Nature Biotechnology: doi: /nbt Supplementary Figure 1. RNAseq expression profiling of selected glycosyltransferase genes in CHO.
|
|
- Georgiana Chambers
- 5 years ago
- Views:
Transcription
1 Supplementary Figure 1 RNAseq expression profiling of selected glycosyltransferase genes in CHO. RNAseq analysis was performed on two common CHO lines (CHO-K1, CHO-GS) and two independent CHO-GS triple mgat4a/4b/5 KO clones (ZFN91-1C8, ZFN91-2A6). The reported RNAseq analysis of CHO-K1 1 is included, and this was largely identical to our analyses except that the relative expression levels were higher. Importantly, a few genes including mgat4b reported not to be expressed in CHO-K1, were found in the present study to be expressed, and in the case of mgat4b this gene was found to be relevant for engineering loss of triantennary N-glycan branching (Fig. 1b). Another important observation was that the expression profiles of glycogenes in the two triple KO clones analysed were identical to those of the parental CHO lines, providing evidence that the gene editing did not alter expression of other glycogenes, although RNAseq data for all mutant clones are required for complete evaluation.
2 Supplementary Figure 2 N-glycan analysis by exoglycosidase treatment. Sialic acid and galactose residues of native N-glycans were sequentially removed by chemical (2% formic acid (v/v), 80 C, 2h) and enzymatic (E. coli βgalactosidase) treatment. Permethylated N-glycans from EPO produced in mgat4a/4b/5 KO CHO cells before (a) and after (b) desialylation/degalactosylation. The major peak detected after treatment (m/z ) (b) corresponds to a fucosylated biantennary N-glycan lacking sialic acids and galactose residues. The minor peak at m/z in (a) also shifts to m/z after the treatment (b), suggesting that one galactose residue is shielded by a terminal N-acetylglucosamine, which is in agreement with a LacNAc extension 2,3. Permethylated N-glycans from EPO produced in B3gnt2 KO CHO cells before (c) and after (d) desialylation/degalactosylation. The two major peaks at m/z and m/z (d) correspond to fucosylated tri- and tetraantennary N-glycans lacking sialic acid and galactose residues. In this genetic background (B3gnt2 KO), the peak at m/z (c) shifts to m/z (d), demonstrating that all galactose residues were exposed to the β-galactosidase enzyme which is in agreement with a triantennary N-glycan structure. The peaks at m/z and m/z (d) could theoretically be LacNAc extensions on triantennary N-glycans, but are most likely due to incomplete β-galactosidase digestion in agreement with the genetic background.
3 Supplementary Figure 3 Analysis of O-glycosylation of EPO expressed in CHO KO clones. The single O-glycan in human EPO at S126 was analysed from tryptic digests of EPO expressed in CHO WT and CHO with six stacked glycogene KO (B3gnt2/st3gal4/6/mgat4A/4B/5) monitoring the EAISPPDAASAAPLR131 glycopeptide. We used isotopomeric dimethyl labeling 4 for relative quantification of the glycosite occupancy and glycan structure. CHO WT and KO EPO tryptic digest samples were labelled with light (L) and medium (M), respectively, and quantification of peak areas of the extracted ion chromatogram (XIC) for the unglycosylated peptide and the glycopeptides with different glycoforms were presented as a bar chart. EPO tryptic peptides from WT and KO samples were loaded on in-house made equilibrated Stage Tip columns followed by by 3 x CV 0.1% FA wash. Digests were labeled on-column by adding 5 CV 30 mm NaBH3CN and 0.2% formaldehyde in 50 mm sodium phosphate buffer ph 7.5 (L-labeling) or 30 mm NaBH3CN and 0.2% D-formaldehyde in 50 mm sodium phosphate buffer ph 7.5 (M-labeling). Columns were washed using 3 CV 0.1% FA and eluted with 0.5 ml 50% MeOH in 0.1% FA. Samples were dried, reconstituted in 0. 1% FA and analyzed by EASYnLC 1000 UHPLC (Thermo Scientific) interfaced via nanospray Flex ion source to an LTQ-Orbitrap Velos Pro spectrometer (Thermo Scientific).
4 Supplementary Figure 4 SDS-PAGE Coomassie analysis of purified recombinant EPO and IgG. Approximately 1 µg of purified EPO (a), and 10 µl of culture supernatant of CHO clones producing high levels of IgG (b) were loaded to each lane.
5 Supplementary Figure 5 Growth characterization of CHO KO clones. (a) Growth and productivity analysis of CHO WT and two KO clones producing EPO. Cells were cultured in CO2 shaker in 30 ml of growth media in 50 ml TPP TubeSpin Bioreactors with 200rpm or 100ml in Corning 500ml culture bottle with 130rpm (Infors, USA). Cell density and viability were measured by trypan blue and productivity characterized by ELISA. Cells were seeded at 0.3x106 cell/ml, cultured for 5 days in fed-batch with additional 10% of feed containing 3% glucose supplied at day 3. Samples for glycan profiling were collected day 5. (b) Growth and productivity analysis of CHO WT and KO clones producing high levels of IgG (1 g/l). The B4galt1/fut8 KO was performed directly on the IgG producing CHO clone, and the KO clone maintained high productivity (g/l) as the WT. Growth of clones and productivity of EPO and IgG were not substantially affected by the glycogene KO. In general most of the CHO KO clones produced EPO yields comparable to WT cells (Supplementary Table 4). Based on purified yields the average yield of all CHO KO clones were 1.8 +/- 1.5 mg/l, and only 3 out of 21 of the initial KO cell lines produced low yields (22-44% of WT). This is remarkable since selection of stacked KO/KI CHO clones as well as EPO (and IgG) transfectants were based on a limited screen without focus on production yields. Thus, it is expected that careful selection of both stacked KO/KI CHO clones as well as screening for high producers will greatly improve yields, demonstrated in panel (b).
