glycopeptide identification with yonic™ software€¦ · 512 da for any glycan with hexnac, hex,...

Protein Metrics – ASMS Asilomar

Glycopeptide Identification with Byonic™ Software

www.proteinmetrics.com

Marshall Bern


Byonic™ is an advanced proteomics search engine

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# 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



5. Glycopeptides!



Glycopeptide search


1. How does Byonic work?

2. How to run Byonic

3. Interesting examples

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Outline for Rest of the Talk


Byonic score is a sum of benefits and penalties

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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


Byonic annotates glycan (and peptide + glycan) peaks that match glycopeptide pieces for any reasonable glycan topology

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• 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)


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


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)


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





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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


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


Glycan database size and modification fine control can have a huge impact on search size!

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…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


Glycan database size and modification fine control can have a huge impact on search size!

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…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


Glycan databases

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• 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, …


One more option, meant for “one-protein” samples

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Glycopeptide assignments will be shown regardless of Byonic score or FDR


One more option, meant for “one-protein” samples

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Glycopeptide assignments will be shown regardless of Byonic score or FDR

Uncheck this box


• “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

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Practical tips for Glycoproteomics





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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 × ?


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+


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+


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!)

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Pep

_1+

c10


• 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


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 ?

glycopeptide identification with yonic™ software€¦ · 512 da for any glycan with hexnac, hex,...

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