Glycoprotein Microheterogeneity via N-Glycopeptide Identification Kevin Brown Chandler, Petr...
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Transcript of Glycoprotein Microheterogeneity via N-Glycopeptide Identification Kevin Brown Chandler, Petr...
Glycoprotein Microheterogeneity via N-Glycopeptide
Identification
Kevin Brown Chandler, Petr Pompach,
Radoslav Goldman, Nathan EdwardsGeorgetown University Medical Center
The challenge
Identify glycopeptides in large-scale tandem mass-spectrometry datasets Many glycopeptide enriched fractions Many tandem mass-spectra / fraction
Good, but not great, instrumentation QStar Elite – CID, good MS1/MS2 resolution
Strive for hypothesis-generating analysis Site-specific glycopeptide characterization Glycoform occupancy in differentiated samples
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Observations
Oxonium ions (204, 366) help distinguish glycopeptides from peptides… …but do little to identify the glycopeptide
Few peptide b/y-ions to identify peptides… …but intact peptide fragments are common
If the peptide can be guessed, then… …the glycan's mass can be determined
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Observations
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Glycopeptide Search Strategy
Glycan-Peptide to Spectrum Matches Multi-Peptide, Multi-Glycan Mass (Single Peptide), Single Glycan Mass, Single Glycan (Topology)
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Compromises
Single protein / Simple protein mixture Few peptides to distinguish
Single N-glycan per peptide Subtraction from precursor
Digest may not resolve site Need peptide/glycan fragments to distinguish
Isobaric peptide-glycan pairs are not resolved Need peptide/glycan fragments to distinguish
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Glycan Databases
Link putative glycan masses to N-linked glycan structures (and organism, etc. ): Human N-linked GlycomeDB Cartoonist structure enumeration CFG Mammalian Array (v5.0) In-house database (Oxford notation)
Database(s) provide "biased" search space: Coverage vs. "Reasonableness" Trade off: Time, Specificity, Biology
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Hap
tog
lob
in (
HP
T_H
UM
AN
)
NLFLNHSE*NATAK
MVSHHNLTTGATLINE
VVLHPNYSQVDIGLIK
Haptoglobin standard
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• N-glycosylation motif (NX/ST)* Site of GluC cleavage
Pompach et al. Journal of Proteome Research 11.3 (2012): 1728–1740.
Haptoglobin standard
11 HILIC fractions enriched for glycopeptides 11 x LC-MS/MS acquisitions (≥ 15k spectra) 2887/3288 MS/MS spectra have oxonium ion(s) 317 have "intact-peptide" fragment ions
263 spectra matched to peptide-glycan pairs 52% matched single-glycan 8% matched multi-peptide
27 distinct (mass) glycans on 11 peptides Glycans identified on all 4 haptoglobin sites
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Algorithms & Infrastructure
Glycan databases indexed by composition, mass, N-linked, and motif/type Formats: IUPAC, Linear Code, GlycoCT_condensed Implemented: GlycomeDB, Cartoonist, CFG Array
Monosaccharide decomposition of glycan mass Böcker et al. Efficient mass decomposition (2005)
χ2 Goodness-of-fit test for precursor cluster Theoretical isotope cluster from composition. ICScore based on χ2 -test p-value.
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False Discovery Rate (FDR)
How confident can we be in these mass-matches?
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False Discovery Rate (FDR)
How confident can we be in these mass-matches?
FDR: 3.9% [ ~ 10 / 263 spectra ]
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False Discovery Rate (FDR)
How confident can we be in these mass-matches?
FDR: 3.9% [ ~ 10 / 263 spectra ]
Estimate the number of errors by searching with non-N-linked motif (decoy) peptides too. Count spectra matched to decoy peptide-glycan pairs. Rescale decoy counts to balance the number of motif
and non-motif peptides. 13
Tuning the filters…
Adjusting thresholds and parameters to Increase specificity (lower FDR, fewer spectra), or Increase sensitivity (more spectra, higher FDR)
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Tuning the filters…
Oxonium ions: Number & intensity Match tolerance
"Intact-peptide" fragments: Number & intensity Match tolerance
Glycan composition: ICScore Constrain search space Match tolerance
Glycan database: Constrain search space Match tolerance
Precursor ion: Non-monoisotopic selection Sodium adducts Charge state
Peptide search space: Semi-specific peptides Non-specific peptides Peptide MW range Variable modifications 15
Tuning the filters…
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Tuning the filters…
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GlycoPeptideSearch (GPS) 1.3
Freely available implementation Windows, Linux
Reads open-format spectra (mzXML, MGF) Pre-indexed Glycan databases
Human & Mammalian GlycomeDB Mammalian CFG Array (v5.0) User-Named (Oxford notation)
In silico digest and N-linked motif identification Automatic target/decoy analysis for FDR
http://edwardslab.bmcb.georgetown.edu/GPS18
Where to from here?
Demonstrate utility on new instrument platforms, proteins, samples
Develop a scoring model for fragments Re-implement Cartoonist demerits Exploit relationships between
MS2 spectra, MSn spectra Explore application to
O-glycopeptides, N-glycans, O-glycans
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Edwards Lab (Georgetown) Kevin Brown Chandler [NSF] (Poster 32)
Goldman Lab (Georgetown) Radoslav Goldman (Poster 6) Petr Pompach Miloslav Sanda (Poster 23)
Marshal Bern (Xerox PARC) Cartoonist, Peptoonist
Rene Ranzinger (CCRC) GlycomeDB
Acknowledgements
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