real high-throughput glycoanalysis using glyxbox a … · => 1d, 2d & dige • lc-ms/ms...

Max Planck Institute Magdeburg Real HT Glycoanalysis Using xCGE-LIF 1 Okt. 3rd, 2012 Max Planck Institute Magdeburg

Oct. 3rd, 2012

CE Pharm

Scottsdale, Arizona

Real High-Throughput Glycoanalysis

using glyXbox

a High Performance System

based on xCGE-LIF

E. Rapp1,2 , R. Hennig 1,2, T. Muth 1,2, R. Kottler 1, U. Reichl 1,3

MAX-PLANCK-INSTITUT

DYNAMIK KOMPLEXER

TECHNISCHER SYSTEME

MAGDEBURG

1 Max Planck Institute for Dynamics of Complex Technical Systems,

Magdeburg/Germany

2 glyXera GmbH, Magdeburg/Germany

3 Otto-von-Guericke University, Chair of Bioprocess Engineering,

Magdeburg/Germany

Max Planck Institute Magdeburg Real HT Glycoanalysis Using xCGE-LIF 2 Okt. 3rd, 2012

Bio/Process-Analytics @ MPI-DCTS

Hardware-, Software- &

Method-Development

Genomics

Qualitative Protein Analysis

Quantitative Protein Analysis

Analysis of Post-Translational Modifications

• Phosphorylation

• Glycosylation

• …

• Glycans

• Glycopeptides

• Glycoproteins

and

• other Carbohydrates (e.g.: Milk oligosaccharides)

Lipidomics

Glycomics,

Glycoproteomics &

Carbohydrate Analytics

• Capillary-(Gel-)Electrophoresis Systems: - CE System with UV & LIF Detection - Multiplexing CGE-LIF Systems (xCGE-LIF)

• HPLC Systems: - HPLC-FLR - nanoHPLC-LIF - HPAEC-PAD

• Diverse Gelelectrophoresis Systems => 1D, 2D & DIGE

• LC-MS/MS Systems: - Online: nanoHPLC-QqTof & nanoHPLC-QIT - Offline: 2D-nanoHPLC & MALDI-Tof/Tof

• GC-MS/MS System

Instrumentation:

Proteomics Metabolomics

Team: 1 Post Doc, 10 PhD Students, 6 Students, 2 TAs

Max Planck Institute Magdeburg Real HT Glycoanalysis Using xCGE-LIF 3

... one Genome …

... different PROTEOMES ... ... BUT …

There is ...

... and by far more GLYCOMES

and GLYCOPROTEOMES:

Okt. 3rd, 2012


Glycoconjugates / Glycosylation

Glycoconjugates

N-Glycosylation

O-Glycosylation Glycolipids

• Glycan attached to nitrogen

atom of the amino acid asparagine

• Glycan attached to oxygen

atom of the amino acids

serin and threonine

• Glycan attached to ceramide

(glycosphingolipids) or

phosphatidylglycerol

(glycophospholipids)

Lip

id B

ila

ye

r

Pro

tein

Pro

tein

Pro

tein

Pro

tein


Antennas Bisecting Core Fucose

Core

Mannose (Man)

Galactose (Gal)

Sialic Acid (SA)

Fucose (Fuc)

N-Acetylglucosamine (GlcNAc)

Typical N-Glycans of Mammalian Cells

High-Mannose Type Complex Type Hybrid Type


Impact of Complex Carbohydrates

• Key-and-Lock principle for receptors and ligands.

• Signal transduction / communication between cells and pathogens.

• Modification of enzyme / protein activities and specificities.

• Potency and specificity of new drugs and vaccines.

• Health-promoting / preventive functions in food, food additives and functional food.

Oligosaccharides, glycolipids, glycans etc. play a central role in many aspects of life:


But, ...

... GLYCOMICS is significantly lagging behind

GENOMICS and PROTEOMICS !!!

mainly due to the absence of HT-analytical methods which can reliably identify and quantify a multitude of glycan structures in complex biological samples.

today’s glycoprofiling methods are too slow, too expensive, and too low throughput.

robust, efficient and fast HT-glycoprofiling technologies urgently need to be established for major fields in life sciences:

• systems biology and disease biomarker discovery / screening.

• development and production of biopharmaceuticals and vaccines.

• development and production of (functional) food.