6 Supplementary Figure 6 Immunocytology of CHO KO clones for N-glycan branching and poly-lacnac biosynthesis. (a) The L-PHA lectin was used to specifically probe β6-branching of N-glycans and only KO of mgat5 produced (mgat4a/4b/5) altered Phytohemagglutinin-L lectin (L-PHA) labeling, while the control lectin Concanavalin A lectin (ConA) that binds to all types of N-glycans was unaffected. This confirms that MGAT4A and 4B forms the β4-branch, while MGAT5 forms the β6-branch, and hence supports the interpretation of the mass spec data presented in Figure 1b regarding branch assignments. (b) A similar strategy was used to probe for poly-lacnac with Lycopersicon esculentum lectin (LEL), where only KO of B3gnt2 resulted in loss of labeling. This data also support the interpretation of the mass spec data presented in Figure 2 regarding assignment of HexHexNAc units to poly-lacnac biantennary structures.
7 Supplementary Figure 7 Immunocytology of CHO KO clones with mutations related to LacNAc formation. (a) The monoclonal antibody 1B2 was used to label presence of LacNAc after removal of sialic acid by neuraminidase pretreatment. Analysis of KO of B4galt1-4 in CHO-GS WT with heterogeneous branching showed that KO of B4galt1 reduced labeling, while only stacked KO of B4galt1/3 completely abolished labeling. (b) The same analysis of KO of B4galt1-4 in CHO-GS engineered with homogenous biantennary N-glycans (KO of mgat4a/4b/5) showed that KO of B4galt1 alone essentially abolished MAb 1B2 labeling, indicating that β4gal-t1 is the main enzyme responsible for galactosylation of biantennary N-glycans. This finding is expected to have implications for recombinant production of therapeutic IgGs in CHO, since these proteins in general are produced with biantennary N- glycans with limited galactosylation 5.
8 Supplementary Figure 8 Immunocytology of CHO KO and KI clones related to sialylation. (a) The antibody 1B2 was used to label exposure of LacNAc as a result of loss of sialylation capacity, and this demonstrated that KO of st3gal3 and st3gal6 did not affect labeling substantially, while KO of st3galt4 and stacked KO of st3galt4/6 resulted in strong labeling. This suggests that the ST3Gal-IV enzyme is the main sialyltransferase responsible for α2,3sialylation of N-glycans. Furthermore, de novo introduction of human ST6Gal-I in the stacked st3galt4/6 KO clone completely abolished MAb 1B2 labeling demonstrating efficient sialylation by this enzyme. (b) To confirm the de novo introduction of human ST6Gal-I resulted in α2,6sialylation we used the Sambucus nigra lectin (SNA), which did not label CHO WT cells as expected and only labeled after introduction of ST6Gal-I.
9 Supplementary Figure 9 Analysis of targeted KI of ST6Gal-I by junction PCR. (a) We used a modified ObLiGaRe targeted KI strategy 6 utilizing two inverted ZFN binding sites flanking the ST6Gal-I full open reading frame in donor plasmid. 5 and 3 junction PCR confirmed targeted integration into the Safeharbor#1 site in the CHO clone with st3gal4/6 KO. (b) 5 and 3 junction PCR confirmed targeted integration in the CHO clone with st3gal4/6/mgat4a/4b/5 KO. The status of allelic copy number of integration was determined by WT allelic PCR. The presence of desired band in WT allelic PCR indicates the presence of Safeharbor#1 site without the integration of targeted KI of gene of interest on at least one of the allele. The results showed biallelic integration of ST6Gal-I at Safeharbor#1 site in the CHO-GS st3gal4/6 KO clone and monoallelic integration in the mgat4a/4b/5/st3gal4/6 KO clone.
10 Supplementary Figure 10 Negative ion ESI-MS glycoprofiling of EPO. Glycoprofiling of EPO produced in CHO WT, B3gnt2/mgat4A/4B/5, and st3gal4/6/mgat4a/4b/5 KO with KI of ST6Gal-I, showing complete loss of poly-lacnac on biantennary N-glycans, and complete de novo capping by α2,6 sialic acid on biantennary N-glycans without poly-lacnac. Negative ion ESI-MS glycoprofiling was used to validate MALDI-TOF based glycan profiling data.
11 Supplementary Figure 11 Negative ion ESI-MS/MS. Negative ion ESI-MS/MS of the precursor ions at m/z (sialylated biantennary N-glycan with core fucose) from EPO produced in CHO with mgat4a/4b/5 KO (top panels) and with both mgat4a/4b/5 and st3gal4/6 KO and KI of ST6Gal-I (bottom panels). The presence of the diagnostic fragment ions at m/z , 8 indicates α2,6 terminal sialylation.
