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Hahne H.,TU Munich | Pachl F.,TU Munich | Ruprecht B.,TU Munich | Maier S.K.,TU Munich | And 8 more authors.
Nature Methods | Year: 2013

We report that low percentages of dimethylsulfoxide (DMSO) in liquid chromatography solvents lead to a strong enhancement of electrospray ionization of peptides, improving the sensitivity of protein identification in bottom-up proteomics by up to tenfold. The method can be easily implemented on any LC-MS/MS system without modification to hardware or software and at no additional cost.

Gupta R.,Max Planck Institute of Biochemistry | Kasturi P.,Max Planck Institute of Biochemistry | Bracher A.,Max Planck Institute of Biochemistry | Loew C.,Max Planck Institute of Biochemistry | And 6 more authors.
Nature Methods | Year: 2011

Maintenance of cellular protein homeostasis (proteostasis) depends on a complex network of molecular chaperones, proteases and other regulatory factors. Proteostasis deficiency develops during normal aging and predisposes individuals for many diseases, including neurodegenerative disorders. Here we describe sensor proteins for the comparative measurement of proteostasis capacity in different cell types and model organisms. These sensors are increasingly structurally destabilized versions of firefly luciferase. Imbalances in proteostasis manifest as changes in sensor solubility and luminescence activity. We used EGFP-tagged constructs to monitor the aggregation state of the sensors and the ability of cells to solubilize or degrade the aggregated proteins. A set of three sensor proteins serves as a convenient toolkit to assess the proteostasis status in a wide range of experimental systems, including cell and organism models of stress, neurodegenerative disease and aging. © 2011 Nature America, Inc. All rights reserved.

Schiller C.B.,Ludwig Maximilians University of Munich | Seifert F.U.,Ludwig Maximilians University of Munich | Linke-Winnebeck C.,Ludwig Maximilians University of Munich | Hopfner K.-P.,Ludwig Maximilians University of Munich | Hopfner K.-P.,Center for Integrated Protein science
Cold Spring Harbor Perspectives in Biology | Year: 2014

DNA double-strand breaks are repaired by two major pathways, homologous recombination or nonhomologous end joining. The commitment to one or the other pathway proceeds via different steps of resection of the DNA ends, which is controlled and executed by a set of DNA double-strand break sensors, endo- and exonucleases, helicases, and DNA damage response factors. The molecular choreographyof the underlying protein machinery is beginning to emerge. In this review, we discuss the early steps of genetic recombination and double-strand break sensing with an emphasis on structural and molecular studies. ©2014 Cold Spring Harbor Laboratory Press; all rights reserved.

Rojowska A.,Ludwig Maximilians University of Munich | Lammens K.,Ludwig Maximilians University of Munich | Seifert F.U.,Ludwig Maximilians University of Munich | Direnberger C.,Ludwig Maximilians University of Munich | And 4 more authors.
EMBO Journal | Year: 2014

The Mre11-Rad50 nuclease-ATPase is an evolutionarily conserved multifunctional DNA double-strand break (DSB) repair factor. Mre11-Rad50's mechanism in the processing, tethering, and signaling of DSBs is unclear, in part because we lack a structural framework for its interaction with DNA in different functional states. We determined the crystal structure of Thermotoga maritima Rad50NBD (nucleotide-binding domain) in complex with Mre11HLH (helix-loop-helix domain), AMPPNP, and double-stranded DNA. DNA binds between both coiled-coil domains of the Rad50 dimer with main interactions to a strand-loop-helix motif on the NBD. Our analysis suggests that this motif on Rad50 does not directly recognize DNA ends and binds internal sites on DNA. Functional studies reveal that DNA binding to Rad50 is not critical for DNA double-strand break repair but is important for telomere maintenance. In summary, we provide a structural framework for DNA binding to Rad50 in the ATP-bound state. Synopsis The SMC protein Rad50 is a key subunit of the MRN complex, a repair factor involved in processing and tethering DNA double-strand breaks. First structural insights into its interaction with DNA show that such binding is important for some but not other functions of the MRN complex. Thermotoga maritima Rad50 in complex with dsDNA offers the first crystal structure view of DNA binding by an SMC protein. dsDNA binds to a strand-loop-helix motif on the Rad50 nucleotide-binding domain. DNA binds between the coiled-coil domains of the ATP-bound dimer of Rad50. The strand-loop-helix motif does not directly recognize DNA ends, but rather binds a DNA duplex internally. In yeast, DNA binding to the strand-loop-helix motif is important for MRN complex function in telomere maintenance but not in DNA double-strand break repair. The first structural insight into DNA binding by an SMC protein reveals the importance of Rad50-DNA interaction for some but not other functions of the MRN complex. © 2014 The Authors.

Marx H.,TU Munich | Lemeer S.,TU Munich | Schliep J.E.,TU Munich | Matheron L.,University Utrecht | And 10 more authors.
Nature Biotechnology | Year: 2013

We present a peptide library and data resource of >100,000 synthetic, unmodified peptides and their phosphorylated counterparts with known sequences and phosphorylation sites. Analysis of the library by mass spectrometry yielded a data set that we used to evaluate the merits of different search engines (Mascot and Andromeda) and fragmentation methods (beam-type collision-induced dissociation (HCD) and electron transfer dissociation (ETD)) for peptide identification. We also compared the sensitivities and accuracies of phosphorylation-site localization tools (Mascot Delta Score, PTM score and phosphoRS), and we characterized the chromatographic behavior of peptides in the library. We found that HCD identified more peptides and phosphopeptides than did ETD, that phosphopeptides generally eluted later from reversed-phase columns and were easier to identify than unmodified peptides and that current computational tools for proteomics can still be substantially improved. These peptides and spectra will facilitate the development, evaluation and improvement of experimental and computational proteomic strategies, such as separation techniques and the prediction of retention times and fragmentation patterns. © 2013 Nature America, Inc. All rights reserved.

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