Bioinformacsof, Glycansand,Glycoproteins,

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Bioinforma)cs of Glycans and Glycoproteins

Marshall Bern

Palo Alto Research Center

Protein Metrics

Cell to cell signaling is mediated by glycosyla1on…

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ABRF Course

1)  Background – Glycomics vs. Proteomics bioinformaFcs

2)  AutomaFc detached N-‐glycan analysis from MS1 – Cartoonist

3)  Semi-‐automaFc glycan analysis from MS1 and MS2 – GlycoWorkbench

4)  AutomaFc glycan analysis from MS2 – SimGlycan

5)  GlycopepFde analysis from MS2 – Byonic

Outline

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ABRF Course






Outline

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  N-‐linked GlycosylaFon –  AOached to Asparagine –  NX{S/T} moFf, X ≠ P (rare case – NXC)

–  1200 – 12,000 Da –  Common core

  O-‐linked GlycosylaFon –  AOached to Serine or Threonine (rare case – Tyrosine)

–  No consensus moFf –  200 – 2000 Da –  At least 7 cores

Two Types of GlycosylaFon

PepFde Chain

Galactose

Mannose

NeuGc NeuAc Fucose

GlcNAc

Symbols for the most common monosaccharides in mammals

GalNAc

5 ABRF Course

Pep)des

1.  19 different residue masses Total pepFde mass uninformaFve

2.  Linear sequence 3.  Only one type of pepFde bond

4.  Any sequence is valid 5.  Good databases exist

Glycans

1.  ~5 monosaccharide masses Total mass informaFve

2.  Branched structure 3.  Various types of linkages

4.  Structures follow certain paOerns 5.  Databases have poor coverage

Proteomics vs. Glycomics

ABRF Course 6

= Easier

= Harder

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Pep)des




Glycans





ABRF Course 7

Different levels of glycan idenFficaFon: Linkage informaFon

Cartoon (branching topology)

ComposiFon

4 HexNAc, 5 Hex, 1 Fuc, 1 NeuAc

α 2-‐6

Pep)des




Glycans




•  ~1000 trypFc pepFdes in human proteome with mass 2351 Daltons

•  ~20 in single sample: LGLLVFPYTHQNWEVQYSR EVTLHLLPGEQLLC[+57]EASTVLK LQEEMLQREEAENTLQSFR etc.

2351 Daltons (not permethylated)


ABRF Course 8

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Pep)des




Glycans




•  ~1000 trypFc pepFdes in human proteome with mass 2351 Daltons

•  ~20 in single sample: LGLLVFPYTHQNWEVQYSR EVTLHLLPGEQLLC[+57]EASTVLK LQEEMLQREEAENTLQSFR etc.

2351 Daltons (not permethylated)

Proteomics uses MS2 for idenFficaFon Glycomics can use either MS1 or MS2 or both


ABRF Course 9

Domon-‐Costello nomenclature •  Draw the glycan with reducing end to the right

•  Cut before – O – leo half = B-‐ion, right half = Y-‐ion •  Cut aoer – O – leo half = C-‐ion, right half = Z-‐ion •  Internal fragments can be YY, ZZ, etc.

•  Cross-‐ring = X 1,5X is most common

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From AB Sciex brochure

ABRF Course

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Outline

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•  Developed at Xerox PARC, mostly by David Goldberg (currently Ebay fellow)

•  Glycomics guidance from Anne Dell, Stuart Haslam at Imperial College

•  Plarorm-‐independent Java sooware, Free!, bundled with PARC mass spectrum browser

•  Works well for MALDI-‐TOF profiles of detached, permethylated, mammalian N-‐glycans

•  Works to some extent for other types of data

Cartoonist Sooware

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1.  Use graph grammar to generate a library of all plausible cartoons (topology only, no linkage informaFon!) ~145,000 N-‐glycan cartoons in library (bigger than current databases)

2.  Find the set (typically 1 – 20) matching each MS peak

3.  Use organism-‐ and Fssue-‐specific hints to select most likely cartoon from each set

Cartoonist uses an “expert systems” approach

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2. Fill antennae with lactosamine (GlcNAc-Gal):

… …

3. Cap with one of:

1. Start with N-glycan core

Empty graph

4. Optionally add: Bisecting GlcNAc, Base fucose, NeuAc NeuGc

1 2 4 3 N-‐Glycan Graph Grammar

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Core

No lactosamines:

GlcNAc-Gal-Gal caps:

Base Fucose

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Organism-‐Specific Hints (PenalFes)

•  Unlikely paOerns for all mammals: –  BisecFng GlcNAc –  Uncapped terminal GlcNAc –  Fucose on antennae, but not on base

•  Specific to Mouse: –  LacdiNAc, Sialyl Lewis-‐X, MulFple-‐fucose antenna

•  Specific to Human: –  Gal-‐alpha-‐Gal cap –  NeuGc (Is this what makes us human??)

