Biochemical Society Transactions
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