Supplementary materials
Verification of pSMART vector insert size
Forward primer SL1 CAGTCCAGTTACGCTGGAGTC
Reverse primer SR2 GGTCAGGTATGATTTAAATGGTCAGT
PCR conditions
95°C for 5 min, and 30 cycles of 95°C for 30 s, annealing at 55°C for 30 s and extension at
68°C for times depending on the expected insert size i.e. 1 Kb insert = 1.1 min, 2 Kb insert =
2.2 min, 4 Kb insert = 4.4 min, 8 Kb insert = 8.8 mins. A final extension step of 68°C for 10
min was included.
PCR products were visualised by agarose (1.5% w/v) gel electrophoresis.
Absolute quantitation
1
x 10000
25000
y = 3E+06x - 5E+06
R² = 0.9791
20000
15000
10000
5000
0
0
20
40
60
80
100
fmol
FIGURE S1. A linear calibration curve for the heavy (15N labelled) peptide
NGFKVPQIGVK (50 amol to 100 fmol on column). Triplicate technical measurements were
made.
2
TABLE SI. Primer sequences for cloning into pjexpress401
Gene
malQ
rseP
ptsA
rffE
dxs
Forward primer
Reverse primer
TATATCTAGATTAGGAGGTATATATCATGG
AAAGCAAACGTCTGGA
TATATCTAGATTAGGAGGTATATATCATGC
TGAGTTTTCTCTGGGA
TATATCTAGATTAGGAGGTATATATCATGG
CCCTGATTGTGGAAT
TATATCTAGATTAGGAGGTATATATCATGT
TGAATAATGCTATGAG
TATATCTAGATTAGGAGGTATATATCATGA
GTTTTGATATTGCCAA
TATAGGATCCCTACTTCTTCTTCG
CTGCAG
TATAGGATCCTCATAACCGAGAG
AAATCAT
TATAGGATCCTTACAGTTCCAGT
TCATG
TATAGGATCCTCACTTTTTCTTTA
CCGGAC
TATAGGATCCTTATGCCAGCCAG
GCCTTGA
Restriction sites are underlined.
3
Dot blot verification
FIGURE S2. Lectin blot verification using membrane induced (with 0.2% w/v L-arabinose)
CLM24 (AcrA, pgl) cells on the left (n=3) and induced CLM24 (AcrA) cells on the right
(n=3). A faint background is apparent in the middle, right colony illustrating that the highest
intensity colony must be chosen for further analysis.
4
TABLE SII. Gene list – The list of genes sequenced from inserts of pSMART vector in selected colonies.
Locus
Gene Symbol
EC
Number
1070261
1070776
3665421
3666548
3250960
3251567
5' End
1
2
3
4
5
6
ycdG
ycdH
rffD
rffE
yhaH
yhaI
predicted transporter
predicted oxidoreductase, flavin:NADH component
UDP-N-acetyl-D-mannosaminuronic acid dehydrogenase
UDP-N-acetyl glucosamine-2-epimerase
predicted inner membrane protein
predicted inner membrane protein
7
lpxB
tetraacyldisaccharide-1-P synthase
203348
204496
8
rnhB
ribonuclease HII, degrades RNA of DNA-RNA hybrids
204493
205089
9
10
yijO
frwD
predicted DNA-binding transcriptional regulator
predicted enzyme IIB component of PTS
3488364
3489543
3489215
3489202
11
pflC
pyruvate formate lyase II activase
3490423
3489545
12
pflD
predicted formate acetyltransferase 2
3492686
3490389
13
frwB
predicted enzyme IIB component of PTS
3493057
3492737
14
frwC
predicted enzyme IIC component of PTS
3494151
3493072
15
ptsA
3494460
3496961
16
fsaB
fused predicted PTS enzymes Hpr component, enzyme I
component, and enzyme IIA component
fructose-6-phosphate aldolase 2
3496973
3497635
17
rof
modulator of Rho-dependent transcription termination
213932
213678
18
19
20
yaeP
yaeQ
yaeJ
conserved hypothetical protein
conserved hypothetical protein
conserved hypothetical protein
214125
214291
214833
213925
214836
215255
1068933
1070282
3664159
3665418
3251325
3251923
Functional category
Amino acid biosynthesis: Aspartate family
Amino acid biosynthesis: Glutamate family
Amino acid biosynthesis: Glutamate family
Amino acid biosynthesis: Serine family
Biosynthesis of cofactors, prosthetic groups, and carriers: Biotin
Biosynthesis of cofactors, prosthetic groups, and carriers: Heme,
porphyrin, and cobalamin
Biosynthesis of cofactors, prosthetic groups, and carriers:
Menaquinone and ubiquinone
Biosynthesis of cofactors, prosthetic groups, and carriers:
Molybdopteri
Biosynthesis of cofactors, prosthetic groups, and carriers: Other
Biosynthesis of cofactors, prosthetic groups, and carriers:
Pantothenate and coenzyme A
Biosynthesis of cofactors, prosthetic groups, and carriers:
Thiamine
Biosynthesis of cofactors, prosthetic groups, and carriers:
Thiamine
Cell envelope: Biosynthesis and degradation of surface
polysaccharides and lipopolysaccharides
Cell envelope: Biosynthesis and degradation of surface
polysaccharides and lipopolysaccharides
Cell envelope: Biosynthesis and degradation of surface
polysaccharides and lipopolysaccharides
Cell envelope: Biosynthesis and degradation of surface
polysaccharides and lipopolysaccharides
Cell envelope: Biosynthesis and degradation of surface
polysaccharides and lipopolysaccharides
Cell envelope: Other
Cell envelope: Other
Cell envelope: Other
5
21
nlpE
lipoprotein involved with copper homeostasis and adhesion
215269
