PNGase F (CarboClip)

Descripción

Deglycosylation Protocol

PNGase F Deglycosylation Protocol

Denaturing deglycosylation of 100 µg of glycoproteins and analysis

1. Add 100 µg of glycoprotein and fill with UltraPure H2O (milliQ, NanoPure, etc) until 70 µl.
2. Add 10 µl of Denaturing Buffer (5% SDS, 400 mM DTT).
3. Incubate at 100°C for 10 minutes.
4. Add 10 µl of reaction buffer (500 mM Sodium Phosphate (pH 7.5)) and 10 µl of 10% NP-40.
5. Add 0.1 µl of Carboclip PNGase F
6. Incubate at 37°C for 30 min.
   (Depending on the glycoprotein up to overnight incubation could be necessary)
7. Analyze by SDS-PAGE

Non-denaturing deglycosylation of Antibody Glycosylation:

Glycan analysis mass spectrometry and MALDI-TOF compatible application.

1. IgG desalting using an Amicon Ultra-0.5 mL Centrifugal Filter device MWCO 30 kDa:
Add your IgG sample to the filter device. If the volume is less than 400 µL, add ultra-pure water up to 400 µL and spin the sample for 5 minutes at 9,000x g at room temperature. Discard the flow-through. Repeat for a total of three times.
Desalting of antibodies is recommended as the presence of salts and detergents could affect the MALDI-TOF-MS acquisition.
2. Antibody Deglycosylation:
Add 3-6 µL of reconstituted CarboClip enzyme for each 50-200 µg of IgG to deglycosylate. Mix the sample by flicking (do not vortex or pipette up and down). Incubate the mixture at 60°C for 1 hour with gentle shaking.
For efficient deglycosylation of the Fab region, IgG denaturation could be necessary.

Citations

This PNGase F has been used in the following Scientific Publications:

Comparative analysis of the human serum N-glycome in lung cancer, COPD and their comorbidity using capillary electrophoresis. Journal of Chromatography B. 2019.
Mészáros, Gábor Járvás, Anna Farkas, Márton Szigeti, Zsuzsanna Kovács, Renáta Kun, Miklós Szabó, Eszter Csánky, András Guttman*

Comparing different domains of analysis for the characterisation of N-Glycans on monoclonal antibodies. Journal of Pharmaceutical Analysis. 2019.
Sara Carillo, Raquel Pérez-Robles, CraigJakes, Meire Ribeiro da Silva, Silvia Millán Martín, AmyFarrell, Natalia Navas, Jonathan Bones*

Sample preparation scale-up for deep N-glycomic analysis of human serum by capillary electrophoresis and CE-ESI-MS. Molecular & Cellular Proteomics. 2019.
Marton Szigeti and Andras Guttman

N-glycosylation of mollusk hemocyanins contributes to their structural stability and immunomodulatory properties in mammals. JBC. 2019.
Michelle L Salazar, José M Jiménez, Javiera Villar, Maira Rivera, Mauricio Báez Sr., Augusto Manubens and María Inés Becker*

Quantitative comparison of the N-glycosylation of therapeutic glycoproteins using the Glycosimilarity Index. A Tutorial. TrAC Trends in Analytical Chemistry. 2019Akos Szekrenyes, MartonSzigeti, Veronika Dvorakova, Gabor Jarvas, AndrasGuttman*

Expression of glycosylated human prolactin in HEK293 cells and related N-glycan composition analysis. AMB Express. 2019.  Silva,Oliveira, Freire, Suzuki, Soares & Bartolini*

Analysis of Fluorophore Labeled N-glycans by the Multicapillary C100ht Biologics Analyzer and HILIC-UPLC: Emphasis on Sialylated Multiantennary Structures. Sciex Technical note Drug Discovery and Development. 2019
Csenge Filep, Beata Borza, Zuzana Demianova, and Andras Guttman*

N-glycosylation analysis of biopharmaceuticals by multicapillary gel electrophoresis: Generation and application of a new glucose unit database. J Pharm Biomed Anal. 2019
Csenge Filep, Beata Borza, Gabor Jarvas and Andras Guttman

