Glycosylation analyses of recombinant proteins by LC-ESI mass spectrometry

Dr Malin Bäckström

Mammalian Protein Expression Core Facility

P4EU meeting Porto Nov 11-12, 2013

MPE - A tissue culture facility for protein

expression in mammalian cells • Culture of cell lines in different

formats

• T flasks, roller bottles, spinner

bottles

• Bioreactors with controlled pH

and oxygen for optimal

production

What MPE core facility offers:Recombinant proteins:

• Generation of expression vectors

• Mutagenesis

• Generation of stable expressing clones in different cell lines

• Production of larger amounts of protein (≤100 mg) by perfusion culture in bioreactors

• Transient protein expression in 1L scale in bioreactors

• Concentration of product down to easy-to-handle volumes

• Simple purification (His-tag, Protein G)

Other:

• Culture of cell lines

• Culture of hybridoma cells for monoclonal antibody production

• Mycoplasma testing of cell lines

Transient transfection of CHO-S, Lec3.2.8.1-S and 293F cells

• CHO-S and Lec3.2.8.1 (adapted to

suspension) are transfected using

NOVACHOice (Merck Millipore) in

Freestyle medium

• 293F cells are transfected using

PEI in Freestyle medium

Production of MUC2 D3 proteins in Lec3.2.8.1 cells – glycosylation deficient CHO cells

Lec3.2.8.1; a CHO mutant

producing high-mannose N-

glycans and GalNAc O-

glycans

(deficient in α-glucosidase I responsible for

N-glycan trimming in the ER) Asn Ser/Thr

N-glycans(removable by EndoH)

O-glycans

Lec3.2.8.1 cells were adapted tosuspension culture at MPE,

for the production of MUC2 D3 domain for structural determination

His-purified proteins from

transient transfection

of Lec3.2.8.1-cells in 1L

bioreactor (dec 2012)

Production of glycosylated recombinant proteins

• N-glycans (in many proteins)

• O-glycans (less common, but very

extensive in some proteins)

• Mammalian cells (CHO, HEK etc)

• Yeast

• Insect cells

MUC1 signal peptide

n tandem repeats

Proteolytic

cleavage site

TM

CT

HGVTSAPDTRPAPGSTAPPA** * **

• Extracellular domain made up by a variable number of multiple tandem

repeats of 20 amino acids

• 5 potential O-glycosylation sites per tandem repeat

MUC1 mucin; a densely O-glycosylated

membrane protein on epithelial surfaces

MUC1 signal peptideExtra-cellular part of MUC1

with 16 tandem repeatsMurine IgG2a Fc (exon1-3)

Recombinantly expressed as

an Ig fusion protein:

Endogenous membrane-

bound form:

Aberrant O-glycosylation of MUC1 in breast carcinoma cells

Normal (Breast) carcinomaGalNAc-Thr/Ser

Galb1-3GalNAc (core 1=T)

GlcNAcb1,6

Galb1-3GalNAc (core 2)

Chain extension

SAa2-3Galb1-3GalNac (sialyl-T)

Chain termination

(further sialylation possible)

Brockhausen et al., 1995, Eur J Biochem

Lloyd et al., 1996, J Biol Chem

Hanisch et al., 1996, Eur J Biochem

Muller and Hanisch, 2002, J Biol Chem

SAa2-6GalNac-Thr/Ser (sialyl-Tn)

Release of O-glycans from blotted protein on PVDF membrane

MS2

MS3

MS4 etc.