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•  Unlikely paOerns for all mammals: –  BisecFng GlcNAc –  Uncapped terminal GlcNAc –  Fucose on antennae, but not on base

•  Specific to Mouse: –  LacdiNAc, Sialyl Lewis-‐X, MulFple-‐fucose antenna

•  Specific to Human: –  Gal-‐alpha-‐Gal cap –  NeuGc (Is this what makes us human??)

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•  Unlikely paOerns for all mammals:

–  BisecFng GlcNAc –  Uncapped terminal GlcNAc

–  Fucose on antennae, but not on base

•  Specific to Mouse:

–  LacdiNac, Sialyl Lewis-‐X, MulFple-‐fucose antenna

•  Specific to Human: –  No NeuGc (Is this what makes us human??)

From a talk by Ajit Varki, UCSD 20 ABRF Course

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PenalFes

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CFG Core C Profiles

ABRF Course

Manual cartoon annotaFons viewed in PARC spectrum browser

•  We typed in the cartoons in our own shorthand, e.g. 1661.8 = n/n///

•  Program matched them to peaks (Reports a warning error if no match)

•  This caught lots of typos / errors

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Cartoonist automaFc annotaFons

Two differences from manual:

•  Two structures for 1661.8 Da

•  Missed the glycan at 1620.8 Da

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Why did Cartoonist miss 1620.8 Da ?

•  Incomplete permethylaFon

What’s at 1606.8 and 1633.9 Da ?

•  Overlapping peak envelopes

•  Most of the Cartoonist code is for recalibraFng mass measurements, matching isotope envelopes to theoreFcal envelopes, separaFng signal from noise …

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•  Go to Tools menu in PARC mass spectrum viewer

•  Auto makes up the cartoons according to “expert system” rules

•  Use Database matches a cartoon list to peaks (the same way we digiFzed CFG profiles) Best for O-‐glycans, non-‐mammalian N-‐glycans, …

How to run Cartoonist?

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Using / creaFng databases

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Outline

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•  Developed at Imperial College (Primary Authors: Alessio Ceroni, Kai Maass, David Damerell)

•  Java code for Windows, Mac OS, and Linux. Free! hOp://www.soopedia.com/get/Science-‐CAD/GlycoWorkbench.shtml

•  FuncFons:

-‐ Match MS1 peaks to glycan structures in a database (GlycomeDB)

(Caveat: Matches everything! No expert system knowledge)

-‐ Match MS2 peaks to fragments of a selected structure

-‐ Draw new structures and calculate masses

GlycoWorkbench

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Canvas for drawing glycan structures

Database of known structures

Spectrum (MS1)

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•  Commercial sooware from Premier Biosoo (Palo Alto, CA)

•  Annual license, Approx. $10K / year

•  FuncFons:

-‐ IdenFfies glycan structures from MS2 fragmentaFon spectra

-‐ Structures are chosen from databases of known glycans (e.g., GlycomeDB)

-‐ High-‐throughput: can run a batch of 10,000 MS2 spectra

-‐ Scoring algorithm: which structure has highest percentage of its fragments matching MS2 peaks ?

SimGlycan

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Import data from MS instrument formats

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Search Parameters: Specify Fragmentation Patterns and Filters Specify fragmentaFon paOerns and database filters

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Results: IdenFfied glycans, related info, and annotated spectra

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Three ways to annotate MS2 spectra: Successive fragment losses, cartoons, or Domon-‐Costello

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Cartoons

Domon-‐Costello B, Y, C, Z, etc.

Successive fragment losses

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Outline

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MALDI-‐TOF profile of detached, permethylated, sodiated N-‐glycans

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Zoom of MS1 Spectrum 2750 – 3100 Da

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MS2 Spectrum

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MS2 peaks are annotated with fragments of structures determined by MS1

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Gives lots of annotaFons – User chooses structure

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Gives lots of annotaFons – User chooses structure

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Aoer the user chooses structure, the sooware will match MS2 peaks to fragments

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Top-‐scoring structure

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Successive fragment losses, Y-‐ions

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Outline

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Byonic Sooware

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•  Developed at Xerox PARC and Protein Metrics Inc, 2005 – 2013 (Primary Authors: Marshall Bern and Yong J. Kil)

•  About $10K perpetual license, $3K annual renewal

•  Will be released as a node in Thermo Fisher’s Proteome Discoverer 2.0

•  FuncFons:

-‐ PepFde / Protein idenFficaFon from MS2 data

-‐ GlycopepFde idenFficaFon

ABRF iPRG 2012 Study on Modified Pep)de Iden)fica)on Byonic outperformed Mascot, OMSSA, X!Tandem, ProteinPilot, SpectrumMill, Andromeda, … on every measure (# IDs, # consensus IDs, # spikes, # modificaFons, site localizaFon, FDR, … )

