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


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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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Evaluation of different matrices for the maldi tof analysis of peptide rna oligonucleotides l.jpg

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


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Dynamics of Spliceosome Assembly


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Composition of human snRNP


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In vitro reconstitution of a U4/U6 sub-snRNP


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Protein 61K shares a homology domain with

snoRNP associated proteins


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Isolation of peptide-RNA oligonucleotide crosslinks

1st purification step


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Isolation of peptide-RNA oligonucleotides crosslinks

2nd purification step


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Purification of peptide-RNA oligonucleotides via RP-HPLC


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MALDI-ToF analysis of peptide-RNA Oligonucleotide

crosslink using CHCA as matrix

CHCA reflectron

CHCA linear


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Enhanced sensitivity in the MALDI-ToF analysis

of crosslinked peptide-RNA oligonucleotides

CHCA linear

DHB reflectron

THAP reflectron


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Enhanced mass accuracy in the MALDI-ToF analysis

of crosslinked peptide-RNA oligonucleotides


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Precise determination of the crosslinked protein and

RNA moiety by MALDI-ToF analysis


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PSD analysis of crosslinked peptide-RNA oligonucleotides


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Highly accurate MALDI-ToF analysis of crosslinked peptide-RNA

oligonucleotides circumvent N-terminal sequencing

RNA sequencing

MALDI-ToF-MS

N-terminal sequencing


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Protein 61K contacts RNA through its conserved domain


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Another example: isolation of crosslinked peptide-RNA oligonucleotides

from native UV irradiated U1 snRNP particles


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Highly accurate MALDI-ToF analysis of peptide-oligonucleotide

crosslinks derived from native UV irradiated U1 snRNP particles


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Location of crosslinked peptide-RNA oligonucleotides

from native UV irradiated U1 snRNP particles


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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


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Acknowledgments

ABI Germany

Volker Kruft

Dietmar Waidelich

MPI of Biopyhysical Chemistry

Steffi Nottrott

Monika Raabe

Holger Stark

Björn Sander


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