Automated N-Glycopeptide Lookup from Glycan

Report
Automated N-Glycopeptide Lookup from Glycan Databases using Tandem Mass Spectra
Kevin B.
1
Chandler ,
1 Georgetown
Petr
1
Pompach ,
2
Bern ,
Marshall
Radoslav
1
Goldman ,
Nathan J.
1
Edwards
University Medical Center, Washington, DC; 2 Palo Alto Research Center, Palo Alto, CA
Background
Glycopeptide Discovery Workflow
• Protein glycosylation is important!
• N-linked glycans are heterogeneous – glycan synthesis
affected by spatial, temporal, physiological, enzymatic context
Sample GlycomeDB1 Link
11 x LC-MS/MS
• Mass spectrometry is used to study N-glycosylation
• Tools are poorly designed for large MS/MS datasets
• Glycopeptide identification from CID MS/MS spectra is
challenging due to low glycopeptide abundance and
oxonium ion fragments.
MS/MS Spectra
3288
w/ glycan oxonium ion
(204, 366) peaks
2303
w/ “peptide” peaks
885
GlycomeDB1
534
Distinct
Glycopetpides
Detection: UV 214 nm. HILIC fractions 16 to 26.
116
• Analysis of human haptoglobin, digest with trypsin and Glu-C –
six putative N-motif peptides.
• Glycopeptide separation by hydrophilic interaction liquid
chromatography (HILIC)
• Eleven glycopeptide fractions analyzed by nanoC18 RP LCMS/MS using a Q-STAR Elite mass-spectrometer.
• IDA: Four most abundant ions with 20 sec exclusion.
• 15,780 MS and 3,468 MS/MS spectra (msconvert)
• Spectra filtered for glycan oxinium ions and peptide + Nlinked core fragments, then mass-lookup in GlycomeDB.
Results
800.78, 5+
[GlcNAc]+
Match:
[GlcNAc+ Man ]+
[GlcNAc – 2H2O ]+
[NeuAc - H2O ]+
VVLHPNYSQVDIGLIK (2+)
[NeuAc]+
[GlcNAc – H2O ]+
[GlcNAc+Gal+NeuAc]+
MVSHHNLTTGATLINE
NLFLNHSENATAK
VVLHPNYSQVDIGLIK (2+)
VVLHPNYSQVDIGLIK (2+)
VVLHPNYSQVDIGLIK
(2+)
KVVLHPNYSQVDIGLIK
Results
• 116 distinct glycan-peptide pairs matched to 534 spectra in
less than 5 minutes.
• Open format (XML) spectra input and Excel output.
• Automated lookup of N-glycopeptides matched most of those
identified by expert manual curation.
• Identified haptoglobin glycopeptides are consistent with
• 56% (299) of filtered spectra matched a single glycan-peptide
pair (< 0.2 Da).
• Nine spectra matched glycan-peptide pairs representing more
than one peptide.
published reports2.
• In future work, we plan to identify more oxonium/characteristic
ions (such as Fucose), match glycopeptide ions’ isotope
clusters in MS spectra, and integrate with Cartoonist3.
• 60 distinct non-isobaric glycans are represented.
V
V
L
H
P
N
Y
S
Q
V
D
I
G
L
I
K
Haptoglobin
Methods
References
Sample Glycopeptide Matches
1. Ranzinger, Herget, von der Lieth, Frank. Nucleic Acids Res. 39(Database
Scan
m/z
Δmass
Peptide
1367 1062.11 -0.003 VVLHPNYSQVD
GlycanID
17745
issue):D373-376 (2011).
2.Fujimura, Shinohara, Tossot, Pang, Kurogochi, Saito, Arai, Sadilek,
Murayama, Dell, Nishimura, Hakomori. Int. J. Cancer 122:39–49 (2008).
3.Goldberg, Sutton-Smith, Paulson, Dell. Proteomics 5:865-875 (2005).
1548 1004.92 0.007 MVSHHNLTTGATLINE 18352
1669 1261.51 -0.008 NLFLNHSE
2113
Acknowledgements
1388 1060.09 0.014 NLFLNHSE
4706
1407 800.781 0.089 VVLHPNYSQVDIGLIK
4706
Kevin B Chandler is supported by a Graduate Research Fellowship from the
National Science Foundation. Nathan J Edwards is supported, in part, by
NIH/NCI/CPTI grant CA126189.

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