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Shabaz Mohammed - One of the best experts on this subject based on the ideXlab platform.

  • benchmarking stable isotope Labeling based quantitative proteomics
    Journal of Proteomics, 2013
    Co-Authors: A. F. Maarten Altelaar, Christian K Frese, Marco L Hennrich, H T M Timmers, Christian Preisinger, Albert J R Heck, A. W. Schram, Shabaz Mohammed
    Abstract:

    Abstract Several quantitative mass spectrometry based technologies have recently evolved to interrogate the complexity, interconnectivity and dynamic nature of proteomes. Currently, the most popular methods use either metabolic or Chemical isotope Labeling with MS based quantification or Chemical Labeling using isobaric tags with MS/MS based quantification. Here, we assess the performance of three of the most popular approaches through systematic independent large scale quantitative proteomics experiments, comparing SILAC, dimethyl and TMT Labeling strategies. Although all three methods have their strengths and weaknesses, our data indicate that all three can reach a similar depth in number of identified proteins using a classical (MS2 based) shotgun approach. TMT quantification using only MS2 is heavily affected by co-isolation leading to compromised precision and accuracy. This issue may be partly resolved by using an MS3 based acquisition; however, at the cost of a significant reduction in number of proteins quantified. Interestingly, SILAC and Chemical Labeling with MS based quantification produce almost indistinguishable results, independent of which database search algorithm used. This article is part of a Special Issue entitled: New Horizons and Applications for Proteomics [EuPA 2012].

  • benchmarking stable isotope Labeling based quantitative proteomics
    Journal of Proteomics, 2013
    Co-Authors: A. F. Maarten Altelaar, Christian K Frese, Marco L Hennrich, H T M Timmers, Christian Preisinger, Albert J R Heck, A. W. Schram, Shabaz Mohammed
    Abstract:

    Abstract Several quantitative mass spectrometry based technologies have recently evolved to interrogate the complexity, interconnectivity and dynamic nature of proteomes. Currently, the most popular methods use either metabolic or Chemical isotope Labeling with MS based quantification or Chemical Labeling using isobaric tags with MS/MS based quantification. Here, we assess the performance of three of the most popular approaches through systematic independent large scale quantitative proteomics experiments, comparing SILAC, dimethyl and TMT Labeling strategies. Although all three methods have their strengths and weaknesses, our data indicate that all three can reach a similar depth in number of identified proteins using a classical (MS2 based) shotgun approach. TMT quantification using only MS2 is heavily affected by co-isolation leading to compromised precision and accuracy. This issue may be partly resolved by using an MS3 based acquisition; however, at the cost of a significant reduction in number of proteins quantified. Interestingly, SILAC and Chemical Labeling with MS based quantification produce almost indistinguishable results, independent of which database search algorithm used. This article is part of a Special Issue entitled: New Horizons and Applications for Proteomics [EuPA 2012].

  • triplex protein quantification based on stable isotope Labeling by peptide dimethylation applied to cell and tissue lysates
    Proteomics, 2008
    Co-Authors: Paul J Boersema, Toon A B Van Veen, Albert J R Heck, Shabaz Mohammed
    Abstract:

    Stable isotope Labeling is at present one of the most powerful methods in quantitative proteomics. Stable isotope Labeling has been performed at both the protein as well as the peptide level using either metabolic or Chemical Labeling. Here, we present a straightforward and cost-effective triplex quantification method that is based on stable isotope dimethyl Labeling at the peptide level. Herein, all proteolytic peptides are Chemically labeled at their α- and e-amino groups. We use three different isotopomers of formaldehyde to enable the parallel analysis of three different samples. These labels provide a minimum of 4 Da mass difference between peaks in the generated peptide triplets. The method was evaluated based on the quantitative analysis of a cell lysate, using a typical "shotgun" proteomics experiment. While peptide complexity was increased by introducing three labels, still more than 1300 proteins could be identified using 60 μg of starting material, whereby more than 600 proteins could be quantified using at least four peptides per protein. The triplex Labeling was further utilized to distinguish specific from aspecific cAMP binding proteins in a Chemical proteomics experiment using immobilized cAMP. Thereby, differences in abundance ratio of more than two orders of magnitude could be quantified.

