Native Ion Mobility–Mass Spectrometry-Enabled Fast Structural Interrogation of Labile Protein Surface Modifications at the Intact Protein Level

Gongyu Li; Ashley Phetsanthad; Min Ma; Qinying Yu; Ashita Nair; Zhen Zheng; Fengfei Ma; Kellen DeLaney; Seungpyo Hong; Lingjun Li

doi:10.1021/acs.analchem.1c04503

Native Ion Mobility–Mass Spectrometry-Enabled Fast Structural Interrogation of Labile Protein Surface Modifications at the Intact Protein Level

Gongyu Li, Ashley Phetsanthad, Min Ma, Qinying Yu, Ashita Nair, Zhen Zheng, Fengfei Ma, Kellen DeLaney, Seungpyo Hong, Lingjun Li

Department of Pharmacy

Research output: Contribution to journal › Article › peer-review

7 Citations (Scopus)

Abstract

Protein sialylation has been closely linked to many diseases including Alzheimer’s disease (AD). It is also broadly implicated in therapeutics operating in a pattern-dependent (e.g., Neu5Ac vs Neu5Gc) manner. However, how the sialylation pattern affects the AD-associated, transferrin-assisted iron/Aβ cellular uptake process remains largely ill-defined. Herein, we report the use of native ion mobility–mass spectrometry (IM–MS)-based fast structural probing methodology, enabling well-controlled, synergistic, and in situ manipulation of mature glycoproteins and attached sialic acids. IM–MS-centered experiments enable the combinatorial interrogation of sialylation effects on Aβ cytotoxicity and the chemical, conformational, and topological stabilities of transferrin. Cell viability experiments suggest that Neu5Gc replacement enhances the transferrin-assisted, iron loading-associated Aβ cytotoxicity. Native gel electrophoresis and IM–MS reveal that sialylation stabilizes transferrin conformation but inhibits its dimerization. Collectively, IM–MS is adapted to capture key sialylation intermediates involved in fine-tuning AD-associated glycoprotein structural microheterogeneity. Our results provide the molecular basis for the importance of sustaining moderate TF sialylation levels, especially Neu5Ac, in promoting iron cellular transportation and rescuing iron-enhanced Aβ cytotoxicity.

Original language	English
Pages (from-to)	2142-2153
Number of pages	12
Journal	Analytical Chemistry
Volume	94
Issue number	4
DOIs	https://doi.org/10.1021/acs.analchem.1c04503
Publication status	Published - 2022 Feb 1

Bibliographical note

Funding Information:
The authors would like to thank Professor Dick Zare from Stanford University and Professor Brandon Ruotolo from the University of Michigan for providing insightful discussions and suggestions. The authors gratefully appreciate Dr. Yu Gao and Dr. Xinyu Zhao (Waisman Center, UW-Madison) for their generous providing of cell lines. This work was funded in part by NIH (R01DK071801, R56DK071801, U01CA231081, R21AG065728, and RF1AG052324), and NSF (CHE-1710140 and CHE-2108223). The Orbitrap instruments were purchased through the support of an NIH shared instrument grant (NIH-NCRR S10RR029531) and the University of Wisconsin–Madison, Office of the Vice Chancellor for Research and Graduate Education with funding from the Wisconsin Alumni Research Foundation. K.D. acknowledges the National Institutes of Health-General Medical Sciences F31 National Research Service Award (1F31GM126870-01A1) for funding. L.L. acknowledges a Vilas Distinguished Achievement Professorship and Charles Melbourne Johnson Distinguished Chair Professorship with funding provided by the Wisconsin Alumni Research Foundation and University of Wisconsin–Madison School of Pharmacy. G.L. thanks the Nankai University start-up funding support through the Fundamental Research Funds for the Central Universities (no. 020/63213057).

Funding Information:
The authors would like to thank Professor Dick Zare from Stanford University and Professor Brandon Ruotolo from the University of Michigan for providing insightful discussions and suggestions. The authors gratefully appreciate Dr. Yu Gao and Dr. Xinyu Zhao (Waisman Center, UW-Madison) for their generous providing of cell lines. This work was funded in part by NIH (R01DK071801, R56DK071801, U01CA231081, R21AG065728, and RF1AG052324), and NSF (CHE-1710140 and CHE-2108223). The Orbitrap instruments were purchased through the support of an NIH shared instrument grant (NIH-NCRR S10RR029531) and the University of Wisconsin?Madison, Office of the Vice Chancellor for Research and Graduate Education with funding from the Wisconsin Alumni Research Foundation. K.D. acknowledges the National Institutes of Health-General Medical Sciences F31 National Research Service Award (1F31GM126870-01A1) for funding. L.L. acknowledges a Vilas Distinguished Achievement Professorship and Charles Melbourne Johnson Distinguished Chair Professorship with funding provided by the Wisconsin Alumni Research Foundation and University of Wisconsin?Madison School of Pharmacy. G.L. thanks the Nankai University start-up funding support through the Fundamental Research Funds for the Central Universities (no. 020/63213057).