12 Supplementary Figure 12 Graphic depiction of the genetic deconstruction of N-glycosylation in CHO established using EPO as N-glycoprotein reporter. The KO screen performed identifies the key glycogenes that control decisive steps in N-glycosylation of proteins in CHO and provides a matrix for genetic deconstruction of N-glycosylation capacity as well as a platform for reconstruction of desirable more homogeneous glycosylation capacities. The deconstruction provides engineering designs for CHO cells with capacity for production of EPO with for example homogeneous biantennary N-glycans capped with α2,3-linked. Two examples of reconstruction based on KI are shown producing homogeneous α2,6-sialylation after KO of α2,3-sialylation capacities with either WT N-glycan branching heterogeneity or homogeneous biantennary structure. We engineered capacity for homogenous N-glycans with and without core Fuc on IgG1 antibodies. Moreover, we engineered CHO cells with capacity for production of homogeneous monoantennary N-glycans, which are useful for direct GlycoPEGylation with a single modification per N-glycan. Several KO CHO lines have potential for production of glycoprotein therapeutics with more homogeneous glycosylation (e.g. biantennary α2,3-sialylated N-glycans for EPO, and biantennary non-galactosylated/sialylated with and without core Fuc for IgG). Reconstruction may address homogeneity as shown, but also branching, poly-lacnac elongation, and any type of capping as indicated. KO of mgat1 and fut8 were reported previously 9-11.
Mammalian-type Glycosylation l in LEXSY
Mammalian-type Glycosylation l in LEXSY Case Study: Recombinant hu Erythropoietin Jena Bioscience GmbH Loebstedter Str. 80 07749 Jena, Germany Tel.: +49-3641-628-5000 Fax: +49-3641-628-5100 628 e-mail:
More informationLudger Guide to Sialylation: II. Highly Sialylated Glycoproteins
Ludger Guide to Sialylation: II Highly Sialylated Glycoproteins Ludger has over 15 years experience providing products and services for the biopharmaceutical industry and in that time we have noticed that
More informationNature Biotechnology: doi: /nbt Supplementary Figure 1
Supplementary Figure 1 The timeline of the NGAG method for extraction of N-linked glycans and glycosite-containing peptides. The timeline can be changed based on the number of samples. Supplementary Figure
More informationCertificate of Analysis
Certificate of Analysis Human IgG Glycoprotein Standard Cat. #: GCP-IGG-50U Batch: B13T-06 Nominal size: 50μg Expiry: Dec 2020 Description: A glycoprotein standard for use during glycan release and labeling.
More informationDetailed Characterization of Antibody Glycan Structure using the N-Glycan Sequencing Kit
be INSPIRED drive DISCOVERY stay GENUINE APPLICATION NOTE Detailed Characterization of Antibody Glycan Structure using the N-Glycan Sequencing Kit Beth McLeod, New England Biolabs, Inc. Materials Remicade
More informationSupplementary figure 1 Supplementary figure 2
Supplementary figure 1 Schematic overview of the Fc-glycan of IgG. The glycan is composed of a constant core domain (highlighted in red) composed of mannose (Man) and N-acetylglucosamine (GlcNAc) residues
More informationTECHNICAL BULLETIN. R 2 GlcNAcβ1 4GlcNAcβ1 Asn
GlycoProfile II Enzymatic In-Solution N-Deglycosylation Kit Product Code PP0201 Storage Temperature 2 8 C TECHNICAL BULLETIN Product Description Glycosylation is one of the most common posttranslational
More informationN-Glycan Sequencing Kit
PROTEIN TOOLS N-Glycan Sequencing Kit Instruction Manual NEB #E577S 2 reactions Version 1. 1/18 be INSPIRED drive DISCOVERY stay GENUINE This product is intended for research purposes only. This product
More informationN-Glycosidase F Deglycosylation Kit
For life science research only. Not for use in diagnostic procedures. FOR IN VITRO USE ONLY. N-Glycosidase F Deglycosylation Kit Kit for the deglycosylation of asparagine-linked glycan chains on glycoproteins.
More informationProfiling the Distribution of N-Glycosylation in Therapeutic Antibodies using the QTRAP 6500 System
Profiling the Distribution of N-Glycosylation in Therapeutic Antibodies using the QTRAP 6500 System Scheduled MRM Pro Algorithm for Increased Efficiency of Targeted Detection Jenny Albanese 1, Christie
More informationSupplementary Figure 1. PD-L1 is glycosylated in cancer cells. (a) Western blot analysis of PD-L1 in breast cancer cells. (b) Western blot analysis
Supplementary Figure 1. PD-L1 is glycosylated in cancer cells. (a) Western blot analysis of PD-L1 in breast cancer cells. (b) Western blot analysis of PD-L1 in ovarian cancer cells. (c) Western blot analysis
More informationDr Mark Hilliard, NIBRT. Waters THE SCIENCE OF WHAT S POSSIBLE TM
RFMS Glycan Characterization Techniques for Biotherapeutics Dr Mark Hilliard, NIBRT Waters THE SCIENCE OF WHAT S POSSIBLE TM The Complexity of Glycosylation Glycosylation is the most common posttranslational
More informationAutomating Mass Spectrometry-Based Quantitative Glycomics using Tandem Mass Tag (TMT) Reagents with SimGlycan
PREMIER Biosoft Automating Mass Spectrometry-Based Quantitative Glycomics using Tandem Mass Tag (TMT) Reagents with SimGlycan Ne uaca2-3galb1-4glc NAcb1 6 Gal NAca -Thr 3 Ne uaca2-3galb1 Ningombam Sanjib
More informationFetuin Glycoprotein Standard
Certificate of Analysis Fetuin Glycoprotein tandard Cat. #: GCP-FET-U-X (GCP-FET-U B7K- *) Batch: BC- Nominal size: * μg Expiry Date: Apr Description: A glycoprotein standard for use during glycan release
More informationOligosaccharide Analysis by High-Performance Anion- Exchange Chromatography with Pulsed Amperometric Detection
Oligosaccharide Analysis by High-Performance Anion- Exchange Chromatography with Pulsed Amperometric Detection Jeff Rohrer, Ph.D. Director, Applications Development, Dionex Products 1 The world leader
More informationAnalysis of N-Linked Glycans from Coagulation Factor IX, Recombinant and Plasma Derived, Using HILIC UPLC/FLR/QTof MS
Analysis of N-Linked Glycans from Coagulation Factor IX, Recombinant and Plasma Derived, Using HILIC UPLC/FLR/QTof MS Ying Qing Yu Waters Corporation, Milford, MA, U.S. A P P L I C AT ION B E N E F I T
More informationIsomeric Separation of Permethylated Glycans by Porous Graphitic Carbon (PGC)-LC-MS/MS at High- Temperatures
Supplementary Information Isomeric Separation of Permethylated Glycans by Porous Graphitic Carbon (PGC)-LC-MS/MS at High- Temperatures Shiyue Zhou 1, Yifan Huang 1, Xue Dong 1, Wenjing Peng 1, Lucas Veillon
More informationCollection of CCP1-binding ACPA from RA serum Serum from RA patients. Serum from healthy donors (HDs) Ammonium sulfate cut.