  • automated online sequential isotope Labeling for protein quantitation applied to proteasome tissue specific diversity
    Molecular & Cellular Proteomics, 2008
    Co-Authors: Reinout Raijmakers, Albert J R Heck, Celia R Berkers, Annemieke De Jong, Huib Ovaa, Shabaz Mohammed
    Abstract:

    Quantitation of protein abundance is a vital component in the proteomic analysis of biological systems, which can be achieved by differential stable isotopic Labeling. To analyze tissue-derived samples, the isotopic Labeling can be performed using Chemical Labeling of the peptides post-digestion. Standard Chemical Labeling procedures often require many manual sample handling steps, reducing the accuracy of measurements. Here, we describe a fully automated, online (in nanoLC columns), Labeling procedure, which allows protein quantitation using differential isotopic dimethyl Labeling of peptide N termini and lysine residues. We show that the method allows reliable quantitation over a wide dynamic range and can be used to quantify differential protein abundances in lysates and, more targeted, differences in composition between purified protein complexes. We apply the method to determine the differences in composition between bovine liver and spleen 20 S core proteasome complexes. We find that although all catalytically active immunoproteasome subunits were up-regulated in spleen (compared with liver), only one of the normal catalytic subunits was down-regulated, suggesting that the tissue-specific immunoproteasome assembly is more diverse than previously assumed.

Albert J R Heck - One of the best experts on this subject based on the ideXlab platform.

  • benchmarking stable isotope Labeling based quantitative proteomics
    Journal of Proteomics, 2013
    Co-Authors: A. F. Maarten Altelaar, Christian K Frese, Marco L Hennrich, H T M Timmers, Christian Preisinger, Albert J R Heck, A. W. Schram, Shabaz Mohammed
    Abstract:

    Abstract Several quantitative mass spectrometry based technologies have recently evolved to interrogate the complexity, interconnectivity and dynamic nature of proteomes. Currently, the most popular methods use either metabolic or Chemical isotope Labeling with MS based quantification or Chemical Labeling using isobaric tags with MS/MS based quantification. Here, we assess the performance of three of the most popular approaches through systematic independent large scale quantitative proteomics experiments, comparing SILAC, dimethyl and TMT Labeling strategies. Although all three methods have their strengths and weaknesses, our data indicate that all three can reach a similar depth in number of identified proteins using a classical (MS2 based) shotgun approach. TMT quantification using only MS2 is heavily affected by co-isolation leading to compromised precision and accuracy. This issue may be partly resolved by using an MS3 based acquisition; however, at the cost of a significant reduction in number of proteins quantified. Interestingly, SILAC and Chemical Labeling with MS based quantification produce almost indistinguishable results, independent of which database search algorithm used. This article is part of a Special Issue entitled: New Horizons and Applications for Proteomics [EuPA 2012].

  • benchmarking stable isotope Labeling based quantitative proteomics
    Journal of Proteomics, 2013
    Co-Authors: A. F. Maarten Altelaar, Christian K Frese, Marco L Hennrich, H T M Timmers, Christian Preisinger, Albert J R Heck, A. W. Schram, Shabaz Mohammed
    Abstract:

    Abstract Several quantitative mass spectrometry based technologies have recently evolved to interrogate the complexity, interconnectivity and dynamic nature of proteomes. Currently, the most popular methods use either metabolic or Chemical isotope Labeling with MS based quantification or Chemical Labeling using isobaric tags with MS/MS based quantification. Here, we assess the performance of three of the most popular approaches through systematic independent large scale quantitative proteomics experiments, comparing SILAC, dimethyl and TMT Labeling strategies. Although all three methods have their strengths and weaknesses, our data indicate that all three can reach a similar depth in number of identified proteins using a classical (MS2 based) shotgun approach. TMT quantification using only MS2 is heavily affected by co-isolation leading to compromised precision and accuracy. This issue may be partly resolved by using an MS3 based acquisition; however, at the cost of a significant reduction in number of proteins quantified. Interestingly, SILAC and Chemical Labeling with MS based quantification produce almost indistinguishable results, independent of which database search algorithm used. This article is part of a Special Issue entitled: New Horizons and Applications for Proteomics [EuPA 2012].

