Mass Spectrometry Identification

点击翻译按钮获取中文摘要

A variety of service and collaborative projects for mass spectrometry analyses have been or are being carried out within the Mass Spectrometry Research and Support Group with approximately 3000 samples analyzed from 39 scientists representing 26 principal investigators or core heads from 7 laboratory branches and the DTT. One large effort is in support of the protein expression function of the Structural Biology Core Laboratory and Dr. Bob Petrovich. The role of the MSRSG 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. 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. 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. Additionally, the use of the crosslinker BS3 with a variety of proteins has been utilized. 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. Histone Proteins. In collaboration 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 in human osteosarcoma cells. Knockout cell lines of the histone protein H1.4 protein (previously determined by MS to be phosphorylated) was compared to a "wild type" cell line. Data were acquired and showed 8 linker histone H1 proteins were confidently identified and quantified in the protein digests of the 3 types of samples. Significant sequence coverage (38-77%) was obtained for all linker histones identified. Label-free quantification using a MS1-based quantification approach confirms initial observations that the cells try to compensate for the loss of H1.4 by upregulating the other histone variants. 17 histone marks were confidently identified, consisting of 6 types of histone modifications: arginine methylation, lysine methylation, lysine acetylation, serine phosphorylation, threonine phosphorylation, and tyrosine phosphorylation. Eleven PTM-containing peptides were confidently quantified across all types of samples. Of these, 9 PTMs were up-regulated and 2 PTMs were down-regulated in the knockout lines relative to the parental wild-type cell line. As an outcome of these efforts, this collaboration has resulted in a publication. 3. 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. While there have been successful analyses on multiple projects, we recently identified chemical crosslinks in the TSEN-CLP1 complex. In efforts to understand the role of the TSEN and tRNA splicing, multiple approaches were used including chemical cross-linking and mass spectrometry as well as cryo-electron microscopy. In addition, in collaboration with the P. Blackshear group, we have analyzed the chemical crosslinks in Gaboon TTP and the CNOT1 proteins.Additionally, in collaboration with the R.S. Williams group, where we have analyzed the intramolecular crosslinks within BS3 treated Senataxin. Finally, in collaboration with the W. Copeland group, we have analyzed the intra- and inter-molecular BS3 crosslinks between polymerase gamma and lonP protease. 4. Characterization of PTMs. In 2023 we collaborated with Ms. Carol Trempus and Dr. Stavros Garantziotis to investigate the phosphorylation of proteins from pdgfr-alpha positive fibroblasts isolated from murine lungs after PBS or bleomycin treatment. 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 bleomycin treatment. We are also currently collaborating with Dr. Carlos Guardia in the analyses of glycosylation and other PTMs on Autophagy-Related Protein 9A and 9B. Moreover, in collaboration with Drs. Anirban Kar and Paul Doestch, we have been analyzing the in vitro acetylation of NTHL1. Acetylation on DNA repair proteins is a dynamic epigenic modification regulated by acetyltransferases and lysine deacetylases. Accumulating evidence has indicated that the aberrant acetylation of DNA repair proteins contributes to the pathogenesis of cancer. Hence, probing the acetylation states of DNA repair proteins might be of great help in better understanding pathogenesis of cancer. NTHL1 is a bifunctional glycosylase involved in base excision repair. We were asked to identify the acetylation sites on four truncated variants of NTHL1 that were overexpressed as fusions to the C-terminus of bacterial MBP. The MBP-NHTL1 fusion proteins were subjected to in vitro acetylation using p300 protein as acetyl transferase before running the protein on an SDS-PAGE gel. Proteins in the gel bands were then reduced, alkylated, and digested with trypsin. Tryptic digests were analyzed using UPLC coupled to an orbitrap high resolution mass spectrometer. Nine lysine acetylation sites were identified in the four types of NHTL-1 constructs whereas 26 sites were detected on MBP. There were no significant differences in lysine acetylation profiles between p300-treated variants and the corresponding non-acetylated control. Other smaller projects include characterization of stable domains, post translational modifications, and extent of chemical labeling for various intramural research groups.