Publisher Copyright:
© 2022 American Chemical Society

All Science Journal Classification (ASJC) codes

Analytical Chemistry

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10.1021/acs.analchem.1c04503

Cite this

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title = "Native Ion Mobility–Mass Spectrometry-Enabled Fast Structural Interrogation of Labile Protein Surface Modifications at the Intact Protein Level",

abstract = "Protein sialylation has been closely linked to many diseases including Alzheimer{\textquoteright}s disease (AD). It is also broadly implicated in therapeutics operating in a pattern-dependent (e.g., Neu5Ac vs Neu5Gc) manner. However, how the sialylation pattern affects the AD-associated, transferrin-assisted iron/Aβ cellular uptake process remains largely ill-defined. Herein, we report the use of native ion mobility–mass spectrometry (IM–MS)-based fast structural probing methodology, enabling well-controlled, synergistic, and in situ manipulation of mature glycoproteins and attached sialic acids. IM–MS-centered experiments enable the combinatorial interrogation of sialylation effects on Aβ cytotoxicity and the chemical, conformational, and topological stabilities of transferrin. Cell viability experiments suggest that Neu5Gc replacement enhances the transferrin-assisted, iron loading-associated Aβ cytotoxicity. Native gel electrophoresis and IM–MS reveal that sialylation stabilizes transferrin conformation but inhibits its dimerization. Collectively, IM–MS is adapted to capture key sialylation intermediates involved in fine-tuning AD-associated glycoprotein structural microheterogeneity. Our results provide the molecular basis for the importance of sustaining moderate TF sialylation levels, especially Neu5Ac, in promoting iron cellular transportation and rescuing iron-enhanced Aβ cytotoxicity.",

author = "Gongyu Li and Ashley Phetsanthad and Min Ma and Qinying Yu and Ashita Nair and Zhen Zheng and Fengfei Ma and Kellen DeLaney and Seungpyo Hong and Lingjun Li",

note = "Funding Information: The authors would like to thank Professor Dick Zare from Stanford University and Professor Brandon Ruotolo from the University of Michigan for providing insightful discussions and suggestions. The authors gratefully appreciate Dr. Yu Gao and Dr. Xinyu Zhao (Waisman Center, UW-Madison) for their generous providing of cell lines. This work was funded in part by NIH (R01DK071801, R56DK071801, U01CA231081, R21AG065728, and RF1AG052324), and NSF (CHE-1710140 and CHE-2108223). The Orbitrap instruments were purchased through the support of an NIH shared instrument grant (NIH-NCRR S10RR029531) and the University of Wisconsin–Madison, Office of the Vice Chancellor for Research and Graduate Education with funding from the Wisconsin Alumni Research Foundation. K.D. acknowledges the National Institutes of Health-General Medical Sciences F31 National Research Service Award (1F31GM126870-01A1) for funding. L.L. acknowledges a Vilas Distinguished Achievement Professorship and Charles Melbourne Johnson Distinguished Chair Professorship with funding provided by the Wisconsin Alumni Research Foundation and University of Wisconsin–Madison School of Pharmacy. G.L. thanks the Nankai University start-up funding support through the Fundamental Research Funds for the Central Universities (no. 020/63213057). Funding Information: The authors would like to thank Professor Dick Zare from Stanford University and Professor Brandon Ruotolo from the University of Michigan for providing insightful discussions and suggestions. The authors gratefully appreciate Dr. Yu Gao and Dr. Xinyu Zhao (Waisman Center, UW-Madison) for their generous providing of cell lines. This work was funded in part by NIH (R01DK071801, R56DK071801, U01CA231081, R21AG065728, and RF1AG052324), and NSF (CHE-1710140 and CHE-2108223). The Orbitrap instruments were purchased through the support of an NIH shared instrument grant (NIH-NCRR S10RR029531) and the University of Wisconsin?Madison, Office of the Vice Chancellor for Research and Graduate Education with funding from the Wisconsin Alumni Research Foundation. K.D. acknowledges the National Institutes of Health-General Medical Sciences F31 National Research Service Award (1F31GM126870-01A1) for funding. L.L. acknowledges a Vilas Distinguished Achievement Professorship and Charles Melbourne Johnson Distinguished Chair Professorship with funding provided by the Wisconsin Alumni Research Foundation and University of Wisconsin?Madison School of Pharmacy. G.L. thanks the Nankai University start-up funding support through the Fundamental Research Funds for the Central Universities (no. 020/63213057). Publisher Copyright: {\textcopyright} 2022 American Chemical Society",

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doi = "10.1021/acs.analchem.1c04503",

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AU - Yu, Qinying

AU - Nair, Ashita

AU - Zheng, Zhen

AU - Ma, Fengfei

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N2 - Protein sialylation has been closely linked to many diseases including Alzheimer’s disease (AD). It is also broadly implicated in therapeutics operating in a pattern-dependent (e.g., Neu5Ac vs Neu5Gc) manner. However, how the sialylation pattern affects the AD-associated, transferrin-assisted iron/Aβ cellular uptake process remains largely ill-defined. Herein, we report the use of native ion mobility–mass spectrometry (IM–MS)-based fast structural probing methodology, enabling well-controlled, synergistic, and in situ manipulation of mature glycoproteins and attached sialic acids. IM–MS-centered experiments enable the combinatorial interrogation of sialylation effects on Aβ cytotoxicity and the chemical, conformational, and topological stabilities of transferrin. Cell viability experiments suggest that Neu5Gc replacement enhances the transferrin-assisted, iron loading-associated Aβ cytotoxicity. Native gel electrophoresis and IM–MS reveal that sialylation stabilizes transferrin conformation but inhibits its dimerization. Collectively, IM–MS is adapted to capture key sialylation intermediates involved in fine-tuning AD-associated glycoprotein structural microheterogeneity. Our results provide the molecular basis for the importance of sustaining moderate TF sialylation levels, especially Neu5Ac, in promoting iron cellular transportation and rescuing iron-enhanced Aβ cytotoxicity.

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Native Ion Mobility–Mass Spectrometry-Enabled Fast Structural Interrogation of Labile Protein Surface Modifications at the Intact Protein Level

Abstract

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