Analysis of Fluorophore Labeled N-glycans by the Multicapillary C100HT Biologics Analyzer and HILIC-UPLC: Emphasis on Neutral Structures. Sciex Technical note Drug Discovery and Development. 2019
Beata Borza, Csenge Filep, Akos Szekrenyes, Zuzana Demianova and Andras Guttman*

A Novel Methanol-Free Platform for Extracellular Expression of rhGM-CSF in Pichia pastoris. Molecular Biotechnology. 2019
Roghayeh Shirvani, Sajjad Yazdanpanah, Mohammad Barshan‑tashnizi*, Maryam Shahali*

TLR4, but Neither Dectin-1 nor Dectin-2, Participates in the Mollusk Hemocyanin-Induced Proinflammatory Effects in Antigen-Presenting Cells From Mammals. Frontiers Immunology. 2019
José M. Jiménez, Michelle L. Salazar, Sergio Arancibia, Javiera Villar, Fabián Salazar, Gordon D. Brown, Ed C. Lavelle, Luisa Martínez-Pomares, Jafet Ortiz-Quintero, Sergio Lavandero, Augusto Manubens and María Inés Becker*

Distinct glycosylation in membrane proteins within neonatal versus adult myocardial tissue. Matrix Biology. 2019
Contessotto P, Ellis B, Jin C, Karlsson NG, Zorlutuna P, Kilcoyne M, Pandit A*.

Parsimonious Charge Deconvolution for Native Mass Spectrometry. Journal of Proteome Research. 2018
Marshall Bern, Tomislav Caval, Yong J. Kil, Wilfred Tang, Christopher Becker, Eric Carlson, Doron Kletter, K. Ilker Sen, Nicolas Galy, Dominique Hagemans, Vojtech Franc, and Albert J. R. Heck*

Quantitative assessment of mAb Fc glycosylation of CQA importance by capillary electrophoresis. Electrophoresis. 2018
Marton Szigeti , Jeff Chapman , Beata Borza and Andras Guttman*

Methods and apparatuses for determining the intact mass of large molecules from mass spectrographic data. Published Patent Application. 2018
Marshall Bern

Participación de los receptores de inmunidad innata Dectina-2, Dectina-1 y TLR4 en la activación de las células presentadoras de antígeno de mamíferos inducida por hemocianinas de moluscos. PhD Thesis. Chile University. 2019.
José Manuel Jiménez Rubilar

Efecto de la eliminación enzimática de N-glúcidos sobre la estructura y propiedades inmunogénicas y antitumorales de las hemocianinas de moluscos en mamíferos. PhD Thesis. Chile University. 2018
Salazar Luco, Michelle Luna, Becker Contreras, María Inés

Applications

This enzyme from Asparia Glycomics has been used for several applications, including:

N-glycomic analysis of therapeutic antibodies and therapeutic glycoproteins, as described by Sciex. 2019

Deglycosylation for Capillary Electrophoresis Separations of Glycans, as described by Mészáros et al.  Journal of Chromatography B. 2019.

Deglycosylation of Membrane proteins, as described by Contessotto et al. Matrix Biol. 2019.

Deglycosylation of Hemocyanins, as described by Jimenez et al. Front. Immunol. 2019.

Removal of N-Linked Glycans for Native Mass Spectrometry, as described by Bern et al. J. Proteome Res. 2018.

User’s guide – please request it at sales@aspariaglycomics.com

Glycan Mass Spectrometry

Glycan Analysis Mass Spectrometry and Analysis of protein deglycosylation by MALDI-TOF.

e.g. Deglycosylation of Antibody under non-denaturing conditions

1. Add 2 µl of PNGase F (CarboClip) to 100 µl of IgG (10 µg/µl) diluted in nanoPure H₂O.
2. Incubate at 60 ºC for 2h.
3. Clean and concentrate the sample using an amicon device
4. Add 1 µl of concentrated IgG to the polished steel MALDI-TOF plate,
5. Add 1 µl of SuperDHB (diluted in ACN at 20 µg/µl).
6. Let it dry and analyze by MALDI-TOF
7. Flex control-ultraflex tof-tof software
   Method: LP_30-210_KDa.par.
   Set laser Attenuator “3_medium”.
   Mass range 68,000 – 170,000.