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 crosslinks1st purification step
Isolation of peptide-RNA oligonucleotides crosslinks
2nd purification step
Purification of peptide-RNA oligonucleotides via RP-HPLC
CHCA reflectron
CHCA linear
MALDI-ToF analysis of peptide-RNA Oligonucleotidecrosslink using CHCA as matrix
CHCA linear
DHB reflectron
THAP reflectron
Enhanced sensitivity in the MALDI-ToF analysisof crosslinked peptide-RNA oligonucleotides
Enhanced mass accuracy in the MALDI-ToF analysisof crosslinked peptide-RNA oligonucleotides
Precise determination of the crosslinked protein andRNA moiety by MALDI-ToF analysis
0 700 1400 2100 2800 3500
Mass (m/z)
0
827.5
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Stitched PS D MC=>MC=>AdvBC(32,0.5,0.1)=>SM21[BP = 3262.5, 827]
y23(+1)
y10(+1)
y17(+1)
2704.0
y18(+1)
- H3PO4
- Adenosin
- Adenin
y14(+1)
y16(+1)
y12(+1)
y13(+1)
y15(+1)
2727.8
2416.6
y5(+1)
y8(+1)
y9(+1)
y11(+1)
y7(+1)
y2(+1)
y6(+1)
PSD analysis of crosslinked peptide-RNA oligonucleotides
MALDI-ToF-MSRNA sequencing
N-terminal sequencing
Highly accurate MALDI-ToF analysis of crosslinked peptide-RNAoligonucleotides circumvent N-terminal sequencing
Protein 61K contacts RNA through its conserved domain
Another example: isolation of crosslinked peptide-RNA oligonucleotidesfrom native UV irradiated U1 snRNP particles
Highly accurate MALDI-ToF analysis of peptide-oligonucleotidecrosslinks derived from native UV irradiated U1 snRNP particles
Location of crosslinked peptide-RNA oligonucleotidesfrom 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 KruftDietmar Waidelich
MPI of Biopyhysical Chemistry
Steffi NottrottMonika Raabe
Holger StarkBjörn Sander