a! Collection of CCP1-binding ACPA from serum Serum from patients Serum from healthy donors (HDs) Ammonium sulfate cut Ammonium sulfate cut CCP1 column x2 Eluates Flow through fraction CCP1-binding Ab!
More informationGlycan and Monosaccharide Workshop Eoin Cosgrave David Wayland Bill Warren
Glycan and Monosaccharide Workshop Eoin Cosgrave David Wayland Bill Warren 2012 Waters Corporation 1 Requests and Questions Optimised sample prep protocol to reduce sample preparation time How can I detect
More informationThank you for joining us! Our session will begin shortly Waters Corporation 1
UPLC and HPLC Separation Strategies for Successful Characterization of Glycans Derived from Therapeutic Proteins Thank you for joining us! Our session will begin shortly 2013 Waters Corporation 1 Friendly
More informationPTM Discovery Method for Automated Identification and Sequencing of Phosphopeptides Using the Q TRAP LC/MS/MS System
Application Note LC/MS PTM Discovery Method for Automated Identification and Sequencing of Phosphopeptides Using the Q TRAP LC/MS/MS System Purpose This application note describes an automated workflow
More informationSupplementary Figure 1 (previous page). EM analysis of full-length GCGR. (a) Exemplary tilt pair images of the GCGR mab23 complex acquired for Random
S1 Supplementary Figure 1 (previous page). EM analysis of full-length GCGR. (a) Exemplary tilt pair images of the GCGR mab23 complex acquired for Random Conical Tilt (RCT) reconstruction (left: -50,right:
More informationSite-Specific Glycan Microheterogeneity of Inter-Alpha-Trypsin Inhibitor Heavy Chain H4
This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. pubs.acs.org/jpr Site-Specific
More informationSimGlycan. A high-throughput glycan and glycopeptide data analysis tool for LC-, MALDI-, ESI- Mass Spectrometry workflows.
PREMIER Biosoft SimGlycan A high-throughput glycan and glycopeptide data analysis tool for LC-, MALDI-, ESI- Mass Spectrometry workflows SimGlycan software processes and interprets the MS/MS and higher
More informationApplication Note. Abstract. Author. Biotherapeutics & Biosimilars. Sonja Schneider Agilent Technologies, Inc. Waldbronn, Germany
Sensitive and Reproducible Glycan Analysis of Human Immunoglobulin G The Agilent 1260 Infi nity Bio-inert Quaternary LC System with an Agilent AdvanceBio 2.7 µm Glycan Mapping Column and Fluorescence Detection
More informationStructural Elucidation of N-glycans Originating From Ovarian Cancer Cells Using High-Vacuum MALDI Mass Spectrometry
PO-CON1347E Structural Elucidation of N-glycans Originating From Ovarian Cancer Cells Using High-Vacuum MALDI Mass Spectrometry ASMS 2013 TP-708 Matthew S. F. Choo 1,3 ; Roberto Castangia 2 ; Matthew E.
