Mass Spectrometry

General

At IBC2, we maintain a proteomics platform allowing our researchers to quantitatively describe signaling networks and to determine how components of signaling complexes are modified in response to pathway perturbations. We perform complementary qualitative and quantitative MS approaches for identification and quantitation of proteins and their post-translational modifications (PTMs, e.g. phosphorylation and ubiquitination), both at small (e.g. interactomes) and large (whole cell- or subcellular proteome) scale.

In general, we follow the so-called bottom-up approach: protein samples are first treated with proteases (e.g. Trypsin and/or LysC) and then analyzed by LC-MS/MS for identification and quantification of the resulting peptides and thus the underlying proteins. Besides label-free protein quantitation based on summed peak intensities of identified peptides, we take advantage of various labelling techniques, such as Tandem Mass Tag (TMT) reagents or Stable Isotope Labelling using Amino acids in Cell culture (SILAC), providing higher throughput and/or more accurate quantification. The generated spectral data is mostly analyzed using MaxQuant (MPI Martinsried) and Proteome Discoverer (Thermo Scientific) in order to obtain the list of identified proteins and their abundances in the samples.

Instrumentation

Q Exactive HF

In 2016, we acquired this quadrupole-Orbitrap with an ultra-high-field analyzer as part of the Z project of SFB 1177 (Molecular and Functional Characterization of Selective Autophagy). It provides high sensitivity and acquisition speed for complex samples, especially in conjunction with the hyphenated UHPLC which allows the use of smaller column particles and longer columns due to higher pressure resistance of the fluidics (up to 1200 bar).

Orbitrap Fusion LUMOS

Since November 2018, the unit houses an Orbitrap Fusion Lumos mass spectrometer, which was partially funded by the DFG (FUGG 161/922-1). This so-called “Tribrid”-MS, adding a third mass analyzer (ion trap) to the above-mentioned quadrupole-Orbitrap configuration, allows us to perform SPS-MS3 analyses providing increased accuracy for TMT-based quantitative proteomics experiments. As for the Q Exactive HF, it is coupled to an EASY nLC 1200 for high sensitivity chromatographic separations.

Two DIONEX U3000 HPLC

(one analytical, one micro) with fraction collection option

Used to prefractionate peptides using highPh-reversed phase chromatography for increased coverage of identified proteins and PTM-sites in proteomics samples.

Team

Dr. Christian Münch

The mass spectrometry unit is headed by Christian, who spent 4,5 years as a postdoctoral researcher in the laboratories of Dr. Wade Harper and Dr. Steve Gygi at Harvard Medical School (Boston, USA). There, he gained extensive experience in the use of TMT (sample preparation, measurement and sample analysis including comprehensive bioinformatic analysis) and broad expertise in the maintenance and use of Thermo Scientific mass spectrometers such as LTQ Velos, Q Exactive, Orbitrap Fusion and Orbitrap Lumos.

Mass Spectrometry Unit team members:

Dr. Georg Tascher (SFB-1177 proteomics staff scientist)
Martin Adrian-Allgood (technician)
Kristina Wagner (technician)
David Krause (IT specialist)

If you wish to set up a new collaboration with us, or are planning mass spectrometry experiments as part of the SFB 1177, always contact us before starting the experiments.
Members of the SFB 1177 with projects requiring MS analysis should contact our service platform team for a first consultation via email. Afterwards, please register to MS Portal and complete the sample submission form.

For external researchers use of the MS platform is possible on the basis of collaboration. Please contact Christian Münch or one of the IBC2 group leaders with whom you wish to collaborate directly to discuss the options.

Publications

Publications employing quantitative proteomics performed during Christian Münch’s postdoctoral tenure at Harvard Medical School with Wade Harper and Steve Gygi

Wiechmann S, Gärtner A, Kniss A, Stengl A, Behrends C, Rogov VV, Rodriguez MS, Dötsch V, Müller S, Ernst A. Site-specific inhibition of the small ubiquitin-like modifier (SUMO)-conjugating enzyme Ubc9 selectively impairs SUMO chain formation. J Biol Chem. 2017 Sep 15;292(37):15340-15351.

