Mass Spectrometry Identification

National Institute Of Environmental Health Sciences

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Abstract

A variety of service and collaborative projects in protein identification have been or are being carried out within the Mass Spectrometry Research and Support Group with approximately 3000 samples analyzed from 40 scientists representing 20 principal investigators or core heads from 6 laboratory branches. 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. Approximately 10 % of IgA1 in archival samples from IgAN patients and controls was found as high molecular mass complexes. Label-free quantification using a MS1-based quantification approach showed significantly higher levels of heterodimeric complexes in patients with IgAN than in HCs. All these complexes dissociated completely to free proteins and IgA when reducing agents were added suggesting that these proteins were covalently linked through disulfide bonds. An SS mapping approach that maximizes the release of SS-containing peptides from non-reduced proteins, while minimizing SS scrambling, was used to identify the inter-chain disulfide bonds within the complexes. With this protocol, we identified several inter-chain SS between IgA and other plasma proteins, including one protein that, according to a recent publication, when associated with IgA in the circulation showed strong positive correlation with mesangial cell injury parameters. 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 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. 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, two recent examples where we have identified chemical crosslinks are in the multimeric Rix7 protein complex and the Twinkle helicase. 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. Additionally, chemical crosslinks in the mitochondrial Twinkle helicase were identified by the MSRSG in efforts to aid the structural characterization of the protein as well as the molecular mechanisms of the disease causing W315L mutation. In collaboration with the R.S. Williams group, we have analyzed the crosslinks of the UL5-UL52-UL8 proteins involved in the HSV1 protein complex and with the P. Blackshear group we have analyzed the crosslinks of TTP with the CNOT1 complex. 4. Characterization of PTMs of mast cells. We are currently collaborating with Drs. Pamela Guerrerio and Tamara Haque of NIAID and their colleagues investigating the phosphorylation post-translation modifications of signaling proteins in cultured primary mouse mast cells. We are using a relative quantification approach to evaluate changes in phosphorylation of proteins in the samples with the hopes that changes in the phosphorylation events shed light on what signal transduction pathways are altered in the samples. Other smaller projects include characterization of stable domains, post translational modifications, and extent of chemical labeling for various intramural research groups.