More informationSupporting Information Parsimonious Charge Deconvolution for Native Mass Spectrometry
Supporting Information Parsimonious Charge Deconvolution for Native Mass Spectrometry Marshall Bern* 1, Tomislav Caval 2, Yong J. Kil 1, Wilfred Tang 1, Christopher Becker 1, Eric Carlson 1, Doron Kletter
More informationSupporting Information for MassyTools-assisted data analysis of total serum N-glycome changes associated with pregnancy
Supporting Information for MassyTools-assisted data analysis of total serum N-glycome changes associated with pregnancy Bas C. Jansen 1, Albert Bondt 1,2, Karli R. Reiding 1, Coen J. de Jong 1, David Falck
More informationApplication Note # ET-17 / MT-99 Characterization of the N-glycosylation Pattern of Antibodies by ESI - and MALDI mass spectrometry
Bruker Daltonics Application Note # ET-17 / MT-99 Characterization of the N-glycosylation Pattern of Antibodies by ESI - and MALDI mass spectrometry Abstract Analysis of the N-glycosylation pattern on
More informationfor the Identification of Phosphorylated Peptides
Application of a Data Dependent Neutral-Loss Experiment on the Finnigan LTQ for the Identification of Phosphorylated Peptides Gargi Choudhary Diane Cho Thermo Electron, San Jose, CA Abstracted from posters
More informationGlycosylation analyses of recombinant proteins by LC-ESI mass spectrometry
Glycosylation analyses of recombinant proteins by LC-ESI mass spectrometry Dr Malin Bäckström Mammalian Protein Expression Core Facility P4EU meeting Porto Nov 11-12, 2013 MPE - A tissue culture facility
More informationCurrent Glycoprotein Analysis. Glycan Characterization: Oligosaccharides. Glycan Analysis: Sample Preparation. Glycan Analysis: Chromatography
Bio Day DENMARK MARCH 2013 Analysis of N-linked Glycans of GlycoProteins marleen_van_wingerden@waters.com Agenda Importance of Glycan Analysis Current Glycoprotein Analysis Glycan Characterization: Oligosaccharides
More informationDouble charge of 33kD peak A1 A2 B1 B2 M2+ M/z. ABRF Proteomics Research Group - Qualitative Proteomics Study Identifier Number 14146
Abstract The 2008 ABRF Proteomics Research Group Study offers participants the chance to participate in an anonymous study to identify qualitative differences between two protein preparations. We used
More informationGlycoproteins and N-glycans from exosomes
Glycoproteins and N-glycans from exosomes Júlia Costa Laboratory of Glycobiology WP3: Exosome specific glycosignatures defining specificity in exosomes targeting GlioEx University Medical Center Hamburg
More informationHigh Throughput Accurate Mass Screening of Monoclonal Antibodies
High Throughput Accurate Mass Screening of Monoclonal Antibodies Jim Blasberg April 28, 211 sigma-aldrich.com Life Science and High Technology Center Life Science and High Technology Center2 Sigma-Aldrich,
More informationDetergentOUT Detergent Removal Systems
252PR-04 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name DetergentOUT Detergent Removal Systems For the Removal of Detergents from Peptide
More informationBIOSYNTHESIS OF CANCER-RELATED CARBOHYDRATE ANTIGENS. Fabio Dall Olio Department of Experimental Pathology University of Bologna, Italy
BIOSYNTHESIS OF CANCER-RELATED CARBOHYDRATE ANTIGENS Fabio Dall Olio Department of Experimental Pathology University of Bologna, Italy TOPICS OF THE LECTURE 1. Structure and function of some representative
More informationSupplementary Materials for
advances.sciencemag.org/cgi/content/full/2/1/e1500678/dc1 Supplementary Materials for Chemical synthesis of erythropoietin glycoforms for insights into the relationship between glycosylation pattern and
More informationChapter 6: Comparison of methods for the analysis of therapeutic immunoglobulin G Fcglycosylation profiles-part 2: mass spectrometric methods
Chapter 6: Comparison of methods for the analysis of therapeutic immunoglobulin G Fcglycosylation profiles-part 2: mass spectrometric methods Dietmar Reusch, 1, Markus Haberger, 1 David Falck, 2 Britta
More informationEnzymatic Removal of N- and O-glycans using PNGase F or the Protein Deglycosylation Mix
be INSPIRED drive DISCOVERY stay GENUINE APPLICATION NOTE Enzymatic Removal of N- and O-glycans using PNGase F or the Protein Deglycosylation Mix Alicia Bielik and Paula Magnelli, New England Biolabs,
More informationDetergentOUT Tween. DetergentOUT GBS10. OrgoSol DetergentOUT
252PR 01 G-Biosciences, St Louis, MO. USA 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name DetergentOUT Detergent Removal Systems For the Removal of Detergents
More informationLudgerPure TM APTS Labelled IgG Glycan Library
Certificate of Analysis LudgerPure TM APTS Labelled IgG Glycan Library Cat. #: CAPTS-IgG-0 Batch #. B-0 Size: approx. 0 pmol Description and: Source A mixture of APTS labelled fucosylated bi-antennary
More informationAnalysis of Serum Haptoglobin Fucosylation in Hepatocellular Carcinoma and Liver Cirrhosis of Different Etiologies
pubs.acs.org/jpr Analysis of Serum Haptoglobin Fucosylation in Hepatocellular Carcinoma and Liver Cirrhosis of Different Etiologies Jianhui Zhu, Zhenxin Lin, Jing Wu, Haidi Yin, Jianliang Dai, Ziding Feng,
More informationQuantitation of Protein Phosphorylation Using Multiple Reaction Monitoring
Quantitation of Protein Phosphorylation Using Multiple Reaction Monitoring Application Note Authors Ning Tang, Christine A. Miller and Keith Waddell Agilent Technologies, Inc. Santa Clara, CA USA This
