Optimizing High-Resolution Mass Spectrometry for the Identification of Low-Abundance Post-Translational Modifications of Intact Proteins.
Lisa E KilpatrickEric L KilpatrickPublished in: Journal of proteome research (2017)
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.
Keyphrases
- liquid chromatography
- high resolution mass spectrometry
- mass spectrometry
- binding protein
- tandem mass spectrometry
- ultra high performance liquid chromatography
- gas chromatography
- high resolution
- simultaneous determination
- protein protein
- bioinformatics analysis
- antibiotic resistance genes
- high performance liquid chromatography
- single molecule
- solid phase extraction
- deep learning
- amino acid
- capillary electrophoresis
- genome wide
- body composition
- dna methylation
- squamous cell carcinoma
- computed tomography
- magnetic resonance