ACS Publications. Most Trusted. Most Cited. Most Read
My Activity
Recently Viewed
You have not visited any articles yet, Please visit some articles to see contents here.
CONTENT TYPES

Figure 1Loading Img
RETURN TO ISSUEPREVResearch ArticleNEXT

Effective Amino Acid Sequencing of Intact Filgrastim by Multimodal Mass Spectrometry with Topdownr

Cite this: J. Am. Soc. Mass Spectrom. 2022, 33, 11, 2087–2093
Publication Date (Web):October 19, 2022
https://doi.org/10.1021/jasms.2c00193
Copyright © 2022 American Society for Mass Spectrometry. Published by American Chemical Society. All rights reserved.
Article Views
189
Altmetric
-
Citations
-
LEARN ABOUT THESE METRICS
Read OnlinePDF (4 MB)
Supporting Info (1)»

Abstract

Abstract Image

Therapeutic proteins, known as biologicals, are an important and growing class of drugs for treatment of a series of human ailments. Amino acid sequence variants of therapeutic proteins can affect their safety and efficacy. Top-down mass spectrometry is well suited for the sequence analysis of intact therapeutic proteins. Fine-tuning of tandem mass spectrometry (MS/MS) fragmentation conditions is essential for maximizing the amino acid sequence coverage but is often time-consuming. We used topdownr, an automated and integrated multimodal approach to systematically assess high mass accuracy MS/MS fragmentation parameters to characterize filgrastim, a 19 kDa recombinant human granulocyte colony-stimulating factor used in treating neutropenia. A total of 276 different MS/MS conditions were systematically tested, including the following parameters: protein charge state, HCD and CID collision energy, ETD reaction time, ETD supplemental activation, and UVPD activation time. Stringent and accurate evaluation and annotation of the MS/MS data was achieved by requiring a fragment ion mass error of 5 ppm, considering reproducible N- and C-terminal fragment ions only, and excluding internal fragment ion assignments. We report the first EThcD and UVPD MS/MS analysis of intact filgrastim, and these two techniques combined resulted in 98% amino acid sequence coverage. By combining all tested fragmentation modes, we obtained near-complete amino acid sequence coverage (99.4%) of intact filgrastim.

Supporting Information

ARTICLE SECTIONS
Jump To

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jasms.2c00193.

  • Supporting figures and tables detailing data processing with ProteomeDiscoverer and topdownr, annotated spectra and sequence maps showing the best or combined conditions in different fragmentation modes, and the impact of varying fragmentation parameters (PDF)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Cited By

This article has not yet been cited by other publications.

Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

You’ve supercharged your research process with ACS and Mendeley!

STEP 1:
Click to create an ACS ID

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

MENDELEY PAIRING EXPIRED
Your Mendeley pairing has expired. Please reconnect

This website uses cookies to improve your user experience. By continuing to use the site, you are accepting our use of cookies. Read the ACS privacy policy.

CONTINUE