More informationHigh Resolution Glycopeptide Mapping of EPO Using an Agilent AdvanceBio Peptide Mapping Column
High Resolution Glycopeptide Mapping of EPO Using an Agilent AdvanceBio Peptide Mapping Column Application Note BioPharma Authors James Martosella, Phu Duong, and Alex Zhu Agilent Technologies, Inc. Abstract
More informationGlycoprotein Deglycosylation Kit Cat. No
Visit our interactive pathways at /pathways User Protocol 362280 Rev. 23 February 2006 RFH Page 1 of 5 Glycoprotein Deglycosylation Kit Cat. No. 362280 Note that this user protocol is not lot-specific
More informationN-Glycan Analysis: From High-Throughput
N-Glycan Analysis: From High-Throughput Screening to In-Depth Characterization Aled Jones, Ph.D. Senior Product Manager ProZyme, Inc., Hayward CA The 20th Symposium on the Interface of Regulatory and Analytical
More informationCharacterization of the Oligosaccharides Associated with the Human Ovarian Tumor Marker CA125*
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 31, Issue of August 1, pp. 28619 28634, 2003 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Characterization
More informationExtended Mass Range Triple Quadrupole for Routine Analysis of High Mass-to-charge Peptide Ions
Extended Mass Range Triple Quadrupole for Routine Analysis of High Mass-to-charge Peptide Ions Application Note Targeted Proteomics Authors Linfeng Wu, Christine A. Miller, Jordy Hsiao, Te-wei Chu, Behrooz
More informationPr oducts List Ludger Ltd Culham Science Centre Oxford OX14 3EB United Kingdom
Pr oducts List 2018 Ludger Ltd Culham Science Centre Oxford OX14 3EB United Kingdom Email: lily.wang@ludger.com cindy.li@ludger.com www.ludger.com www.ludgersh.com Enzymes for Glycan Release E-PNG01 PNGase
More informationMass Spectrometry. Mass spectrometer MALDI-TOF ESI/MS/MS. Basic components. Ionization source Mass analyzer Detector
Mass Spectrometry MALDI-TOF ESI/MS/MS Mass spectrometer Basic components Ionization source Mass analyzer Detector 1 Principles of Mass Spectrometry Proteins are separated by mass to charge ratio (limit
More informationSupporting information
Electronic Supplementary Material (ESI) for ChemComm. This journal is The Royal Society of Chemistry 2014 Supporting information Glycan Reductive Isotope-coded Amino Acid Labeling (GRIAL) for Mass Spectrometry-based
More informationComparison of Relative Quantification of Monoclonal Antibody N-glycans Using Fluorescence and MS Detection
Comparison of Relative Quantification of Monoclonal ntibody N-glycans Using Fluorescence and MS Detection pplication Note iotherapeutics & iologics uthors scar Potter and Gregory Staples gilent Technologies,
More informationThe addition of sugar moiety determines the blood group
The addition of sugar moiety determines the blood group Sugars attached to glycoproteins and glycolipids on the surfaces of red blood cells determine the blood group termed A, B, and O. The A and B antigens
More informationDetergentOUT GBS10 Spin Plates
G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name DetergentOUT GBS10 Spin Plates 96-Well Plates for the Removal of Detergents from Peptide
More informationOnline 2D-LC Analysis of Complex N-Glycans in Biopharmaceuticals Using the Agilent 1290 Infinity 2D-LC Solution
Online D-LC Analysis of Complex N-Glycans in Biopharmaceuticals Using the Agilent 19 Infinity D-LC Solution Comprehensive and Multiple Heart-Cutting D-LC Analysis for Highest Resolution Application Note
More informationA Fully Integrated Workflow for LC-MS/MS Analysis of Labeled and Native N-Linked Glycans Released From Proteins
A Fully Integrated Workflow for LC-MS/MS Analysis of Labeled and Native N-Linked Glycans Released From Proteins Udayanath Aich, 1 Julian Saba, 2 Xiaodong Liu, 1 Srinivasa Rao, 1 Yury Agroskin, 1 and Chris
More informationMolecular Cell, Volume 46. Supplemental Information
Molecular Cell, Volume 46 Supplemental Information Mapping N-Glycosylation Sites across Seven Evolutionary Distant Species Reveals a Divergent Substrate Proteome Despite a Common Core Machinery Dorota
More informationNature Methods: doi: /nmeth Supplementary Figure 1
Supplementary Figure 1 Subtiligase-catalyzed ligations with ubiquitin thioesters and 10-mer biotinylated peptides. (a) General scheme for ligations between ubiquitin thioesters and 10-mer, biotinylated
More informationQuantitative chromatin proteomics reveals a dynamic histone. post-translational modification landscape that defines asexual
Quantitative chromatin proteomics reveals a dynamic histone post-translational modification landscape that defines asexual and sexual Plasmodium falciparum parasites Nanika Coetzee 1, Simone Sidoli 2,
More informationUNIVERSITY OF YORK BIOLOGY. Glycobiology
Examination Candidate Number: This paper has two parts: UNIVERSITY OF YORK BSc Stage 3 Degree Examinations 2017-18 Department: BIOLOGY Title of Exam: Glycobiology Time allowed: 2 hours Total marks available
More informationModification of sialic acids on solid-phase: accurate characterization of. protein sialylation
Supporting Information for Modification of sialic acids on solid-phase: accurate characterization of protein sialylation Shuang Yang 1,3, Lei Zhang 1, Stefani Thomas 1, Yingwei Hu 1, Shuwei Li 2, John
More informationon Non-Consensus Protein Motifs Analytical & Formulation Sciences, Amgen. Seattle, WA
N-Linked Glycosylation on Non-Consensus Protein Motifs Alain Balland Analytical & Formulation Sciences, Amgen. Seattle, WA CASSS - Mass Spec 2010 Marina Del Rey, CA. September 8 th, 2010 Outline 2 Consensus
