Mass Spectrometry Characterization

National Institute Of Environmental Health Sciences

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Abstract

Mass spectrometry has been used to determine the extent of modification and the specific sites of modification on biomolecules. MS-based approaches have many advantages, including generally rapid analyses without radiolabeling. The MS analysis of a variety of proteins has been investigated using mass spectrometry. Products and digests have been analyzed by both positive and negative ion MALDI mass spectrometry and LC in combination with electrospray mass spectrometry. In addition, we are currently analyzing the use of the crosslinker BS3 with a variety of proteins. 1. Glycosylation Characterization Studies. We are currently performing two projects related to glycosylation characterization. One project involves a recombinant SARS-CoV-2 spike glycoprotein which has recently been successfully expressed by the Structural Biology Core Laboratory at NIEHS. We have been asked to characterize the glycan structures and the degree of sequon occupancy on the glycoprotein. Our preliminary data indicate that all the sites on the recombinant protein are fully occupied and we are currently investigating the distribution of various glycoforms on each of 22 glycan sites by high resolution mass spectrometry. The other project involves an extramural collaborative effort between the Division of Renal Diseases and Hypertension from the University of Minnesota at Minneapolis and our group at NIEHS. Abnormalities in O-glycosylation of circulating IgA1 are implicated in the pathogenesis of IgAN. This was initially demonstrated by the altered binding of lectins with specificity for O-linked glycans and confirmed later by mass spectrometry. We aim to identify potential sources of discrepancy between the two analytical methods. 2. Histone Proteins. We have been collaborating with the Archer laboratory in an effort to examine the role of H1 in controlling gene expression and protein levels when we knock out the H1 gene. Knockout cell lines of the histone protein H1.4 protein (as it was determined by MS previously that it was phosphorylated) was compared to a wild-type cell line. Data were acquired and are being analyzed to determine if other histone levels change and/or their PTMS change if the H1.4 isoform is knocked out. Also in collaboration with the Archer laboratory, the MSRSG evaluated changes in core and linker histone marks associated with proteosome inhibition. 13 histones were identified in these studies and 177 histone marks were identified in the histone H1 variants, consisting of 5 types of histone modifications: arginine methylation, lysine methylation, lysine acetylation, serine phosphorylation, and threonine phosphorylation. More than 133 PTMs-containing peptides were quantified and statistical testing using ANOVA and Tukeys HSD post-hoc test showed that very few histone marks were significantly changed. 3. Protein Crosslinking. Multiple projects are underway to characterize protein complexes by mass spectrometry in conjunction with chemical cross-linking. Most of these experiments have been conducted using BS3 as the cross-linking reagent followed by trypsin digestion and nanoLC-ESI-MS performed on a Q-Exactive Plus mass spectrometer. While there have been successful analyses on multiple projects, most recently we have determined the crosslinks in the multimeric Rix7 protein complex. In efforts to understand the role of the N-terminal domain in the Rix7 structure and this domains contribution to the proteins function, multiple approaches were used including chemical cross-linking and mass spectrometry as well as cryo-electron microscopy. 4. Characterization of RNA cleavage events. In collaboration with the Stanley lab at the NIEHS the MSRSG has characterized the RNA cleavage specificity for the NSP15 endoribonuclease from SARS-CoV-2. This work also characterized the nature of the resulting 3 ends of the cleavage products. Other smaller projects include characterization of stable domains, post translational modifications, and extent of chemical labeling for various intramural research groups (Hall, Shaw, S. Williams, Wilson).