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Evaluation of Different Matrices for the MALDI-ToF Analysis of Peptide-RNA Oligonucleotides

Evaluation of Different Matrices for the MALDI-ToF Analysis of Peptide-RNA Oligonucleotides. Christof Lenz 1 , Reinhard Lührmann 2 and Henning Urlaub 2

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Evaluation of Different Matrices for the MALDI-ToF Analysis of Peptide-RNA Oligonucleotides

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  1. Evaluation of Different Matrices for the MALDI-ToF Analysis of Peptide-RNA Oligonucleotides Christof Lenz1, Reinhard Lührmann2 and Henning Urlaub2 1Applied Biosystems, Paul-Ehrlich-Str. 17, D-63225 Langen, Germany; 2Max-Planck-Institute for Biophysical Chemistry, Dept. of Cellular Biochemistry, Am Fassberg 11, D-37077Göttingen, Germany

  2. Dynamics of Spliceosome Assembly

  3. Composition of human snRNP

  4. In vitro reconstitution of a U4/U6 sub-snRNP

  5. Protein 61K shares a homology domain with snoRNP associated proteins

  6. Isolation of peptide-RNA oligonucleotide crosslinks 1st purification step

  7. Isolation of peptide-RNA oligonucleotides crosslinks 2nd purification step

  8. Purification of peptide-RNA oligonucleotides via RP-HPLC

  9. MALDI-ToF analysis of peptide-RNA Oligonucleotide crosslink using CHCA as matrix CHCA reflectron CHCA linear

  10. Enhanced sensitivity in the MALDI-ToF analysis of crosslinked peptide-RNA oligonucleotides CHCA linear DHB reflectron THAP reflectron

  11. Enhanced mass accuracy in the MALDI-ToF analysis of crosslinked peptide-RNA oligonucleotides

  12. Precise determination of the crosslinked protein and RNA moiety by MALDI-ToF analysis

  13. PSD analysis of crosslinked peptide-RNA oligonucleotides

  14. Highly accurate MALDI-ToF analysis of crosslinked peptide-RNA oligonucleotides circumvent N-terminal sequencing RNA sequencing MALDI-ToF-MS N-terminal sequencing

  15. Protein 61K contacts RNA through its conserved domain

  16. Another example: isolation of crosslinked peptide-RNA oligonucleotides from native UV irradiated U1 snRNP particles

  17. Highly accurate MALDI-ToF analysis of peptide-oligonucleotide crosslinks derived from native UV irradiated U1 snRNP particles

  18. Location of crosslinked peptide-RNA oligonucleotides from native UV irradiated U1 snRNP particles

  19. Conclusions • 2 step purification procedure for the isolation of crosslinked peptide-RNA oligonucleotides from reconstituted and native UV-irradiated RNP particles • Highly accurate MALDI-ToF analysis using DHB and THAP allowed for the identification of both the crosslinked peptide and the RNA moiety • PSD analysis of the crosslinked peptide-RNA oligonucleotide using THAP revealed sequence information of the crosslinked components • MALDI-ToF analysis circumvents the need for N-terminal peptide and RNA sequencing to identify the actual crosslinking sites • MALDI-ToF analysis of crosslinked complexes identifies hitherto unknown protein-RNA binding regions • Determination of the precise crosslinking sites adds valuable information about the orientation of proteins within RNP complexes

  20. Acknowledgments ABI Germany Volker Kruft Dietmar Waidelich MPI of Biopyhysical Chemistry Steffi Nottrott Monika Raabe Holger Stark Björn Sander

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