More informationTECHNICAL BULLETIN. Enzymatic Protein Deglycosylation Kit. Catalog Number EDEGLY Storage Temperature 2 8 C
Enzymatic Protein Deglycosylation Kit Catalog Number EDEGLY Storage Temperature 2 8 C TECHNICAL BULLETIN Product Description The EDEGLY kit contains all the enzymes and reagents needed to completely remove
More informationSupporting information
Supporting information Figure legends Supplementary Table 1. Specific product ions obtained from fragmentation of lithium adducts in the positive ion mode comparing the different positional isomers of
More informationImprovement of Prostate Cancer Diagnosis by Detecting PSA Glycosylation-Specific Changes
Ivyspring International Publisher 1190 Theranostics Research Paper 2016; 6(8): 1190-1204. doi: 10.7150/thno.15226 Improvement of Prostate Cancer Diagnosis by Detecting PSA Glycosylation-Specific Changes
More informationSupplementary Materials for
immunology.sciencemag.org/cgi/content/full/2/16/eaan6049/dc1 Supplementary Materials for Enzymatic synthesis of core 2 O-glycans governs the tissue-trafficking potential of memory CD8 + T cells Jossef
More informationSupplementary Figures
Supplementary Figures Supplementary Fig. 1. Galectin-3 is present within tumors. (A) mrna expression levels of Lgals3 (galectin-3) and Lgals8 (galectin-8) in the four classes of cell lines as determined
More informationGlycosylation of liver acute-phase proteins in pancreatic cancer and chronic pancreatitis
432 DOI 10.1002/prca.200900150 Proteomics Clin. Appl. 2010, 4, 432 448 RESEARCH ARTICLE Glycosylation of liver acute-phase proteins in pancreatic cancer and chronic pancreatitis Ariadna Sarrats 1, Radka
More informationThermo Fisher Scientific, Sunnyvale, CA, USA; 2 Thermo Fisher Scientific, San Jose, CA, USA
An Ultra High Resolution Glycan Column for Isomeric Separation and the Structural Identification of Labeled N-Glycans from Proteins Including Antibodies Udayanath Aich, 1 Julian Saba, 2 Rosa Viner, 2 Shanhua
More informationDetailed Analysis of Polyclonal IgG with Emphasis on Sialylation and Sub-Classes After Fractionation on a Sialic-Acid Binding Lectin
Universität für Bodenkultur Wien Institute of Biochemistry Detailed Analysis of Polyclonal IgG with Emphasis on Sialylation and Sub-Classes After Fractionation on a Sialic-Acid Binding Lectin by Friedrich
More informationa RT:. - 1.1 1 95 9 85 8 27.64 27.87 28.1 27.42 28.24 38.98 Low ph RPLC NL: 2.29E9 TIC MS TrypticBSA_Dig est_replicate1_ 2Mar18_Preci ous_18-1-6 75 7 65 48.75 65.8 Relative Abundance 6 55 5 45 28.54 48.98
More information1. Sample Introduction to MS Systems:
MS Overview: 9.10.08 1. Sample Introduction to MS Systems:...2 1.1. Chromatography Interfaces:...3 1.2. Electron impact: Used mainly in Protein MS hard ionization source...4 1.3. Electrospray Ioniztion:
More informationIsomer Separation of Positively Labeled N-glycans by CE-ESI-MS
Isomer Separation of Positively Labeled N-glycans by CE-ESI-MS G.S.M. Kammeijer Center for Proteomics and Metabolomics CE IN THE BIOTECHNOLOGY & PHARMACEUTICAL INDUSTRIES 19 TH SYMPOSIUM ON THE PRACTICAL
More informationN-glycans modulate the function of human corticosteroidbinding
N-glycans modulate the function of human corticosteroidbinding globulin Author Sumer-Bayraktar, Zeynep, Kolarich, Daniel, Campbell, Matthew P., Ali, Sinan, Packer, Nicolle H., Thaysen-Andersen, Morten
More informationApplication Note # LCMS-89 High quantification efficiency in plasma targeted proteomics with a full-capability discovery Q-TOF platform
Application Note # LCMS-89 High quantification efficiency in plasma targeted proteomics with a full-capability discovery Q-TOF platform Abstract Targeted proteomics for biomarker verification/validation
More informationSupplementary Material
Supplementary Material Nuclear import of purified HIV-1 Integrase. Integrase remains associated to the RTC throughout the infection process until provirus integration occurs and is therefore one likely
More informationCharacterization of Disulfide Linkages in Proteins by 193 nm Ultraviolet Photodissociation (UVPD) Mass Spectrometry. Supporting Information
Characterization of Disulfide Linkages in Proteins by 193 nm Ultraviolet Photodissociation (UVPD) Mass Spectrometry M. Montana Quick, Christopher M. Crittenden, Jake A. Rosenberg, and Jennifer S. Brodbelt
More informationApplying a Novel Glycan Tagging Reagent, RapiFluor-MS, and an Integrated UPLC-FLR/QTof MS System for Low Abundant N-Glycan Analysis
Applying a Novel Glycan Tagging Reagent, RapiFluor-MS, and an Integrated UPLC-FLR/QTof MS System for Low Abundant N-Glycan Analysis Ying Qing Yu Waters Corporation, Milford, MA, USA APPLICATION BENEFITS
More informationDetermination of Amantadine Residues in Chicken by LCMS-8040
Liquid Chromatography Mass Spectrometry Determination of Amantadine Residues in Chicken by LCMS-8040 A method for the determination of amantadine in chicken was established using Shimadzu Triple Quadrupole
More informationProducts Price List. Euros Ludger Ltd Culham Science Centre Oxford OX14 3EB United Kingdom
Products Price List Euros 2018 Ludger Ltd Culham Science Centre Oxford OX14 3EB United Kingdom Tel: +44 1865 408 554 Fax: +44 870 163 4620 Email: info@ludger.com www.ludger.com Enzymes for Glycan Release
More informationIntroduction to Proteomics 1.0