Polajnar M, Dietz MS, Heilemann M, Behrends C. Expanding the host cell ubiquitylation machinery targeting cytosolic Salmonella. EMBO Rep. 2017 Sep;18(9):1572-1585.

Aguilera-Gomez A, Zacharogianni M, van Oorschot MM, Genau H, Grond R, Veenendaal T, Sinsimer KS, Gavis EA, Behrends C, Rabouille C. Phospho-Rasputin Stabilization by Sec16 Is Required for Stress Granule Formation upon Amino Acid Starvation. Cell Rep. 2017 Aug 29;20(9):2277.

Ilina EI, Armento A, Sanchez LG, Reichlmeir M, Braun Y, Penski C, Capper D, Sahm F, Jennewein L, Harter PN, Zukunft S, Fleming I, Schulte D, Le Guerroué F, Behrends C, Ronellenfitsch MW, Naumann U, Mittelbronn M. Effects of soluble CPE on glioma cell migration are associated with mTOR activation and enhanced glucose flux. Oncotarget. 2017 Jun 27;8(40):67567-67591.

Grumati P, Morozzi G, Hölper S, Mari M, Harwardt MI, Yan R, Müller S, Reggiori F, Heilemann M, Dikic I. Full length RTN3 regulates turnover of tubular endoplasmic reticulum via selective autophagy. Elife. 2017 Jun 15;6. pii: e25555.

Nayak A, Reck A, Morsczeck C, Müller S. Flightless-I governs cell fate by recruiting the SUMO isopeptidase SENP3 to distinct HOX genes. Epigenetics Chromatin. 2017 Mar 23;10:15.

Drané P, Brault ME, Cui G, Meghani K, Chaubey S, Detappe A, Parnandi N, He Y, Zheng XF, Botuyan MV, Kalousi A, Yewdell WT, Münch C, Harper JW, Chaudhuri J, Soutoglou E, Mer G, Chowdhury D. TIRR regulates 53BP1 by masking its histone methyl-lysine binding function.

Nature. 2017 Mar 9;543(7644):211-216.

Jung J, Nayak A, Schaeffer V, Starzetz T, Kirsch AK, Müller S, Dikic I, Mittelbronn M, Behrends C. Multiplex image-based autophagy RNAi screening identifies SMCR8 as ULK1 kinase activity and gene expression regulator. Elife. 2017 Feb 14;6. pii: e23063.

Reiterer V, Figueras-Puig C, Le Guerroue F, Confalonieri S, Vecchi M, Jalapothu D, Kanse SM, Deshaies RJ, Di Fiore PP, Behrends C, Farhan H. The pseudophosphatase STYX targets the F-box of FBXW7 and inhibits SCFFBXW7 function. EMBO J. 2017 Feb 1;36(3):260-273.

Fiskin E, Bhogaraju S, Herhaus L, Kalayil S, Hahn M, Dikic I. Structural basis for the recognition and degradation of host TRIM proteins by Salmonella effector SopA. Nat Commun. 2017 Jan 13;8:14004.

Bhogaraju S, Kalayil S, Liu Y, Bonn F, Colby T, Matic I, Dikic I. Phosphoribosylation of Ubiquitin Promotes Serine Ubiquitination and Impairs Conventional Ubiquitination. Cell. 2016 Dec 1;167(6):1636-1649.e13.

Yang W, Nagasawa K, Münch C, Xu Y, Satterstrom K, Jeong S, Hayes SD, Jedrychowski MP, Vyas FS, Zaganjor E, Guarani V, Ringel AE, Gygi SP, Harper JW, Haigis MC. Mitochondrial Sirtuin Network Reveals Dynamic SIRT3-Dependent Deacetylation in Response to Membrane Depolarization. Cell. 2016 Nov 3;167(4):985-1000.e21.

Raman N, Weir E, Müller S. The AAA ATPase MDN1 Acts as a SUMO-Targeted Regulator in Mammalian Pre-ribosome Remodeling. Mol Cell. 2016 Nov 3;64(3):607-615.

Kunz K, Wagner K, Mendler L, Hölper S, Dehne N, Müller S. SUMO Signaling by Hypoxic Inactivation of SUMO-Specific Isopeptidases. Cell Rep. 2016 Sep 13;16(11):3075-3086.

