Glycosylation analyses of recombinant proteins by LC-ESI ... · (Sewell et al., J Biol Chem, 2006)...
Transcript of Glycosylation analyses of recombinant proteins by LC-ESI ... · (Sewell et al., J Biol Chem, 2006)...
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