Abstract
High magnetic field Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers provide extremely high mass resolution (resolving power of ~200,000 at 400 m/z) protein detection across a broad mass range, enabling analysis of fine structure of isotopic peak clusters that is missed in other types of mass spectrometers. The protocol detailed here describes preparation of cellular extracts for purification of DNA-binding proteins using multiple chromatographic chemistries via fast protein liquid chromatography (FPLC), and identification and quantitation of the protein isoforms and their post-translational modifications by liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FT-ICR-MS). This protocol benefits from selectively purifying proteins for identification and quantitation by high resolution FT-ICR, which has the resolution to definitively distinguish between acetylation and trimethylation post-translational modification (PTM) additions.
Keywords: FT-ICR, Magnetic resonance mass spectrometry, Intact mass, Post-translational modifications, Chromatin protein
Background
Post-translational modifications (PTMs) of proteins play very important roles in regulation of cellular processes and function. The characterization of PTMs is challenging because of the large number of potential modifications and number of amino acid residues that can be modified. This can be particularly true for proteins with a limited number of proteolytic sites that cannot be analyzed in proteolytically digested forms and require an intact protein MS analysis. To achieve this goal, this protocol describes a combination of protein separation by HPLC and high accuracy/resolution mass spectrometry. The HPLC separation helps to; 1) eliminate various salts and other interfering molecules from the protein of the interest, 2) separate various PTM isoforms of the protein, and 3) improve ionization of the protein. A high accuracy/resolution MS instrument is, on the other hand, necessary to identify specific charge envelopes and isotope pattern distribution that are the important features essential for an accurate PTM determination. Although results of the intact protein MS analysis depend on many factors including a molecular mass, amino acid composition and tertiary structure of proteins, usually it can only be achieved by two types of MS instruments: Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR-MS) or recently developed generations of the Orbitrap-based mass spectrometer. Of those, FT-ICR-MS is preferred as data can be acquired at higher resolution and accuracy. On the other hand, this instrument requires liquid helium to keep the magnet temperature low which increases maintenance cost. Electrospary Ionization (ESI) used in mass spectrometry of intact proteins generates many different charge states of the protein. This represents a potential problem in detection of PTMs of the protein as peaks corresponding to individual proteoforms must be resolved. Therefore, only mass spectrometers with high resolving power such as FT-ICR-MS can be used. Depending on a magnetic field, they can have the resolving power at 1,000,000 and still accuracy between 0.05 and 1 ppm.
Materials and Reagents
Equipment
Software
Procedure
A workflow of the protocol from preparation of cellular extracts to identification and quantitation of the protein isoforms and their post-translational modifications by FT-ICR-MS is shown in Figure 1. Figure 1. Schematic workflow of the steps of this protocol
Data analysis
FT-ICR-MS data analysis can be found in Johnson et al., 2019, materials and methods: FT-ICR-MS section. Data analysis must be determined on a protein-by protein basis as many parameters may play role for specific results depending on protein(s) and PTM(s) of interest.
Notes
Recipes
Acknowledgments
The protocol described above was adapted from work described in a recent publication (Johnson et al., 2019).
Competing interests
The authors declare no competing interests.
References
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