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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

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


Dynamics of Spliceosome Assembly


Composition of human snRNP


In vitro reconstitution of a U4/U6 sub-snRNP


Protein 61K shares a homology domain with

snoRNP associated proteins


Isolation of peptide-RNA oligonucleotide crosslinks

1st purification step


Isolation of peptide-RNA oligonucleotides crosslinks

2nd purification step


Purification of peptide-RNA oligonucleotides via RP-HPLC


MALDI-ToF analysis of peptide-RNA Oligonucleotide

crosslink using CHCA as matrix

CHCA reflectron

CHCA linear


Enhanced sensitivity in the MALDI-ToF analysis

of crosslinked peptide-RNA oligonucleotides

CHCA linear

DHB reflectron

THAP reflectron


Enhanced mass accuracy in the MALDI-ToF analysis

of crosslinked peptide-RNA oligonucleotides


Precise determination of the crosslinked protein and

RNA moiety by MALDI-ToF analysis


PSD analysis of crosslinked peptide-RNA oligonucleotides


Highly accurate MALDI-ToF analysis of crosslinked peptide-RNA

oligonucleotides circumvent N-terminal sequencing

RNA sequencing

MALDI-ToF-MS

N-terminal sequencing


Protein 61K contacts RNA through its conserved domain


Another example: isolation of crosslinked peptide-RNA oligonucleotides

from native UV irradiated U1 snRNP particles


Highly accurate MALDI-ToF analysis of peptide-oligonucleotide

crosslinks derived from native UV irradiated U1 snRNP particles


Location of crosslinked peptide-RNA oligonucleotides

from native UV irradiated U1 snRNP particles


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


Acknowledgments

ABI Germany

Volker Kruft

Dietmar Waidelich

MPI of Biopyhysical Chemistry

Steffi Nottrott

Monika Raabe

Holger Stark

Björn Sander


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