  • use of stable isotope dimethyl Labeling coupled to selected reaction monitoring to enhance throughput by multiplexing relative quantitation of targeted proteins
    Analytical Chemistry, 2012
    Co-Authors: Thin Thin Aye, Albert J R Heck, Teck Yew Low, Yngvild Bjorlykke, Harald Barsnes, Frode S Berven
    Abstract:

    In this manuscript, we present a proof-of-concept study for targeted relative protein quantitation workflow using Chemical Labeling in the form of dimethylation, coupled with selected reaction monitoring (dimethyl-SRM). We first demonstrate close to complete isotope incorporation for all peptides tested. The accuracy, reproducibility, and linear dynamic range of quantitation are further assessed based on known ratios of nonhuman standard proteins spiked into human cerebrospinal fluid (CSF) as a model complex matrix. Quantitation reproducibility below 20% (CV < 20%) was obtained for analyte concentrations present at a dynamic range of 4 orders of magnitude lower than that of the background proteins. An error of less than 15% was observed when measuring the abundance of 44 out of 45 major human plasma proteins. Dimethyl-SRM was further examined by comparing the relative quantitation of eight proteins in human CSF with the relative quantitation obtained using synthetic heavy peptides coupled to stable isotop...

  • triplex protein quantification based on stable isotope Labeling by peptide dimethylation applied to cell and tissue lysates
    Proteomics, 2008
    Co-Authors: Paul J Boersema, Toon A B Van Veen, Albert J R Heck, Shabaz Mohammed
    Abstract:

    Stable isotope Labeling is at present one of the most powerful methods in quantitative proteomics. Stable isotope Labeling has been performed at both the protein as well as the peptide level using either metabolic or Chemical Labeling. Here, we present a straightforward and cost-effective triplex quantification method that is based on stable isotope dimethyl Labeling at the peptide level. Herein, all proteolytic peptides are Chemically labeled at their α- and e-amino groups. We use three different isotopomers of formaldehyde to enable the parallel analysis of three different samples. These labels provide a minimum of 4 Da mass difference between peaks in the generated peptide triplets. The method was evaluated based on the quantitative analysis of a cell lysate, using a typical "shotgun" proteomics experiment. While peptide complexity was increased by introducing three labels, still more than 1300 proteins could be identified using 60 μg of starting material, whereby more than 600 proteins could be quantified using at least four peptides per protein. The triplex Labeling was further utilized to distinguish specific from aspecific cAMP binding proteins in a Chemical proteomics experiment using immobilized cAMP. Thereby, differences in abundance ratio of more than two orders of magnitude could be quantified.

  • automated online sequential isotope Labeling for protein quantitation applied to proteasome tissue specific diversity
    Molecular & Cellular Proteomics, 2008
    Co-Authors: Reinout Raijmakers, Albert J R Heck, Celia R Berkers, Annemieke De Jong, Huib Ovaa, Shabaz Mohammed
    Abstract:

    Quantitation of protein abundance is a vital component in the proteomic analysis of biological systems, which can be achieved by differential stable isotopic Labeling. To analyze tissue-derived samples, the isotopic Labeling can be performed using Chemical Labeling of the peptides post-digestion. Standard Chemical Labeling procedures often require many manual sample handling steps, reducing the accuracy of measurements. Here, we describe a fully automated, online (in nanoLC columns), Labeling procedure, which allows protein quantitation using differential isotopic dimethyl Labeling of peptide N termini and lysine residues. We show that the method allows reliable quantitation over a wide dynamic range and can be used to quantify differential protein abundances in lysates and, more targeted, differences in composition between purified protein complexes. We apply the method to determine the differences in composition between bovine liver and spleen 20 S core proteasome complexes. We find that although all catalytically active immunoproteasome subunits were up-regulated in spleen (compared with liver), only one of the normal catalytic subunits was down-regulated, suggesting that the tissue-specific immunoproteasome assembly is more diverse than previously assumed.

Anne S Ulrich - One of the best experts on this subject based on the ideXlab platform.

  • Chemical Labeling strategy with r and s trifluoromethylalanine for solid state 19f nmr analysis of peptaibols in membranes
    Journal of the American Chemical Society, 2009
    Co-Authors: Daniel Maisch, Parvesh Wadhwani, Sergii Afonin, Christoph Bottcher, Beate Koksch, Anne S Ulrich
    Abstract:

    Substitution of a single Aib-residue in a peptaibol with (R)- and (S)-trifluoromethylalanine yields two local orientational constraints θ by solid state 19F NMR. The structure of the membrane-perturbing antibiotic alamethicin in DMPC bilayers was analyzed in terms of two angles τ and ρ from six such constraints, showing that the N-terminus (up to a kink at Pro14) is folded as an α-helix, tilted away from the membrane normal by 8°, and assembled as an oligomer. The new 19F NMR label CF3-Ala has thus been demonstrated to be highly sensitive, virtually unperturbing, and ideally suited to characterize peptaibols in membranes.

