Deglycosylation by the Acidic Glycosidase PNGase H+ Enables Analysis of N-Linked Glycoproteins by Hydrogen/Deuterium Exchange Mass Spectrometry

Research output: Contribution to journal › Journal article › Research › peer-review

Standard

Deglycosylation by the Acidic Glycosidase PNGase H+ Enables Analysis of N-Linked Glycoproteins by Hydrogen/Deuterium Exchange Mass Spectrometry. / Comamala, Gerard; Madsen, Jeppe B; Voglmeir, Josef; Du, Ya-Min; Jensen, Pernille F; Østerlund, Eva C; Trelle, Morten B; Rand, Kasper D.

In: Journal of the American Society for Mass Spectrometry, Vol. 31, No. 11, 2020, p. 2305-2312.

Research output: Contribution to journal › Journal article › Research › peer-review

Harvard

Comamala, G, Madsen, JB, Voglmeir, J, Du, Y-M, Jensen, PF, Østerlund, EC, Trelle, MB & Rand, KD 2020, 'Deglycosylation by the Acidic Glycosidase PNGase H+ Enables Analysis of N-Linked Glycoproteins by Hydrogen/Deuterium Exchange Mass Spectrometry', Journal of the American Society for Mass Spectrometry, vol. 31, no. 11, pp. 2305-2312. https://doi.org/10.1021/jasms.0c00258

APA

Comamala, G., Madsen, J. B., Voglmeir, J., Du, Y-M., Jensen, P. F., Østerlund, E. C., Trelle, M. B., & Rand, K. D. (2020). Deglycosylation by the Acidic Glycosidase PNGase H+ Enables Analysis of N-Linked Glycoproteins by Hydrogen/Deuterium Exchange Mass Spectrometry. Journal of the American Society for Mass Spectrometry, 31(11), 2305-2312. https://doi.org/10.1021/jasms.0c00258

Vancouver

Comamala G, Madsen JB, Voglmeir J, Du Y-M, Jensen PF, Østerlund EC et al. Deglycosylation by the Acidic Glycosidase PNGase H+ Enables Analysis of N-Linked Glycoproteins by Hydrogen/Deuterium Exchange Mass Spectrometry. Journal of the American Society for Mass Spectrometry. 2020;31(11):2305-2312. https://doi.org/10.1021/jasms.0c00258

Author

Comamala, Gerard ; Madsen, Jeppe B ; Voglmeir, Josef ; Du, Ya-Min ; Jensen, Pernille F ; Østerlund, Eva C ; Trelle, Morten B ; Rand, Kasper D. / Deglycosylation by the Acidic Glycosidase PNGase H+ Enables Analysis of N-Linked Glycoproteins by Hydrogen/Deuterium Exchange Mass Spectrometry. In: Journal of the American Society for Mass Spectrometry. 2020 ; Vol. 31, No. 11. pp. 2305-2312.

Bibtex

@article{561dd7276f85496aab02a0a28de86962,
title = "Deglycosylation by the Acidic Glycosidase PNGase H+ Enables Analysis of N-Linked Glycoproteins by Hydrogen/Deuterium Exchange Mass Spectrometry",
abstract = "Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become an important method to study the structural dynamics of proteins. However, glycoproteins represent a challenge to the traditional HDX-MS workflow for determining the deuterium uptake of the protein segments that contain the glycan. We have recently demonstrated the utility of the glycosidase PNGase A to enable HDX-MS analysis of N-glycosylated protein regions. Here, we have investigated the use of the acidic glycosidase PNGase H+, which has a pH optimum at 2.6, to efficiently deglycosylate N-linked glycosylated peptides during HDX-MS analysis of glycoproteins. Our results show that PNGase H+ retains high deglycosylation activity at HDX quench conditions. When used in an HDX-MS workflow, PNGase H+ allowed the extraction of HDX data from all five glycosylated regions of the serpin α1-antichymotrypsin. We demonstrate that PNGase A and PNGase H+ are capable of similar deglycosylation performance during HDX-MS analysis of α1-antichymotrypsin and the IgG1 antibody trastuzumab (TZ). However, PNGase H+ provides broader specificity and greater tolerance to the disulfide-bond reducing agent TCEP, while PNGase A offers advantages in terms of commercial availability and purity. Overall, our findings demonstrate the unique features of PNGase H+ for improving conformational analysis of glycoproteins by HDX-MS, in particular, challenging glycoproteins containing both glycosylations and disulfide bonds.",
author = "Gerard Comamala and Madsen, {Jeppe B} and Josef Voglmeir and Ya-Min Du and Jensen, {Pernille F} and {\O}sterlund, {Eva C} and Trelle, {Morten B} and Rand, {Kasper D}",
year = "2020",
doi = "10.1021/jasms.0c00258",
language = "English",
volume = "31",
pages = "2305--2312",
journal = "Journal of The American Society for Mass Spectrometry",
issn = "1044-0305",
publisher = "Springer Nature",
number = "11",

