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Protein Metrics – ASMS Asilomar
Glycopeptide Identification with Byonic™ Software
www.proteinmetrics.com
Marshall Bern
Protein Metrics – ASMS Asilomar
Byonic™ is an advanced proteomics search engine
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#
Sp
ec
tra
# No Mods
# Common Mods(^q,^c,m,n,q)
Byonic
ABRF iPRG Study on PTM finding, 2012
1. Higher Sensitivity
2. Topdown Proteomics
IgG1 Fc with glycan and M[+16]’s
Protein Metrics – ASMS Asilomar3
4. Multiple Identifications per MS2 scan
216 Da = Characteristic peak for phospho-Tyrosine
3. “Expert system” annotation
Byonic™ is an advanced proteomics search engine
Protein Metrics – ASMS Asilomar4
5. Glycopeptides!
Byonic™ is an advanced proteomics search engine
Protein Metrics – ASMS Asilomar
Protein Metrics – ASMS Asilomar
Glycopeptide search
Protein Metrics – ASMS Asilomar
1. How does Byonic work?
2. How to run Byonic
3. Interesting examples
7
Outline for Rest of the Talk
Protein Metrics – ASMS Asilomar
Byonic score is a sum of benefits and penalties
8
204 Da for HexNAc:Small benefit if observedBig penalty if not observed
~y12 means loss of full glycany12 means glycan on
Mouse brain synaptosome, run on Q-ExactiveData from Kati Medzihradszky, UCSF
AT1B2_MOUSE peptide with man6
Y1 ion:Big benefit if observedBig penalty if not observed
Protein Metrics – ASMS Asilomar
Byonic annotates glycan (and peptide + glycan) peaks that match glycopeptide pieces for any reasonable glycan topology
9
• Byonic will annotate an HCD peak at 657 Da for any glycan with HexNAc, Hex, NeuAc;512 Da for any glycan with HexNAc, Hex, and Fuc; etc.
• Byonic will annotate Pep+HexNAc for any glycopeptide with HexNAc(So watch out α-Dystroglycan!)
Mouse brain Contactin-1 glycopeptide
512 Da = Diagnostic peak for antennal fucose
HexNAc(5)Hex(4)Fuc(2)
Protein Metrics – ASMS Asilomar10
Is it possible to localize O-glycans with CID ?
rEPO made in HEK cellsRun on Orbitrap VelosHCD NCE = 35% Data from Khoo, Academia Sinica, Taiwan
~y11 + HexNAc
~y12 + HexNAc
~y11 + HexNAc Hex
~y11 + 2 HexNAc
Pep_1+
Pep – 18
• ~y11 + 2 HexNAc rules out two HexNAc-Hex-NeuAc’s
• Byonic placed the O-glycan correctly (AASAA not AISPP) on 178 of 185 observations of the peptide (but with low DeltaMod values)
• This is a lucky peptide: only one glycosite, only two S/T’s, nicely placed proline
HexNAc(2) Hex(2) NeuAc(2) = 1312.455 Da
Protein Metrics – ASMS Asilomar
Unlucky PeptideHuman Ig Alpha-1 (not very purified)Run on Orbitrap Fusion, HCD NCE = 30%
Data from Rosa Viner, Thermo Fisher
Total Monosaccharide Composition:HexNAc(4)Hex(4)NeuAc(3)
Protein Metrics – ASMS Asilomar
Unlucky PeptideHuman Ig Alpha-1 (not very purified)Run on Orbitrap Fusion, ETD
Data from Rosa Viner, Thermo Fisher
• Total Monosaccharide Composition: HexNAc(5)Hex(5)NeuAc(2)
• z = 6+ (C’s are carbamidomethyl Cys)
In ETD, we observe various total monosaccharide compositions up to HexNAc(11)Hex(11)NeuAc(1) 4308 Da
Protein Metrics – ASMS Asilomar
1. How does Byonic work?
2. How to run Byonic
3. Interesting examples
13
Outline for Rest of the Talk
Protein Metrics – ASMS Asilomar
Modification Fine Control
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At most 3 common modifications per peptide
Allow up to three M[+16]’s but only one W[+32]
At most 1 rare modification per peptide
Protein Metrics – ASMS Asilomar
Glycan modifications follow the same rules
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Rare1 at most one per peptide
A peptide can have one man5, one man6, and one man7, along with a rare modification
Pre-defined database of ~300 N-glycan compositions
Protein Metrics – ASMS Asilomar
Glycan database size and modification fine control can have a huge impact on search size!
16
…GK.LICTTAVPWNASWSNK.SLEDIWDNMTWMQWER.EIDNYTNT…
HIV gp120 sequence:
• 300 N-glycans, all rare1, no missed cleavages LICT…NK peptide gives ~600 glycoforms
• 300 N-glycans, common1, no missed cleavages LICT…NK peptide gives 300 × 300 = 90,000 glycoforms
• 300 N-glycans, common1, one missed cleavage LICT…ER peptide gives 300 × 300 × 300 = 2.7 x 107 glycoforms
Protein Metrics – ASMS Asilomar
Glycan database size and modification fine control can have a huge impact on search size!