Glycomics & Glycoproteomics

Toolbox

Glycan Analysis Glycopeptide analysis

In-gel-geglycosylation

with endoglycosidase

Glycans

Glycopeptide trapping and

separation by (2D)-nano-HPLC

Separated glycopeptides

directly spotted onto

MALDI targets via micro-

fraction-collector

Online detection by

ESI-(MS/)MS

Optional on

target deglycos.

with endo-

glycosidase

Identification of glycosylation sites

proteolytic digestion

Consecutive eluting

glycopeptides

non labelled

Glycan trapping and

separation

by (2D)-nano-HPLC

directly spotted onto

MALDI targets

Offline detection by

MALDI-MS(/MS)

Structural elucidation of glycans

pos. labelled

neg. Labelled

Database

Search

Database

Search


MALDI or nanoESI-

MS(/MS)


MALDI-MS(/MS)

2AB/AA-glycans

HPLC

glycan

fingerprint

Seq. Exoglyco-

sidase Digest.

HPLC

glycan

fingerprint

APTS-glycans

CGE

glycan

fingerprint

Seq. Exoglyco-

sidase Digest.

CGE

glycan

fingerprint

Protein Analysis

Online detection by

ESI-(MS/)MS

GLYCOMICS

GLYCOPROTEOMICS


xCGE-LIF Based Glycomics

Glyco-

proteins

In-gel / In-solution

Deglycosylation

with Endoglycosidase

Glycans

Structural elucidation

Fluorescent Labelling

with APTS

Database Search

APTS-Glycans

Protein

Analysis

CGE-LIF

Analysis

Sequencial

Exoglycosidase

Digestion (SED)

CGE-LIF

Analysis

"Fingerprint"

Comparison

Electropherogram

Overlay


xCGE-LIF for Glycoanalysis:

Principles and Advantages

--

Time

- --- --

Time

- - --

separation by m/z separation by

molecular shape

• Only ion migration

• NO sample carryover

• Fully automated multicapillary array systems available => xCGE-LIF with up to 96 capillaries

AND


HILIC-FLR vs. xCGE-LIF

Separation power, performance and sensitivity:

Separation of two aliquots of the same sample: the "blood-plasma glycome"

xCGE-LIF (sample amount 2 nL)

HILIC-FLR (sample amount 7500 nL)

Separation power more than one order of magnitude better !

Sensitivity up to three orders of magnitude higher !


Separation Power of Glycoanalysis

via xCGE-LIF

A2FG2S1(2,3)

A2FG2

A2FG2S2(2,3)

A2FG2S2(2,6)

Sig

na

l In

ten

sit

y i

n R

FU

(O

ffs

et

20

00

RF

U)

Migration Time in MTU‘‘


A first powerful "real" HT glycoanalysis-tool

(method, software with GUI & database):

the "glyXbox"

Sample preparation methods

Glycoanalysis method with:

• Automated parallel separation and sensitive

detection (xCGE-LIF)

• Automated data-processing (glyXdata)

• Automated data-analysis (glyXtool)

Glycodatabase:

an oligosaccharide / glycan database (glyXbase)

The system is ready for take-off !

System (Method, Software & Database)

for Automated HT Glycoanalysis via xCGE-LIF

(Ongoing project of @ glyXera, PhD thesis of R. Hennig & T. Muth)

www.glyxera.com

Max Planck Institute Magdeburg Real HT Glycoanalysis Using xCGE-LIF 14 Okt. 3rd, 2012 14

Protein Separation by 1D/2D-GE

& Cut out of Bands/Spots:

1c

Fluorescent Labeling of Released

N-Glycans with APTS

3

APTS

Reducing

Agent

Acid

APTS

Capillary

DNA

Sequencer

xCGE-LIF Analysis,

Normalization & Data Evaluation

6

Using glyXbox for

Automated HT-Glycoanalysis via xCGE-LIF

Addition of

Internal Standards

5

Sample Sample/Standard

Mix

+ Internal Standard

Protein Precipitation

(e.g. to Extract Proteins from

Citrate Blood Plasma)

1a Protein Purification & Concentration

by Affinity-SPE

(e.g.: Protein A Beads)

1b

Post Labeling Clean-Up 4

- SEC

- HILIC-SPE

- NONE

On-Membrane, In-solution or In-Gel,

Deglycosylation (by PNGase F):

PNGase F

2

glyXbox

Schwarzer J, Rapp E, Reichl U, Electrophoresis 2008, 29, 4203-4214.

Ruhaak L, Hennig R, Huhn C, Borowiak M, Dolhain R, Deelder A, Rapp E, Wuhrer M, J Proteome Res 2010, 9, 6655-6664.

Rapp, E, Hennig R, Borowiak M, Kottler R, Reichl U, Glycoconjugate Journal 2011, 28, 234-235.


(Ongoing project @ glyXera, PhD thesis of R. Hennig & PhD thesis of T. Muth)

Software-Development for

Automated HT Glycoanalysis via xCGE-LIF


xCGE-LIF Analysis of N-Glycan Pools

(Normalized Electropherogram = “ Fingerprint”)

• Limit of detection: 50 attomole on column.

