Supplementary data for
Flagellin glycosylation in Paenibacillus alvei CCM 2051T
1
Bettina Janesch, 2,3Falko Schirmeister, 4Daniel Maresch, 4Friedrich Altmann,
1
1
Paul Messner, 2Daniel Kolarich*and1Christina Schäffer*
Department of NanoBiotechnology, NanoGlycobiology unit, Universität für Bodenkultur
Wien, Muthgasse 11, A-1190 Vienna, Austria.
2
Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces,14424
Potsdam, Germany.
3
Freie Universität Berlin, Institute of Chemistry and Biochemistry, Arnimallee 22, 14195
Berlin, Germany.
4
Department of Chemistry, Division of Biochemistry, Universität für Bodenkultur Wien,
Muthgasse 18, A-1190 Vienna, Austria.
* To whom correspondence should be addressed: Christina Schäffer: Tel: +43-1-47654-2203;
Fax: +43-1-4789112; e-mail: christina.schaeffer@boku.ac.at; Daniel Kolarich: Tel: +49 30
838-59306, Fax: +49 30 838-59302, e-mail: daniel.kolarich@mpikg.mpg.de
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Fig S1. Exemplary MALDI-TOF MS spectrum of the tryptic peptides of the 33-35-kDa band.
Signals labelled with a red asterisks correspond to flagellin peptides.
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Fig S2. Top: Base peak chromatogram of an exemplary nanoLC-ESI-IT-MSMS analysis and
extracted ion chromatograms of the identified glycopeptides. The glycopeptide sequences are
shown in each panel. The chromatograms of 880.4 m/z and 922.8 m/z belong to the same
glycopeptide just with a difference of one additional lysine residue at the C-terminus of the
latter peptide. Both methionine residues of these glycopeptides were detected in their oxidized
form. Two major peaks of 829.7 m/z are present in the chromatogram. The peak at 30.3 min
corresponds to the indicated glycopeptide of flagellin. The peak at 24.8 min represents a
singly charged signal of a tryptic peptide from a ribosomal protein of Paenibacillus alvei.
Bottom: The MS/MS spectra of the four detected glycopeptides are shown. While the first
two spectra allowed a detailed identification of the glycopeptides (see Figure 2 in main
article), the two last spectra (880.0 m/z and 922.7 m/z) do not exhibit many fragments to
unambiguously confirm the peptide sequence. Nonetheless, these spectra contain the same
signature glycan peaks (384.1 m/z and 569.1 m/z) as well as fragment peaks corresponding to
the
glycopeptide
minus
saccharide
residues.
MSELMVQGANEVLTTTDAK:
2+
1319.6 [M+2H] −(Hex HexNAc
+
H2O)>>1128.0 [M+2H]2+;1319.6 [M+2H]2+−(Hex HexNAc2)
>>
1035.6 [M+2H]2+.
3
MSELMVQGANEVLTTTDAKK:
1383.6 [M+2H]2+−(Hex HexNAc
+
H2O)
2+
2+
>>1192.1 [M+2H] ; 1383.6 [M+2H] −(Hex HexNAc2) >> 1099.6 [M+2H]2+. Furthermore,
the identification of two forms just differing in the m/z of a lysine residue provide further
evidence that the detected signals are very likely corresponding to the tryptic glycopeptides
MSELMVQGANEVLTTTDAK and MSELMVQGANEVLTTTDAKK, respectively.
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Fig S3. Precursor and fragment spectra of the two glycopeptides for which the site of
glycosylation could not be clearly assigned. Nevertheless their glycopeptide identity could be
confirmed and they could be associated with the flagellin protein. These spectra contain clear
signals corresponding to the oxonium ions of the glycan (384.1 m/z and 569.1 m/z) as well as
fragment peaks corresponding to the glycopeptide minus saccharide residues.
MSELMVQGANEVLTTTDAK:
1319.6 [M+2H]2+−(Hex HexNAc + H2O)
2+
2+
>>1128.0 [M+2H] ;
1319.6 [M+2H] −(Hex HexNAc2)
>>
1035.6 [M+2H]2+.
MSELMVQGANEVLTTTDAKK:
1383.6 [M+2H]2+−(Hex HexNAc + H2O)
>>
2+
2+
1192.1 [M+2H] ; 1383.6 [M+2H] −(Hex HexNAc2) >> 1099.6 [M+2H]2+. Both methionine
residues in these glycopeptides were detected in their oxidized from.
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