Biocenter, Area Biochemistry and Chemistry

Division of Clinical Biochemistry

Ludger Hengst
suppl. Direktor

Secretary office:
Rosanna Nagele
Tel: 0043 (0)512 9003 70300
Fax: 0043 (0)512 9003 73110
E-mail: Rosanna.Nagele@i-med.ac.at

 

Protein Analysis Group

Our group concentrates on the development of rapid and efficient methods for the separation and characterization of proteins and their post-translational modifications with closest attention to the family of histone and HMG proteins. Histone analysis  was always a driving force behind the biological research in this field, because it constantly provided a better insight into the complexity of this protein family. It is precisely this complexity of histones that, because of their varied modifications and their interplay, today gives us new information for such important biochemical processes as, for example, the activation and repression of genes. Because of the intricacy of histones it is not amazing that an entire repertoire of analytical methods is used for their separation. For this purpose a set of separation methods based on capillary electrophoresis (CE), reversed-phase chromatography, hydrophilic interaction liquid chromatography (HILIC) and mass spectrometry was developed in our lab.
At present our main research interest focuses on modifications of linker histones. One of the most important modification is the phosphorylation on certain serine and threonine residues located in the C- and N-terminal domains. Phosphorylation is cell cycle dependent, and individual H1 subtypes differ in their degree of phosphorlyation, which is usually lowest in G1 phase, rises continuously during S and G2 reaching a maximum during mitosis. Using HILIC and MS various phosphorylated H1 forms were separated and their phosphorylation sites and patterns identified. From these data particular site-specific phospho-antibodies were generated as tools for analysing the involvement of particular site-specific phosphorylated H1 subtypes in processes like cell cycle progression, gene expression, DNA replication or repair.

hilic_immunology_a

Fig.1: Left: HILIC separation of non-, mono- di-, and triphosphorylated forms of H1.5 from interphase cells. HILIC enables even the separation of distinctly site-specifically phosphorylated proteins, e.g. the two mono-phosphorylated forms p1g from p1m.
Right: Immunofluorescence images of interphase cells. red= a-tubulin; green=specific phospho-sites of histone H1.5.

MSMS_immunology

Fig.2: Left: Identification of phosphorylated Thr10 (fragment 1-20) of H1.5 by nanoLC-MS/MS.
Right: Immunofluorescence image of cells in mitosis or G1 phase: red=a-tubulin; green=T10p

Group members

Head
Herbert Lindner
Scientific staff
Leopold Kremser
Bettina Sarg
Heribert Talasz
Technician 
Astrid Devich
Bettina Gadner
Klaus Faserl
Andreas Kuen
Michael Brunner (apprentice)
Simon Kirchmair (apprentice)
Melanie Stadler (apprentice)

 

 

Main Aims and Projects

  • Development of multidimensional LC/CE-MS based methods
  • Generation of further site-specific phospho antibodies
  • PTM identifications of various nuclear proteins
  • Identification of novel phospho histone binding proteins
  • Identification of histone modification patterns at the nucleosomal level

Main technologies

  • ESI- and MALDI-TOF Mass Spectrometry
  • HPLC, e.g. RPC, HILIC, IEC, GPC
  • Capillary electrophoresis
  • Phosphoproteomics
  • Chromatin Immunoprecipitation
  • Coimmunoprecipitation
  • Generation of site-specific phospho antibodies

Grants

  • ESF: EUROCORES Progamm EuroDYNA 03-DYNA-F-23
  • ÖNB-Jubiläumsfonds

Publications (selected)