• Linear dynamic range: 4 orders of magnitude.

• Good reproducibility with respect to relative peak heights.

• RSD for migration times of more than 36 consecutive runs < 0,03%. (xCGE-LIF analyses of 3 techn. replicates à 12 repeated runs)

• Longterm RSD (about two years) for migration times < 0,5%.

Overlay of 12 "fingerprints" of the N-glycan pool of a mAB:

(LOD and reproducibility of CGE-LIF utilizing a "Capillary DNA Sequenzer")

Sig

nal In

ten

sit

y in

RF

U



Growing Glycan / Oligosaccharide Library for Structural Elucidation via “Migration-Time-Matching”

Excerpt from

in-house library:

At present:

• About 200 entries.

• Normalized migration times for two different gel matrices.

• Human milk oligosaccharide database started.

(Ongoing project @ glyXera, PhD thesis of R. Hennig & R. Kottler)

tmig in MTU‘‘


N-glycan

Composition/Structure

N-glycan Release

HILIC-HPLC Fractionation

N-glycan Purification

m/z

m/z

m/z

m/z

m/z

m/z

m/z

MS -

Spectra

CGE-LIF

Data tmig

tmig

tmig

tmig

tmig

tmig

tmig

Normalized Migration

Time

Annotation Glycan Peaks

Ruhaak LR, Hennig R, Huhn C, Borowiak M, Dolhain RJEM, Deelder AM, Rapp E, Wuhrer M: Optimized Workflow for Preparation of APTS-Labeled N-Glycans Allowing High-Throughput Analysis of Human Plasma Glycomes using 48-Channel Multiplexed CGE-LIF. Journal of Proteome Research (2010) 9: 6655–6664.

Database Built-Up by Combining

MALDI-TOF-MS & xCGE-LIF Analysis


1435.51663.6

1825.7

1954.7

2028.8

2383.9

267_78_L22.d: +MS

1663.6 1850.7

281_78_L8.d: +MS

1258.5 1501.6

1647.6

1783.7

1850.7

2076.8

285_78_L4.d: +MS

1273.41501.6

1647.7

1783.7 1866.7 2053.8

288_78_L1.d: +MS

0

2

4

6

7x10

Intens.

0.00

0.25

0.50

0.75

1.00

1.25

1.50

7x10

0

2

4

6

7x10

0

1

2

3

4

5

7x10

1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 m/z

Analyzing All Fractions via

MALDI-TOF-MS & xCGE-LIF

... exemplarily shown for the human blood serum “glycome” :


1783.7

1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000

1663.600 1647.600 [M + Na]+

[M + K]+

1273.444

1257.444 [M + Na]+

[M + K]+ 1850.645 [M + Na]+

1866.645 [M + K]+

*

APTS

APTS

APTS

E.G.: Fraction No. 37


CE-ESI-MS

20.0 25.0 30.0

Time [min]

0.0

0.2

0.4

0.6

0.8

1.0

4 x10

Intens.

FA2G2S1 (G2F-1SA)

APTS

FA2G2 (G2F)

APTS

FA2G1(G1F)

APTS

FA2G0 (G0F)

APTS

FA2G2S1 (G2F-1SA)

APTS

FA2G0 (G0F)

APTS

FA2G2 (G2F)

APTS

FA2G1(G1F)

APTS

xCGE-LIF

Database Built-Up by Correlating

CE-ESI-MS & xCGE-LIF Analysis of Labeled Samples

... exemplarily shown for IgG

(Project of R. Hennig - in coop. with Christian Neusüß @ applied University of Aalen / Germany)


Application of glyXbox for

Automated HT Blood Plasma Glycomics

(Project of R. Hennig & M. Borowiak - in coop. with Manfred Wuhrer @ LUMC (Leiden/NL))

Separation of about 4700 samples in 48 hours !

Migration Time Units [MTU]

Rel.

Flu

ore

scen

ce U

nit

s

Sam

ple

No

.

1

384

Inte

nsit

y i

n R

FU




Automated data processing !

Re

lati

ve

Flu

ore

sc

en

ce U

nit

s

Sa

mp

le N

o.

Normalized Migration Time Units [MTU ’’]





Separation and analysis is 90 - 450 times faster than comparable methods

due to multiplexing with up to 96 capillaries in parallel and

automated data processing !