O-glycans are released chemically

using NaBH4/KOH

O-glycans on MUC1 in the prostate cancer cell line DU-145

(Bäckström et al., J Proteome Res, 2009)

Modification of O-glycans by co-transfection with specific glycosyltranserases

(Sewell et al., J Biol Chem, 2006)

Structure of O-glycans and O-glycan occupany in MUC1-Ig from CHO and CHO/ST6GalNAcI cells(Sewell et al., J Biol Chem, 2006)

(Sewell et al., J Biol Chem, 2006)

Cleavage of protein into glycopeptides to analyze the glycan site occupancy

Here: clostripain

Other common enzymes: trypsin, Asp-N etc

(Sewell et al., J Biol Chem, 2006)

LC-MS with ECD fragmentation to analyze which sites are glycosylated

Sihlbom et al., 2009

Glycobiology

Now: ETD dissociation more commonly used for this purpose

N-glycans can have high-mannose, hybrid or complex structures

Full-length CD83

CD83EX-Ig

TM

murine IgG2a Fc

N-79 N-96 N-117

Signal peptide

1 19 145-166V Ig domain 28-114

144myc

EK

hCD83ext (E.coli)

23 128

His6

CHO/CD83FL CD83EX-Ig 293/CD83FL

(Western blot anti-CD83)

N-glycosylation of overexpressed dendritic cell glycoprotein CD83

N-glycans on CD83EX-Ig from CHO K1 released by PNGAseF

RT: 18.02 - 32.06

19 20 21 22 23 24 25 26 27 28 29 30 31 32

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

893.42

731.33

1258.83

675.331258.92

1038.83

812.33

NeuAcGalGalNAc-ol

(O-glycan)

994.83

N-glycans on CD83EX-Ig from Lec3.2.8.1

RT: 18.02 - 32.06

19 20 21 22 23 24 25 26 27 28 29 30 31 32

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

1235.58

819.33

1073.33

NL:1.27E4

Base Peak F: ITMS - p ESI Full ms [380.00-2000.00] MS

mb_090323_04

N-glycans on CD83EX-Ig; summary

CHO K1 Lec3.2.8.1

High-mannose

Hybrid

Complex

none

none

none

Potential oligosaccharide structures of N-glycans on CD83FL

Summary N-glycans CD83

CD83 N-glycans

0

1000

2000

3000

4000

5000

6000

0 2 4 6

Sialic acids (n)

Mw

(D

a)

CD83EX-Ig

Lec3.2.8.1

CD83-Ig CHO K1

CD83FL HEK293

Methodology for glycosylation analysis

• N-glycans: Released by PNGAse F

• O-glycans: Chemical release

• After purification the oligosaccharides are

analysed using graphite columns and negative ion

mode by LC-ESI MS and MSn

• Glycopeptides are analysed in positive ion mode

with milder fragmentation techniques (ECD, ETD

etc) to localize glycan substitutions

O-glycosylation in insect cells

• PSGL-1-Ig fusion protein with many O-

glycans (mucin-like)

• Expressed in Trichoplusia ni (Hi-5) and

Spodoptera frugiperda (Sf9) cell lines

• O-glycans were released and analyzed

by LC-ESI MS

• Gaunitz et al., Glycobiology, 2013

Characterisation of O-linked glycosylation in different expression systems

Insect (Hi-5)

Yeast (Pichia)

CHO

O-glycosylation in insect cellsMonosaccharide Sf9 (mole/mole protein) Hi-5 (mole/mole protein)

Monosaccharide analysis

with HPAEC-PAD

Fucose 54 29

Galactosamine 56 13

Glucosamine 76 72

Galacatose 29 22

Glucose 11 3

Mannose 99 39

Galacturonic acid 2 2

Glucoronic acid 17 13

Iduronic acid - -

Sialic acid analysis with

DMB HPLC

Neu5Gc 0.03 0.06

Neu5Ac 0.06 0.03

Novel termination and elongation in insect cell linesComposition

Hex-HexNAc-dHex-HexA (M - nH)n - (Min) (M + Na)+ (M + H)+ (M + Na)+ (M + 2 Na -H)+

Reducing end GalNAcol cores:

1.1.0.0 a) Galβ3GalNAcol 384.3- 0.9 534.4 n.f b) 408.3 n. f

0.1.0.1 HexA-GalNAcol 398.3- 1.2 548.3 n.f n.f 444.2

0.1.1.1 HexA-(Fuc-)GalNAcol c) 544.2- 12.5 722.4 n.f n.f 590.3

Extensions:

0.2.0.1 Δ d)HexNAc-HexA-GalNol

Δ 559.3- 16.0 n.f n.f n.f n.f

1.1.0.1 HexA-Galβ3GalNAcol 560.3- 13.8 752.5 n.f n.f n.f

1.1.0.1 Hex-(HexA-)GalNAcol 560.3- 13.8 752.5 n.f n.f n.f

1.2.0.0 HexNAc-(Hex-)GalNAcol 587.3- 12.0 n.f n.f n.f n.f

1.2.0.0 HexNAc-Hex-GalNAcol 587.3- 12.0 n.f n.f n.f n.f

0.2.0.1 HexNAc-HexA-GalNAcol 601.3- 15.2 793.5 n.f 625.3 647.3

0.2.1.1 HexNAc-HexA-(Fuc-)GalNAcol 747.4- 17.4 967.6 n.f n.f 793.3

Tentative structureM/z

LC-MSRT

M/z

ESI-MSM/z LC-MS pos mode

O

O

+

0.3.0.1Δ

HexNAc-4HexNAc-HexA-GalNolΔ

762.3- 16.2 n.f n.f n.f n.f

1.2.0.1 Galβ3(HexNAc-HexA)GalNAcol 763.3- 20.0 997.6 n.f n.f n.f

1.2.0.1 HexNAc-HexA-Galβ3GalNAcol 763.3- 23.0 997.6 n.f n.f n.f

0.3.0.1 HexNAc-4HexNac-HexA-GalNAcol e) 804.4- 15.7 1038.6 n.f 828.4 850.4

0.3.1.1 HexNAc-4HexNac-HexA-(Fuc-)GalNAcol 950.4- 17.2 1212.7 n.f 974.5 996.4

Extensions + Decorations:

0.2.0.1-S1 f) S{HexNAc-HexA-GalNAcol 681.3

- 18.5 n.f n.f n.f n.f

0.2.0.1-PC1

g) PC{HexNAc-HexA-GalNAcol 766.3- 14.8 n.f 768.5 n.f n.f

0.3.0.1-PC1

PC{HexNAc-4HexNAc-HexA-GalNAcol 969.3- 13.9 n.f 971.4 993.5 1015.4

0.3.1.1-PC1

PC{HexNAc-HexNAc-HexA-(Fuc-)GalNAcol 1115.4- 11.3 n.f 1117.5 1139.4 1161.4

0.3.2.1-PC1

h) n.d 1261.4- 13.2 1263.6 n.f n.f n.f

+ + S + PC

2.2.0.0 Hex-HexNAc-(Hex-)GalNAcol 749.3- 14.6 n.f n.f n.f n.f

+

Table 3

O-glycome of Sf9

Oligosaccharides released from PSGL-1/mIgG2b

produced in Sf9 cells.

a) Reducing end GalNAcol is included as HexNAc. Identification of Hex-HeNAcol as Galβ3GalNAcol

was based on fragmentation pattern similarity in UniCarb-DB. Structures starting with HexNAcol or Hex-HexNAcol

are assumed to represent GalNAcol and Gal β3GalNAcol.

b) n.f, not found

c) dHex was identified as fucose based on monosaccharide analysis

d) Δ , GalNol, Galactosamine may represent biological biosynthesis or be caused by chemical workup of sample

e) 1,4 linkage was identified for each structure with individual diagnostic MS 2 ions 0,2A or 0,2A - H20

f) S , sulfate

g) PC, phosphocholine

h) n.d, not determined

O

β 3

Thank you!

• Mammalian Protein Expression core facilityDr Elisabeth Thomsson

Richard Lymer

• Mucin Biology Group, University of GothenburgProf Gunnar C Hansson

• Glycoinflammatory Group, University of

GothenburgDr Niclas G Karlsson