Faserl K, Sarg B, Maurer V, Lindner H (2017): Exploiting charge differences for the analysis of challenging post-translational modifications by capillary electrophoresis-mass spectrometry. J Chromatography A in press.
Halfinger B, Hammerer-Lercher A, Amplatz B, Sarg B, Kremser L, and Lindner H (2016): Unraveling the Molecular Complexity of O-glycosylated Endogenous (N-terminal) pro B-Type Natriuretic Peptide Forms in Blood Plasma of Patients with Severe Heart Failure. Clin Chem. 63(1):359-368.
Sarg B, Lopez R, Lindner H, Ponte I, Suau P, Roque A. (2015): Identification of novel post-translational modifications in linker histones from chicken erythrocytes. J Proteomics 15;113:162-77.
Faserl K, Kremser L, Müller M, Teis D, Lindner HH. (2015): Quantitative proteomics using ultralow flow capillary electrophoresis-mass spectrometry. Anal Chem. 2015 May 5;87(9):4633-40.
Sarg B, Lopez R, Lindner H, Ponte I, Suau P, Roque A. (2014): Sequence conservation of linker histones between chicken and mammalian species. Data Brief. Oct 15;1:60-4.
Kostova NN, Srebreva L, Markov DV, Sarg B, Lindner HH, Rundquist I. (2013): Histone H5-chromatin interactions in situ are strongly modulated by H5 C-terminal phosphorylation. Cytometry A 83: 273-9.
Sarg B, Faserl K, Kremser L, Halfinger B, Sebastiano R, Lindner HH. (2013): Comparing and Combining CE-ESI-MS and nano-LC-ESI-MS for the Characterization of Post-translationally Modified Histones. Mol Cell Proteomics. 12(9):2640-56.
Halfinger B, Sarg B, Amann A, Hammerer-Lercher A, Lindner HH. (2011): Unmasking low-abundance peptides from human blood plasma and serum samples by a simple and robust two-step precipitation/immunoaffinity enrichment method. Electrophoresis. 2011 Jun; 32(13):1706-14.
Gréen A, Sarg B, Green H, Lonn A, Lindner HH, Rundquist I (2011): Histone H1 interphase phosphorylation becomes largely established in G1 or early S phase and differs in G1 between T-lymphoblastoid cells and normal T cells. Epigenetics & Chromatin 4:15
Faserl K, Sarg B, Kremser L and Lindner H (2011): Optimization and Evaluation of a Sheathless Capillary Electrophoresis-Electrospray Ionization-Mass Spectrometry (CE-ESI-MS) Platform for Peptide Analysis: Comparison to LC-ESI-MS. Anal Chem 83(19):7297-305
Halfinger B, Sarg B, Lindner HH (2011) Evaluation of non-reductive β-elimination/Michael addition for glycosylation site determination in mucin-like O-glycopeptides. Electrophoresis 32(24):3546-53.
Faserl K, Golderer G, Kremser L, Lindner H, Sarg B, Wildt L, Seeber B: Polymorphism in vitamin D-binding protein as a genetic risk factor in the pathogenesis of endometriosis. J Clin Endocrinol Metab. 96, E233-41 (2011).
Talasz H, Sarg B. and Lindner HH: Site-specifically phosphorylated forms of H1.5 and H1.2 localized at distinct regions of the nucleus are related to different processes during the cell cycle. Chromosoma 118, 693-709 (2009).
Sarg B, Chwatal S, Talasz H and Lindner HH: Testis-specific Linker Histone H1t Is Multiply Phosphorylated during Spermatogenesis: Identification of Phosphorylation Sites. J Biol Chem 284, 3610-8 (2009).
Lindner HH: Analysis of histones, histone variants, and their posttranslationally modified forms. Electrophoresis 29, 2516-2532 (2008).
Sarg B and Lindner H: Capillary electrophoresis of post-translationally modified proteins and peptides: Handbook of Capillary and Microchip Electrophoresis and Associated Microtechniques,Third Edition edited by James P. Landers, Chapter 23, 707-722 (2008).
Hammerer-Lercher A, Halfinger B, Sarg B, Mair J, Puschendorf B, Griesmacher A, Guzman NA, Lindner HH: Analysis of Circulating Forms of proBNP and NT-proBNP in Patients with Severe Heart Failure. Clin Chem. 54, 858-865 (2007).
Rundquist I and Lindner HH: Analyses of linker histone--chromatin interactions in situ. Biochem Cell Biol. 84(4): 427-36 (2006).
Sarg B, Helliger W, Talasz H, Forg B, and Lindner HH: Histone H1 phosphorylation occurs site-specifically during interphase and mitosis. Identification of a novel phosphorylation site on histone H1. J.Biol.Chem. 281, 6573-6580 (2006).
Sarg B, Green A, Soderkvist P, Helliger W, Rundquist I, and Lindner HH: Characterization of sequence variations in human histone H1.2 and H1.4 subtypes. FEBS J. 272, 3673-3683 (2005).
Talasz H, Lindner HH, Sarg B, and Helliger W: Histone h4-lysine 20 monomethylation is increased in promoter and coding regions of active genes and correlates with hyperacetylation J.Biol.Chem. 280, 38814-38822 (2005).
Sarg B, Helliger W, Koutzamani E, and Lindner HH: Histone H4 hyperacetylation precludes histone H4 lysine 20 trimethylation. J.Biol.Chem.279, 53458-64 (2004).
Ott HW, Lindner H, Sarg B, Mueller-Holzner E, Abendstein B, Bergant A, Fessler S, Schwaerzler P, Zeimet A, Marth C, and Illmensee K: Calgranulins in cystic fluid and serum from patients with ovarian carcinomas. Cancer Res. 63, 7507-7514 (2003).
Talasz H, Helliger W, Puschendorf B, Sarg B and Lindner HH: Hyperphosphorylation of histone H2A.X and dephosphorylation of histone H1 subtypes in the course of apoptosis. Cell Death and Differentiation, 9, 27-39 (2002).
Sarg B, Koutzamani E, Helliger W, Rundquist I, and Lindner HH: Postsynthetic trimethylation of histone H4 at lysine 20 in mammalian tissues is associated with aging.J. Biol. Chem. 277, 39195-39201 (2002).

 

Protein Microanalysis Facility

Group members are also allocated to the Protein Micro-Analysis Facility established at Innsbruck Medical University is dedicated to provide investigators with equipment, expertise and custom services for the detection, characterization and quantification of proteins and peptides on a recharge basis. The facility maintains a suite of  state of the art instrumentation including a MALDI TOF/TOF 4800 plus Analyzer (Applied Biosystems), a Hybrid FT-Mass Spectrometer LTQ Orbitrap XL (ThermoScientific), a Procise 492 Protein Sequencer (Applied Biosystems), Nano-LC Gradient Systems UltiMate 3000 (Dionex), a Probot Microfraction Collector (LC-Packings) for on-line MALDI target preparations. Various Capillary Electrophoresis and HPLC Systems and, in addition,  a Solaar M6 Dual Zeeman Spectrometer (ThermoScientific) for trace element analysis are operated in the facility.










 

 

 

 

 

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