Mauthe M, Langereis M, Jung J, Zhou X, Jones A, Omta W, Tooze SA, Stork B, Paludan SR, Ahola T, Egan D, Behrends C, Mokry M, de Haan C, van Kuppeveld F, Reggiori F. An siRNA screen for ATG protein depletion reveals the extent of the unconventional functions of the autophagy proteome in virus replication. J Cell Biol. 2016 Aug 29;214(5):619-35.

Münch C, Harper JW. Mitochondrial unfolded protein response controls matrix pre-RNA processing and translation. Nature. 2016 Jun 30;534(7609):710-3.

Fiskin E, Bionda T, Dikic I, Behrends C. Global Analysis of Host and Bacterial Ubiquitinome in Response to Salmonella Typhimurium Infection. Mol Cell. 2016 Jun 16;62(6):967-981.

Richter B, Sliter DA, Herhaus L, Stolz A, Wang C, Beli P, Zaffagnini G, Wild P, Martens S, Wagner SA, Youle RJ, Dikic I. Phosphorylation of OPTN by TBK1 enhances its binding to Ub chains and promotes selective autophagy of damaged mitochondria. Proc Natl Acad Sci U S A. 2016 Apr 12;113(15):4039-44.

Lamb CA, Nühlen S, Judith D, Frith D, Snijders AP, Behrends C, Tooze SA. TBC1D14 regulates autophagy via the TRAPP complex and ATG9 traffic. EMBO J. 2016 Feb 1;35(3):281-301.

Jung J, Genau HM, Behrends C. Amino Acid-Dependent mTORC1 Regulation by the Lysosomal Membrane Protein SLC38A9. Mol Cell Biol. 2015 Jul;35(14):2479-94.

Genau HM, Huber J, Baschieri F, Akutsu M, Dötsch V, Farhan H, Rogov V, Behrends C. CUL3-KBTBD6/KBTBD7 ubiquitin ligase cooperates with GABARAP proteins to spatially restrict TIAM1-RAC1 signaling. Mol Cell. 2015 Mar 19;57(6):995-1010.

McEwan DG, Popovic D, Gubas A, Terawaki S, Suzuki H, Stadel D, Coxon FP, Miranda de Stegmann D, Bhogaraju S, Maddi K, Kirchof A, Gatti E, Helfrich MH, Wakatsuki S, Behrends C, Pierre P, Dikic I. PLEKHM1 regulates autophagosome-lysosome fusion through HOPS complex and LC3/GABARAP proteins. Mol Cell. 2015 Jan 8;57(1):39-54.

Raman N, Nayak A, Muller S. mTOR signaling regulates nucleolar targeting of the SUMO-specific isopeptidase SENP3. Mol Cell Biol. 2014 Dec;34(24):4474-84.

Spang N, Feldmann A, Huesmann H, Bekbulat F, Schmitt V, Hiebel C, Koziollek-Drechsler I, Clement AM, Moosmann B, Jung J, Behrends C, Dikic I, Kern A, Behl C. RAB3GAP1 and RAB3GAP2 modulate basal and rapamycin-induced autophagy. Autophagy. 2014 10(12):2297-309.

Lu J, He L, Behrends C, Araki M, Araki K, Jun Wang Q, Catanzaro JM, Friedman SL, Zong WX, Fiel MI, Li M, Yue Z. NRBF2 regulates autophagy and prevents liver injury by modulating Atg14L-linked phosphatidylinositol-3 kinase III activity. Nat Commun. 2014 May 22;5:3920.

Popovic D, Akutsu M, Novak I, Harper JW, Behrends C, Dikic I. Rab GTPase-activating proteins in autophagy: regulation of endocytic and autophagy pathways by direct binding to human ATG8 modifiers. Mol Cell Biol. 2012 May;32(9):1733-44.

Behrends C, Sowa ME, Gygi SP, Harper JW. Network organization of the human autophagy system. Nature. 2010 Jul 1;466(7302):68-76.

Ordureau, A., Münch, C., and Harper, J.W. (2015). Quantifying Ubiquitin Signaling. Mol. Cell 58, 660–676.