  • Chemical Labeling strategy with r and s trifluoromethylalanine for solid state 19f nmr analysis of peptaibols in membranes
    Journal of the American Chemical Society, 2009
    Co-Authors: Daniel Maisch, Parvesh Wadhwani, Sergii Afonin, Christoph Bottcher, Beate Koksch, Anne S Ulrich
    Abstract:

    Substitution of a single Aib-residue in a peptaibol with (R)- and (S)-trifluoromethylalanine yields two local orientational constraints theta by solid state (19)F NMR. The structure of the membrane-perturbing antibiotic alamethicin in DMPC bilayers was analyzed in terms of two angles tau and rho from six such constraints, showing that the N-terminus (up to a kink at Pro14) is folded as an alpha-helix, tilted away from the membrane normal by 8 degrees, and assembled as an oligomer. The new (19)F NMR label CF(3)-Ala has thus been demonstrated to be highly sensitive, virtually unperturbing, and ideally suited to characterize peptaibols in membranes.

So Iwata - One of the best experts on this subject based on the ideXlab platform.

  • band 3 the human red cell chloride bicarbonate anion exchanger ae1 slc4a1 in a structural context
    Biochimica et Biophysica Acta, 2016
    Co-Authors: Reinhart A. F. Reithmeier, Yilmaz Alguel, Antreas C Kalli, Joseph R Casey, Mark S P Sansom, So Iwata
    Abstract:

    The crystal structure of the dimeric membrane domain of human Band 31, the red cell chloride/bicarbonate anion exchanger 1 (AE1, SLC4A1), provides a structural context for over four decades of studies into this historic and important membrane glycoprotein. In this review, we highlight the key structural features responsible for anion binding and translocation and have integrated the following topological markers within the Band 3 structure: blood group antigens, N-glycosylation site, protease cleavage sites, inhibitor and Chemical Labeling sites, and the results of scanning cysteine and N-glycosylation mutagenesis. Locations of mutations linked to human disease, including those responsible for Southeast Asian ovalocytosis, hereditary stomatocytosis, hereditary spherocytosis, and distal renal tubular acidosis, provide molecular insights into their effect on Band 3 folding. Finally, molecular dynamics simulations of phosphatidylcholine self-assembled around Band 3 provide a view of this membrane protein within a lipid bilayer.

  • Band 3, the human red cell chloride/bicarbonate anion exchanger (AE1, SLC4A1), in a structural context.
    Biochimica et Biophysica Acta, 2016
    Co-Authors: Reinhart A. F. Reithmeier, Yilmaz Alguel, Antreas C Kalli, Joseph R Casey, Mark S P Sansom, So Iwata
    Abstract:

    The crystal structure of the dimeric membrane domain of human Band 31, the red cell chloride/bicarbonate anion exchanger 1 (AE1, SLC4A1), provides a structural context for over four decades of studies into this historic and important membrane glycoprotein. In this review, we highlight the key structural features responsible for anion binding and translocation and have integrated the following topological markers within the Band 3 structure: blood group antigens, N-glycosylation site, protease cleavage sites, inhibitor and Chemical Labeling sites, and the results of scanning cysteine and N-glycosylation mutagenesis. Locations of mutations linked to human disease, including those responsible for Southeast Asian ovalocytosis, hereditary stomatocytosis, hereditary spherocytosis, and distal renal tubular acidosis, provide molecular insights into their effect on Band 3 folding. Finally, molecular dynamics simulations of phosphatidylcholine self-assembled around Band 3 provide a view of this membrane protein within a lipid bilayer.

Chun-xiao Song - One of the best experts on this subject based on the ideXlab platform.

  • 5 hydroxymethylcytosine signatures in cell free dna provide information about tumor types and stages
    bioRxiv, 2017
    Co-Authors: Chun-xiao Song, Senlin Yin, Amanda Wheeler, Yu Chen, Y Zhang, Bin Liu, Junjie Xiong, Weihan Zhang, Zongguang Zhou, Biao Dong
    Abstract:

    5-Hydroxymethylcytosine (5hmC) is an important mammalian DNA epigenetic modification that has been linked to gene regulation and cancer pathogenesis. Here we explored the diagnostic potential of 5hmC in circulating cell-free DNA (cfDNA) using a sensitive Chemical Labeling-based low-input shotgun sequencing approach. We sequenced cell-free 5hmC from 49 patients of seven different cancer types and found distinct features that could be used to predict cancer types and stages with high accuracy. Specifically, we discovered that lung cancer leads to a progressive global loss of 5hmC in cfDNA, whereas hepatocellular carcinoma and pancreatic cancer lead to disease-specific changes in the cell-free hydroxymethylome. Our proof of principle results suggest that cell-free 5hmC signatures may potentially be used not only to identify cancer types but also to track tumor stage in some cancers.