Introduction to Proteomics 1.0 CMSP Workshop Pratik Jagtap Managing Director, CMSP Objectives Why are we here? For participants: Learn basics of MS-based proteomics Learn what s necessary for success using
More informationProducts Price List. US Dollars Ludger Ltd Culham Science Centre Oxford OX14 3EB United Kingdom
Products Price List US Dollars 2017 Ludger Ltd Culham Science Centre Oxford OX14 3EB United Kingdom Tel: +44 1865 408 554 Fax: +44 870 163 4620 Email: info@ludger.com www.ludger.com Enzymes for Glycan
More informationBiosynthesis of N and O Glycans
TechNote #TNGL101 Biosynthesis of N and O Glycans These suggestions and data are based on information we believe to be reliable. They are offered in good faith, but without guarantee, as conditions and
More informationStructural Characterization of Prion-like Conformational Changes of the Neuronal Isoform of Aplysia CPEB
Structural Characterization of Prion-like Conformational Changes of the Neuronal Isoform of Aplysia CPEB Bindu L. Raveendra, 1,5 Ansgar B. Siemer, 2,6 Sathyanarayanan V. Puthanveettil, 1,3,7 Wayne A. Hendrickson,
More informationRobust extraction, separation, and quantitation of structural isomer steroids from human plasma by SPE-UHPLC-MS/MS
TECHNICAL NOTE 21882 Robust extraction, separation, and quantitation of structural isomer steroids human plasma by SPE-UHPLC-MS/MS Authors Jon Bardsley 1, Kean Woodmansey 1, and Stacy Tremintin 2 1 Thermo
More informationQuantification with Proteome Discoverer. Bernard Delanghe
Quantification with Proteome Discoverer Bernard Delanghe Overview: Which approach to use? Proteome Discoverer Quantification Method What When to use Metabolic labeling SILAC Cell culture systems Small
More informationSupplementary Figure 1. AdipoR1 silencing and overexpression controls. (a) Representative blots (upper and lower panels) showing the AdipoR1 protein
Supplementary Figure 1. AdipoR1 silencing and overexpression controls. (a) Representative blots (upper and lower panels) showing the AdipoR1 protein content relative to GAPDH in two independent experiments.
More informationSUPPORTING INFORMATION FOR: CONCENTRATIONS OF POLYBROMINATED DIPHENYL ETHERS, HEXABROMOCYCLODODECANES AND TETRABROMOBISPHENOL-A IN BREAST MILK FROM
SUPPORTING INFORMATION FOR: CONCENTRATIONS OF POLYBROMINATED DIPHENYL ETHERS, HEXABROMOCYCLODODECANES AND TETRABROMOBISPHENOL-A IN BREAST MILK FROM UNITED KINGDOM WOMEN DO NOT DECREASE OVER TWELVE MONTHS
More informationSupporting Information
Supporting Information Development of a High Coverage Pseudotargeted Lipidomics Method Based on Ultra-High Performance Liquid Chromatography-Mass Spectrometry Qiuhui Xuan 1,2#, Chunxiu Hu 1#, Di Yu 1,2,
More informationSupplementary Figure 1. Properties of various IZUMO1 monoclonal antibodies and behavior of SPACA6. (a) (b) (c) (d) (e) (f) (g) .
Supplementary Figure 1. Properties of various IZUMO1 monoclonal antibodies and behavior of SPACA6. (a) The inhibitory effects of new antibodies (Mab17 and Mab18). They were investigated in in vitro fertilization
More informationWhat sort of Science is Glycoscience? (Introductory lecture)
Glycosciences: Glycobiology & Glycochemistry e-learning course What sort of Science is Glycoscience? (Introductory lecture) Paula Videira Faculdade de Ciências Médicas Nova University, Lisbon Portugal
More informationOligosaccharide Profiling of O-linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2-AA)
Oligosaccharide Profiling of O-linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2-AA) Elisabeth A. Kast and Elizabeth A. Higgins GlycoSolutions Corporation, Worcester, MA Data originally presented
More informationSupporting Information. Lysine Propionylation to Boost Proteome Sequence. Coverage and Enable a Silent SILAC Strategy for
Supporting Information Lysine Propionylation to Boost Proteome Sequence Coverage and Enable a Silent SILAC Strategy for Relative Protein Quantification Christoph U. Schräder 1, Shaun Moore 1,2, Aaron A.
More informationNature Methods: doi: /nmeth.3177
Supplementary Figure 1 Characterization of LysargiNase, trypsin and LysN missed cleavages. (a) Proportion of peptides identified in LysargiNase and trypsin digests of MDA-MB-231 cell lysates carrying 0,
More informationThe distribution of log 2 ratio (H/L) for quantified peptides. cleavage sites in each bin of log 2 ratio of quantified. peptides
Journal: Nature Methods Article Title: Corresponding Author: Protein digestion priority is independent of their abundances Mingliang Ye and Hanfa Zou Supplementary Figure 1 Supplementary Figure 2 The distribution
More informationApplication of a new capillary HPLC- ICP-MS interface to the identification of selenium-containing proteins in selenized yeast
Application of a new capillary HPLC- ICP-MS interface to the identification of selenium-containing proteins in selenized yeast Application note Food supplements Authors Juliusz Bianga and Joanna Szpunar
More informationSupplementary Figure 1
Supplementary Figure 1 Isolation of mt-trnas and RNA-MS analysis of mt-trna Asn from M. nudus (a)m. nudus mt-trnas were isolated by RCC and resolved by 10% denaturing PAGE. The gel was stained with SYBR
More informationDon t miss a thing on your peptide mapping journey How to get full coverage peptide maps using high resolution accurate mass spectrometry
Don t miss a thing on your peptide mapping journey How to get full coverage peptide maps using high resolution accurate mass spectrometry Kai Scheffler, PhD BioPharma Support Expert,LSMS Europe The world
More information