Mass Spectrometry Identification

National Institute Of Environmental Health Sciences

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Abstract

A variety of service and collaborative projects for mass spectrometry analyses have been or are being carried out within the Mass Spectrometry Research Center with approximately 3000 samples analyzed from about 50 scientists representing about 25 principal investigators or core heads from 7 laboratory branches and the Division of Translational Toxicology. One effort is in support of the protein expression function of the Structural Biology Core Laboratory and Dr. Bob Petrovich. The role of the MSRC is to confirm gene expression at the protein level prior to the Structural Biology Core Laboratory handing materials over to their users. We have also put effort into the development of lipidomics. This project involves the identification/characterization as well as the quantitation of compounds; thus, this project is included in both the identification/characterization project and the quantitation project. Extensive lipidomic analyses have been performed in collaboration with the laboratory of Dr. John Cidlowski, Dr. Charly Guardia, and Dr. David Kurtz, In addition, 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. Protein characterization by mass spectrometry has been performed on a variety of proteins. Most of this work has been performed with electrospray mass spectrometry in conjunction with UPLC. Additionally, several proteins’ molecular architecture has been analyzed utilizing XL-MS techniques. 1. Proteomic analysis of IgA-protein aggregates. We are currently involved in a collaborative effort between the Division of Renal Diseases and Hypertension from the University of Minnesota at Minneapolis. IgA nephropathy is the most common form of glomerulonephritis worldwide. The hallmark of the disease is deposition of IgA1 in the glomerular mesangium. These deposited IgA1 are mainly polymeric in nature and include heteromeric complexes of IgA covalently bound to other plasma proteins. Identification of the key constituents of these protein-protein complexes may lead to better understanding of the pathophysiology of this disease. Approximately 10% of IgA1 in archival samples from IgAN patients and controls were found as high molecular mass complexes. Immunoblotting demonstrated 1:1 complex between IgA and albumin, alpha-1-antitrypsin, or alpha-1-microglobulin. Some complexes dissociated completely to free proteins and IgA1 when reducing agents were added, suggesting that these proteins were covalently linked through disulfide bonds (SS bonds). We were able to identify several inter-chain SS between IgA1 and other plasma proteins. A nonreducible thioether bridge, resulting of elimination of one of the sulfur atoms from the disulfide bond, was also identified in one of the heteromeric protein complexes. 2. Protein Crosslinking. Multiple projects have been analyzed to characterize protein complexes by mass spectrometry in conjunction with chemical cross-linking. 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. There have been successful analyses on multiple projects. In collaboration with Drs. Traci Hall and Chen Qiu, we have recently identified chemical crosslinks in the DND1-NANOS3 complex. In collaboration with Drs. Scott Williams and Oya Bermek, we have identified intramolecular and intermolecular crosslinks in the herpesvirus replication machinery. Additionally, in collaboration with Drs. Robin Stanley and Jacob Gordon, we have identified crosslinks in the human RNase PNK complex. 3. Characterization of PTMs. We have collaborated with Drs. John Cidlowski and Matias Grodzielski to investigate the phosphorylation of proteins from platelets harvested from either untreated mice or mice that have been chronically exposed to dexamethasone. We used a relative quantification approach to evaluate changes in phosphorylation of proteins in the samples with the hopes that changes in the phosphorylation events might shed light on what signal transduction pathways were altered by corticosteroid treatment. We are also currently collaborating with Drs. Carlos Guardia and Ruchir Bobde on the analyses of glycosylation and other PTMs on Syncytin-1. Furthermore, we are collaborating with Drs. Jason Watts and Deepti Shrivastava on the identification of sites of modification on MEF3A in response to osmotic stress. Moreover, in collaboration with Drs. Carmen Williams and Wendy Jefferson, we have identified sites in proteins where arginine is converted to citrulline in response to the estrus cycle and PADI knockout. 4. Characterization of the products generated by RNA processing enzymes. Using a combination of nuclease assays, 19F NMR spectroscopy, mass spectrometry, and single-particle cryo-EM, we found that the Nsp15 ribonuclease cleaves more effectively when U is unpaired and already flipped. Our results suggest that Nsp15's cleavage efficiency correlates with U's tendency to spontaneously flip, indicating that Nsp15's activity during infection may target bulged or accessible Us in the coronaviral genomic RNA, providing insights into Nsp15's role in immune evasion. Other smaller projects include characterization of stable domains, post translational modifications, and extent of chemical labeling for various intramural research groups.