  • detection of mismatched 5 hydroxymethyluracil in dna by selective Chemical Labeling
    Methods, 2015
    Co-Authors: Miao Yu, Chun-xiao Song, Chuan He
    Abstract:

    How DNA demethylation is achieved in mammals is still under extensive investigation. One proposed mechanism is deamination of 5-hydroxymethylcytosine to form 5-hydroxymethyluracil (5hmU), followed by base excision repair to replace the mismatched 5hmU with cytosine. In this process, 5hmU:G mispair serves as a key intermediate and its localization and distribution in mammalian genome could be important information to investigate the proposed pathway. Here we describe a selective Labeling method to map mismatched 5hmU. After converting other cytosine modifications to 5-carboxylcytosines, a biotin tag is installed onto mismatched 5hmU through β-glucosyltransferase-catalyzed glucosylation and click chemistry. The enriched 5hmU-containing DNA fragments can be subject to subsequent sequencing to reveal the distribution of 5hmU:G mispair with base-resolution information acquired.

  • Chemical modification assisted bisulfite sequencing cab seq for 5 carboxylcytosine detection in dna
    Journal of the American Chemical Society, 2013
    Co-Authors: Chun-xiao Song, Keith E. Szulwach, Zhipeng Wang, Payton Weidenbacher, Peng Jin
    Abstract:

    5-Methylcytosine (5mC) in DNA can be oxidized stepwise to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) by the TET family proteins. Thymine DNA glycosylase can further remove 5fC and 5caC, connecting 5mC oxidation with active DNA demethylation. Here, we present a Chemical modification-assisted bisulfite sequencing (CAB-Seq) that can detect 5caC with single-base resolution in DNA. We optimized 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)-catalyzed amide bond formation between the carboxyl group of 5caC and a primary amine group. We found that the modified 5caC can survive the bisulfite treatment without deamination. Therefore, this Chemical Labeling coupled with bisulfite treatment provides a base-resolution detection and sequencing method for 5caC.

  • detection of 5 hydroxymethylcytosine in a combined glycosylation restriction analysis cgra using restriction enzyme taqαi
    Bioorganic & Medicinal Chemistry Letters, 2011
    Co-Authors: Chun-xiao Song, Qing Dai
    Abstract:

    Abstract 5-Hydroxymethylcytosine (5-hmC) is a newly discovered DNA base in mammalian cells that is believed to be another important epigenetic modification. Here we report the use of a methylation-insensitive restriction enzyme Taq α I coupled with selective Chemical Labeling of 5-hmC in a combined glycosylation restriction analysis (CGRA) to detect 5-hmC in TCGA sequences. This method, differentiates fully versus hemi-hydroxymethylated cytosine in the CpG dinucleotide, adds a new tool to facilitate biological studies of 5-hmC.

  • Selective Chemical Labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine
    Nature Biotechnology, 2011
    Co-Authors: Chun-xiao Song, Keith E. Szulwach, Chengqi Yi, Qing Dai, Chih-hsin Chen, Yujing Li, Xuekun Li, Ye Fu, Wen Zhang, Xing Jian
    Abstract:

    In contrast to 5-methylcytosine (5-mC), which has been studied extensively, little is known about 5-hydroxymethylcytosine (5-hmC), a recently identified epigenetic modification present in substantial amounts in certain mammalian cell types. Here we present a method for determining the genome-wide distribution of 5-hmC. We use the T4 bacteriophage β-glucosyltransferase to transfer an engineered glucose moiety containing an azide group onto the hydroxyl group of 5-hmC. The azide group can be Chemically modified with biotin for detection, affinity enrichment and sequencing of 5-hmC-containing DNA fragments in mammalian genomes. Using this method, we demonstrate that 5-hmC is present in human cell lines beyond those previously recognized. We also find a gene expression level-dependent enrichment of intragenic 5-hmC in mouse cerebellum and an age-dependent acquisition of this modification in specific gene bodies linked to neurodegenerative disorders.