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