Optimizing High-Resolution Mass Spectrometry for the Identification of Low-Abundance Post- Translational Modifications of Intact Proteins

Published

September 1, 2017

Author(s)

Lisa E. Kilpatrick, Eric L. Kilpatrick

Abstract

Intact protein analysis by liquid chromatography-mass spectrometry (LC-MS) is now possible due to the improved capabilities of mass spectrometers yielding greater resolution, mass accuracy, and extended mass ranges. Concurrent measurement of post-translational modifications (PTMs) during LC-MS of intact proteins is advantageous while monitoring critical proteoform status, such as for clinical samples or during production of reference materials. However, difficulties exist for PTM identification when the protein is large or contains multiple modification sites. In this work, analyses of low abundance proteoforms of proteins of clinical or therapeutic interest, including C-reactive protein, vitamin D-binding protein, transferrin, and immunoglobulin G (NISTmAb), were performed on an Orbitrap Elite mass spectrometer. This work investigated the effect of various instrument parameters including source temperatures, in-source CID, microscan type and quantity, resolution, and automatic gain control on spectral quality. The signal-to-noise ratio was found to be a suitable spectral attribute which facilitated identification of low abundance PTMs. Source temperature and CID voltage were found to require specific optimization for each protein. This study identifies key instrumental parameters requiring optimization for improved detection of a variety of PTMs by LC-MS and establishes a methodological framework to ensure robust proteoform identifications, the first step in their ultimate quantification.

Citation

ACS Journal of Proteome Research

Volume

16

Issue

9

Pub Type

Journals

Download Paper

https://doi.org/10.1021/acs.jproteome.7b00244

Local Download

Keywords

Orbitrap, LC-MS, proteoform, intact protein, post-translational modification

Citation

Kilpatrick, L. and Kilpatrick, E. (2017), Optimizing High-Resolution Mass Spectrometry for the Identification of Low-Abundance Post- Translational Modifications of Intact Proteins, ACS Journal of Proteome Research, [online], https://doi.org/10.1021/acs.jproteome.7b00244 (Accessed March 22, 2025)