H5N4S2

APTS

H6N5S3F1

APTS

H5N4S2

APTS

H5N4S2F1

APTS

H5N5S2F1

APTS

H6N5S3

APTS

3x

H7N6S3

APTS

3x

H7N6S3F1

APTS

2x

H6N5S2F1

APTS

x2

H5N4S1

APTS

H5N4S1

APTS

H7N6S2

APTS

2x

H5N4S1F1

APTS

H5N2

APTS

H5N5S1F1

APTS

H7N6S2F1

APTS

2x

H5N4S1F1

APTS

H6N5S2

APTS

H6N2

APTS

H6N5S1

APTS

H5N4S1

APTS

H6N5S1F1

APTS

H7N6S1

APTS

H4N4F1

APTS

H4N4F1

APTS

H4N5F1

APTS

H4N5

APTS

H5N4F1

APTS

H5N5F1

APTS

H9N2

APTS

H3N5

APTS

Sig

na

l In

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Automated HT Glycoprofiling of Human IgG

Automated separation and analysis of about 1800 samples

in the framework of a joint GWAS study !

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

190

200

Sa

mp

le N

o.

Normalized Migration Time Units [MTU ’’]

(Project of M. Borowiak & R. Hennig - within the EU-FP7-projekt “HighGlycan” (coord. by M.Wuhrer @ LUMC (Leiden/NL))




(Project of M. Borowiak & R. Hennig - within the EU FP7 projekt “HighGlycan” (coord. by M.Wuhrer @ LUMC (Leiden/NL))

Automated data processing, peak picking & integration and database matching!

Migration Time in MTU’’

Sign

al In

ten

sity

in R

FU




500 1000 1500 2000 2500 3000 3500 4000 4500 5000

0

2000

4000

6000

8000

10000

12000

K196

K160

K129

Migration Time in MTU’’

S

ign

al In

ten

sity

in R

FU

(Project of M. Borowiak T.Muth & R. Hennig - within EU-FP7 “HighGlycan” (coord. by M.Wuhrer @ LUMC (Leiden/NL))

Comparing different donors:

Large differences in the IgG N-glycan “fingerprints” !



Automated HT Milk Oligosaccharide Analysis

(Human milk oligosaccharide-fingerprints from two donors; Ongoing project of R. Kottler - in coop. with MILUPA/DANONE) S

ign

al

Inte

nsit

y i

n R

FU



Separation Power of xCGE-LIF

Used for Milk Oligosaccharide Analysis

Sig

nal

Inte

nsit

y i

n R

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Increasing Milk Oligosaccharide Library for Structural Elucidation via “Migration-Time-Matching”

HMOS Structur NZ

fraction

Migration Time

in MTU‘‘

Lacto-N-neo-tetraose

(LNneoT) NZ 3 88,3

Lacto-N-tetraose

(LNT) NZ 3 90,3

Lacto-N-fuco-pentaose II

(LNFP II) NZ 4 109,71

Lacto-N-fuco-pentaose V

(LNFP V) NZ 4 111,7

Lacto-N-fuco-pentaose I

(LNFP I) NZ 4 113,4

Lacto-N-fuco-pentaose III

(LNFP III) NZ 4 115,46

Glucose (Glu)

Galactose (Gal)

Fucose (Fuc)

N-acetylglucosamine

(GlucNAc)


3x

3x

2x

Verify annotated structures by

(sequential) exoglycosidase

digestion:


Automated Glycoprofiling of recombinant EPO (Project of R. Hennig)


2x


Extended Structural Analysis of recombinant EPO (Using exoglycosidase sequencing in combination with xCGE-LIF based glycoprofiling)


Conclusions

• System & Method allow fast and easy characterization of

N-glycosylation patterns (qualitative & quantitative)

and other carbohydrate pools.

• Highly sensitive - high resolution - “real” high throughput

system & method for profiling glycoproteins

and other carbohydrate mixtures.

• N-Glycans and other carbohydrates can be analyzed on three levels:

• Fingerprint Analysis

• Glycoprofiling

• Extended Structural Analysis


Outlook

• Extension of N-glycan and HMOS libraries and generation of other

oligosaccharide libraries (e.g. O-glycans)

• Applying this method to other fields:

- Glycome GWAS Studies

(e.g. in the context of the "HighGlycan" EU-consortium)

- Biopharmaceuticals & Biosimilars

(i.e.: recombinant glycoproteins, vaccines, …)

- Functional food (e.g. infant nutrition) &food additives

- Large scale clinical studies

- Early diagnosis of diseases (e.g.: diabetes, cancer, …)

- Doping analysis (e.g.: EPO glycosylation => “fingerprints”)

- ...

• Commercialized via: www.glyxera.com


Thanks to ...

… YOU ... our cooperation partners:

… the A-Team !!! esp.: • René Hennig • Thilo Muth • Robert Kottler • Matthias Borowiak

… and the glyXera staff:

www.glyxera.com

real high-throughput glycoanalysis using glyxbox a … · => 1d, 2d & dige • lc-ms/ms...

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