17
…HVK.HYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPR.LSH…
Human Ig Alpha-1 Sequence:
• 10 O-glycans, common5, Total common max = 5 HYT…PR peptide gives (12 choose 5) × 510 = 7,434,375,000 glycoforms
• 100 different sums of O-glycan masses, rare1 HYT…PR peptide gives 1200 glycoforms (but no site localization)
• Can search anything in between – e.g., try to resolve the O-glycan on only the first and last S/T
Protein Metrics – ASMS Asilomar
Glycan databases
18
• Use any text editor to write/edit
• Use literature, prior knowledge, glycomics experiments, etc.
• Setting the database to common1is the same as setting each glycan in it to common1.
• Big impact on performance!
• Byonic keywords:
Hex, HexNAc, NeuAc, NeuGc, Fuc, dHex,
GlcNAc, GalNAc, Man, Glc, Gal, Pent, Xyl,
GlcA, IdoA, Sodium, Na, Sulfo, Methyl,
Acetyl, Phospho, DiNAcBac, Pseudaminic,
Legionaminic, Etn, pEtn, …
Protein Metrics – ASMS Asilomar
One more option, meant for “one-protein” samples
19
Glycopeptide assignments will be shown regardless of Byonic score or FDR
Protein Metrics – ASMS Asilomar
One more option, meant for “one-protein” samples
20
Glycopeptide assignments will be shown regardless of Byonic score or FDR
Uncheck this box
Protein Metrics – ASMS Asilomar
• “Right size” the protein database
• Include all the proteins in the sample, but not too many more.
• “Right size” the glycan database by iterative search
• Start from a small glycan database and work up.
• Start large and work down by removing glycans with little support.
• Be careful with “Total common max” – start small and work up
• Manually check all glycopeptide assignments prone to error
• Glycans containing 2 or more Fuc’s or 1 Fuc and 1 NeuGc
• Glycopeptides with close-together oxidations and glycosylations
• Glycopeptides with 2 or more potential glycosylation sites
21
Practical tips for Glycoproteomics
Protein Metrics – ASMS Asilomar
1. How does Byonic work?
2. How to run Byonic
3. Interesting examples
22
Outline for Rest of the Talk
Protein Metrics – ASMS Asilomar
Two full-size N-glycans on one peptide …
Hex
NA
c2H
ex
Hex
NA
c3H
ex
Hex
NA
c4H
ex
Hex
NA
c5H
ex
Total HexNAc(4) Hex(10)man5 + man5 ? man4 + man6 ?
Precursor10x bigger
Total HexNAc(9) Hex(11) NeuAc(4)
+ ?
ETDBlood plasma, conA enrichedTMT-tagged to enhance chargeData from Rosa Viner, Thermo
HCDrEPO made in HEK cells2 × ?
Protein Metrics – ASMS Asilomar24
Bacterial GlycosylationCampylobacter jejuni, run on Orbitrap Velos with HCD fragmentation, HCD NCE = 45% Data from Nick Scott, UBC Nothaft et al, Mol Cell Proteomics 11 (11), 2012
HexNAc(5)Hex(1) diNAcBacillosamine(1)
Glycan contains diNAcBac Byonic looks for Y1 = Peptide + diNAcBac (228 Da)
Gly
cop
ep_2
+
Y1_
1+
Protein Metrics – ASMS Asilomar25
O-glycosylation (and N-terminal alkylation!?) on MHC class I peptides
[+28]IPRPPIT[+365]QSSL from RNA-binding protein 27(cytoplasm and nucleus!)
[+28]IPRPPIT[+656]QSSL
Human B cell line, run on Orbitrap Elite with EThcD fragmentation, HCD NCE = 32% Data from Albert Heck, Utrecht Mommen et al, PNAS 111 (12), 2014
Note: [+14/+28]RPPIT[+203]QSSL (called O-GlcNAc) found in leukemia samples [Hunt lab, ASMS 2014]
Pe
p_
1+
Pep
_1+
Protein Metrics – ASMS Asilomar
O-glycosylation (without N-terminal alkylation) on MHC class I peptides
Human B cell line, run on Orbitrap Elite with EThcD fragmentation, HCD NCE = 32% Data from Albert Heck, Utrecht Mommen et al, PNAS 111 (12), 2014
IPRPPIT[+365]QSSL from RNA-binding protein 27(cytoplasm and nucleus!)
26
Pep
_1+
c10
Protein Metrics – ASMS Asilomar
• Byonic offers:
• Glycopeptide search in a full-service proteomics search engine
• Support for all types of fragmentation and mass analysis
• Special features for one-protein samples
• Byonic is keeping up with MS hardware.
• If you can fragment it, we can identify it!
Summary
27www.proteinmetrics.com
Protein Metrics – ASMS Asilomar
IPRPPIT[+203]QSSLO-GlcNAc
IPRPPIT[+365]QSSLGlcNAc-Gal
Yewdell, et al., Making sense of mass destruction: quantitating MHC class 1 antigen presentation 3, 952 – 961, 2003
GlcNAc or GalNAc ?
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