0 1000 2000 3000 4000 5000 6000 7000

7175

5v

5828

8v

3356

4

9312

8i

1121

1

5840

9

8713

3i

9415

8i

9705

3i

4242

4i

7777

7i

2306

8

4010

4i

8704

8i

9265

3

3428

4i

2311

7

7456

4

1415

1

5278

1

4760

3

1415

2

4551

1

1182

1

# S

pect

ra

# No Mods # Common Mods (^q,^c,m,n,q) # Nterm Mods

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Byonic Input

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NGlycan keyword tells Byonic to allow modificaFon only on NX{S/T}

Predefined tables of common glycan masses

“Blind” modificaFon search

Byonic Input

•  HCD (or QTOF) gives mainly glycan fragments (e.g., 204.087 Da for HexNAc) ETD gives pepFde fragments, but almost no glycan fragmentaFon

•  Idea: Glycan peak in HCD scan triggers an ETD scan

•  Idea: TMT0 (Tandem mass tag) for adding charge and improving ETD spectra

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Rosa Viner et al, Thermo Fisher Product-‐Dependent ETD on Orbitrap

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Glycoproteomics Example •  ConA-‐enriched blood serum, TMT0, SAX, HCD-‐pd-‐ETD on Orbitrap Velos

•  Search allowed common modificaFons + ~400 predefined N-‐glycan masses

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2535 ETD spectra ~70 glycoproteins, ~110 N-‐glyco sites, ~700 disFnct glycopepFdes

Byonic Glycoproteomics Search

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Byonic Glycoproteomics Search

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2535 ETD spectra ~70 glycoproteins, ~110 N-‐glyco sites, ~700 disFnct glycopepFdes

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ETD spectrum of a pepFde with two N-‐glycans N[+224]LFN[+3153]HSEN[+1914]ATAK[+224]5+ haptoglobin

1914 Da = 4 HexNAc, 5 Hex, 1 NeuAc 3153 Da = 5 HexNAc, 6 Hex, 2 Fuc, 3 NeuAc

ABRF Course

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HCD Spectrum

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[+224]VSN[+1914]QTLSLFFTVLQDVPVR4+

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ETD spectrum of a pepFde with four O-‐Glycans

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[+224]SYIS[+656]S[+656]QT[+947]NDT[+947]HK[+224]R5+

Glycophorin-‐A, TMT0 labeled, HCD-‐pd-‐ETD, Orbitrap Elite Chia-‐Wei Lin, KH Khoo, Academia Sinica, Taiwan

No peaks in between

947 Da 656 Da

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Note: O-‐Glycans fly off in HCD, so ETD is needed for site localizaFon

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Summary

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Glycomics is much less developed, especially O-‐glycosylaFon

Strength: “Thinks” like a glycomics expert Weakness: LiOle support for MS2

Strength: Complete package Weakness: Not high-‐throughput

Strength: Commercial support

Strength: All-‐round good proteomics tool Only tool for glycoproteomics

Weakness: Glycoproteomics sees only the most abundant species

Weakness: Requires MS2

ABRF Course

Acknowledgments

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References

•  Grant support for development of Cartoonist: NIH R01 GM085718

•  Grant for development of Byonic: NIH R21 GM094557, NSF CompuFng InnovaFons Fellowship

•  Slides and help with SimGlycan: Sanjib Meitei, Arun Apte, Premier Biosoo

•  Current Cartoonist team: Doron KleOer, Alex Brito, Mudita Singhal, PARC

•  Cartoonist: Goldberg D, SuOon-‐Smith M, Paulson J, Dell A. AutomaFc annotaFon of matrix-‐assisted laser desorpFon/ionizaFon N-‐glycan spectra. Proteomics. 2005; 5:865–75.

•  GlycoWorkbench: Ceroni A, Maass K, Geyer H, Geyer R, Dell A, Haslam SM. GlycoWorkbench: a tool for the computer-‐assisted annotaFon of mass spectra of glycans. J Proteome Res. 2008; 7:1650–9.

•  SimGlycan: Apte A, Meitei NS, BioinformaFcs in Glycomics: Glycan CharacterizaFon with Mass Spectrometry Data using SimGlycan in Func1onal Glycomics: Methods and Protocols, Editor: Jianjun Li, Springer Protocols

•  GlycomeDB: Ranzinger R, Herget S, von der Leith C-‐W, Frank M. GlycomeDB– a unified database for for carbohydrate structures, Nucleic Acids Research, Jan 2011, D373-‐D376.

•  Byonic: Bern M, Cai Y, Goldberg D. Lookup peaks: a hybrid of de novo sequencing and database search for protein idenFficaFon by tandem mass spectrometry. Anal. Chem. 2007; 79(4), 1393-‐1400.

Bioinformacsof, Glycansand,Glycoproteins,

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