Top-Down Mass Spectrometry of Neutrophil Myeloperoxidase

Top-down/native MS was performed of (i) the intact α-chain of nMPO and (ii) intact nMPO. For (i) nMPO was reduced, desalted and injected on a C4 LC column connected to an Agilent 6538 quadrupole-time-of-flight mass spectrometer operating in high-resolution positive polarity mode. Mass spectra were deconvoluted using MassHunter vB.06 (Agilent Technologies). Assignments were guided by the LC-MS/MS peptide data, see Table S3A and ii) intact nMPO was infused into a modified Q-Exactive (Thermo Scientific) operating in positive ion polarity via nano-ESI using custom-made gold-coated capillaries (75 (link)). Data were processed with Xcalibur v2.2 (Thermo Scientific), spectra deconvoluted with UniDec (76 (link)) and annotated using in-house software.
Intact nMPO was analysed using single-molecule mass photometry as described (44 (link)). Coverslips were assembled for sample delivery using silicone CultureWell gaskets (Grace Bio-Labs). Data were acquired, processed and analysed using in-house software (43 (link)), see Extended experimental procedures for details.

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Tjondro H.C., Ugonotti J., Kawahara R., Chatterjee S., Loke I., Chen S., Soltermann F., Hinneburg H., Parker B.L., Venkatakrishnan V., Dieckmann R., Grant O.C., Bylund J., Rodger A., Woods R.J., Karlsson-Bengtsson A., Struwe W.B, & Thaysen-Andersen M. (2020). Hyper-truncated Asn355- and Asn391-glycans modulate the activity of neutrophil granule myeloperoxidase. The Journal of Biological Chemistry, 296, 100144.

Publication 2020

MassHunter vB.06 Q-Exactive Xcalibur v2.2 CultureWell gaskets

Capillaries Delivery Gold Mass spectra Ms ms Peptide Photometry Silicone

Corresponding Organization : Macquarie University

Other organizations : Cordlife (Singapore), University of Oxford, University of Melbourne, University of Gothenburg, University of Georgia, Chalmers University of Technology

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Variable analysis

independent variables

Reduction and desalting of the intact α-chain of nMPO
Infusion of intact nMPO into the modified Q-Exactive (Thermo Scientific) operating in positive ion polarity via nano-ESI

dependent variables

Mass spectra obtained from the Agilent 6538 quadrupole-time-of-flight mass spectrometer
Mass spectra obtained from the modified Q-Exactive (Thermo Scientific) operating in positive ion polarity via nano-ESI
Single-molecule mass photometry analysis of intact nMPO

control variables

C4 LC column connected to the Agilent 6538 quadrupole-time-of-flight mass spectrometer
Custom-made gold-coated capillaries (75 µm) used for nano-ESI
Coverslips assembled for sample delivery using silicone CultureWell gaskets (Grace Bio-Labs)

controls

No positive or negative controls were explicitly mentioned.

Annotations

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