}

RIS

TY - JOUR

T1 - Deglycosylation by the Acidic Glycosidase PNGase H+ Enables Analysis of N-Linked Glycoproteins by Hydrogen/Deuterium Exchange Mass Spectrometry

AU - Comamala, Gerard

AU - Madsen, Jeppe B

AU - Voglmeir, Josef

AU - Du, Ya-Min

AU - Jensen, Pernille F

AU - Østerlund, Eva C

AU - Trelle, Morten B

AU - Rand, Kasper D

PY - 2020

Y1 - 2020

N2 - Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become an important method to study the structural dynamics of proteins. However, glycoproteins represent a challenge to the traditional HDX-MS workflow for determining the deuterium uptake of the protein segments that contain the glycan. We have recently demonstrated the utility of the glycosidase PNGase A to enable HDX-MS analysis of N-glycosylated protein regions. Here, we have investigated the use of the acidic glycosidase PNGase H+, which has a pH optimum at 2.6, to efficiently deglycosylate N-linked glycosylated peptides during HDX-MS analysis of glycoproteins. Our results show that PNGase H+ retains high deglycosylation activity at HDX quench conditions. When used in an HDX-MS workflow, PNGase H+ allowed the extraction of HDX data from all five glycosylated regions of the serpin α1-antichymotrypsin. We demonstrate that PNGase A and PNGase H+ are capable of similar deglycosylation performance during HDX-MS analysis of α1-antichymotrypsin and the IgG1 antibody trastuzumab (TZ). However, PNGase H+ provides broader specificity and greater tolerance to the disulfide-bond reducing agent TCEP, while PNGase A offers advantages in terms of commercial availability and purity. Overall, our findings demonstrate the unique features of PNGase H+ for improving conformational analysis of glycoproteins by HDX-MS, in particular, challenging glycoproteins containing both glycosylations and disulfide bonds.

AB - Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become an important method to study the structural dynamics of proteins. However, glycoproteins represent a challenge to the traditional HDX-MS workflow for determining the deuterium uptake of the protein segments that contain the glycan. We have recently demonstrated the utility of the glycosidase PNGase A to enable HDX-MS analysis of N-glycosylated protein regions. Here, we have investigated the use of the acidic glycosidase PNGase H+, which has a pH optimum at 2.6, to efficiently deglycosylate N-linked glycosylated peptides during HDX-MS analysis of glycoproteins. Our results show that PNGase H+ retains high deglycosylation activity at HDX quench conditions. When used in an HDX-MS workflow, PNGase H+ allowed the extraction of HDX data from all five glycosylated regions of the serpin α1-antichymotrypsin. We demonstrate that PNGase A and PNGase H+ are capable of similar deglycosylation performance during HDX-MS analysis of α1-antichymotrypsin and the IgG1 antibody trastuzumab (TZ). However, PNGase H+ provides broader specificity and greater tolerance to the disulfide-bond reducing agent TCEP, while PNGase A offers advantages in terms of commercial availability and purity. Overall, our findings demonstrate the unique features of PNGase H+ for improving conformational analysis of glycoproteins by HDX-MS, in particular, challenging glycoproteins containing both glycosylations and disulfide bonds.

U2 - 10.1021/jasms.0c00258

DO - 10.1021/jasms.0c00258

M3 - Journal article

C2 - 32955262

VL - 31

SP - 2305

EP - 2312

JO - Journal of The American Society for Mass Spectrometry

JF - Journal of The American Society for Mass Spectrometry

SN - 1044-0305

IS - 11

ER -

ID: 249769918