215979
Cell envelope: Other
22
23
cysD
iap
sulfate adenylyltransferase, subunit 2
aminopeptidase in alkaline phosphatase isozyme conversion
2874985
2875237
2874077
2876274
Cell envelope: Other
Cell envelope: Other
24
25
26
27
28
gltP
yjcO
fdhF
nrfD
nrfE
4298071
4300604
4302845
4294066
4295102
4299384
4299915
4300698
4295022
4296760
Cell envelope: Other
Cell envelope: Other
Cell envelope: Other
Cellular processes: Chemotaxis and motility
Cellular processes: Chemotaxis and motility
29
nrfF
4296753
4297136
Cellular processes: Chemotaxis and motility
30
nrfG
4297133
4297729
Cellular processes: Detoxification
31
32
33
34
potA
pepT
fdnG
fdnH
1187171
1187421
1549115
1552175
1186035
1188647
1552162
1553059
Cellular processes: Pathogenesis
Cellular processes: Toxin production and resistance
Cellular processes: Toxin production and resistance
Central intermediary metabolism: Amino sugars
35
36
37
38
39
40
41
42
rpoB
rplL
eco
cdsA
yaeL
yaeT
yagQ
yagR
3455436
3456121
2307239
195677
196546
197928
297950
300158
3451408
3455756
2307727
196534
197898
200360
296994
297960
Central intermediary metabolism: Nitrogen metabolism
Central intermediary metabolism: Nitrogen metabolism
Central intermediary metabolism: Other
Central intermediary metabolism: Other
Central intermediary metabolism: Sulfur metabolism
DNA metabolism: Degradation of DNA
DNA metabolism: DNA replication, recombination, and repair
DNA metabolism: DNA replication, recombination, and repair
43
44
frvR
hyfF
glutamate/aspartate:proton symporter
conserved hypothetical protein
formate dehydrogenase-H, selenopolypeptide subunit
formate-dependent nitrite reductase, membrane subunit
heme lyase (NrfEFG) for insertion of heme into c552,
subunit NrfE
heme lyase (NrfEFG) for insertion of heme into c552,
subunit NrfF
heme lyase (NrfEFG) for insertion of heme into c552,
subunit NrfG
polyamine transporter subunit
peptidase T
formate dehydrogenase-N, alpha subunit, nitrate-inducible
formate dehydrogenase-N, Fe-S (beta) subunit, nitrateinducible
RNA polymerase, beta subunit
50S ribosomal subunit protein L7/L12
ecotin, a serine protease inhibitor
CDP-diglyceride synthase
zinc metallopeptidase
conserved hypothetical protein
conserved hypothetical protein
predicted oxidoreductase with molybdenum-binding
domain
predicted regulator
hydrogenase 4, membrane subunit
3546826
2605573
3548574
2607153
45
hyfG
hydrogenase 4, subunit
2607143
2608810
Energy metabolism: Amino acids and amines
Energy metabolism: Biosynthesis and degradation of
polysaccharides
Energy metabolism: Biosynthesis and degradation of
polysaccharides
6
46
rpsG
30S ribosomal subunit protein S7
4166335
4166874
47
fusA
protein chain elongation factor EF-G
4166902
4169016
48
chiA
periplasmic endochitinase
4170563
4173256
49
50
51
52
53
bfd
bfr
yjeE
amiB
glnD
bacterioferritin-associated ferredoxin
bacterioferritin, iron storage and detoxification protein
ATPase with strong ADP affinity
N-acetylmuramoyl-l-alanine amidase II
uridylyltransferase
4173425
4173691
4400265
4400745
188650
4173619
4174167
4400726
4402082
185978
Energy metabolism: Biosynthesis and degradation of
polysaccharides
Energy metabolism: Biosynthesis and degradation of
polysaccharides
Energy metabolism: Biosynthesis and degradation of
polysaccharides
Energy metabolism: Electron transport
Energy metabolism: Electron transport
Energy metabolism: Electron transport
Energy metabolism: Electron transport
Energy metabolism: Electron transport
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
map
methionine aminopeptidase
predicted defective phage integrase
flagellar basal-body component
flagellar basal-body MS-ring and collar protein
adhesin
conserved hypothetical protein
predicted class II aldolase
conserved hypothetical protein
predicted DNA-binding transcriptional regulator
ornithine carbamoyltransferase 2, chain F
IS1 transposase InsAB
IS1 repressor protein InsA
valyl-tRNA synthetase
DNA polymerase III, chi subunit
aminopeptidase A, a cyteinylglycinase
predicted peptidase
predicted porin protein
methyl-accepting chemotaxis protein II
transaldolase B
189506
2014639
2015151
2015366
2047048
2860991
2862249
2862892
288386
289529
290376
290570
4488517
4488960
4490631
1576916
1579333
1974405
8238
188712
2014917
2014837
2017024
2054151
2862157
2862887
2863668
287628
288525
289873
290295
4485662
4488517
4489120
1574121
1576961
1972744
9191
Energy metabolism: Electron transport
Energy metabolism: Electron transport
Energy metabolism: Electron transport
Energy metabolism: Electron transport
Energy metabolism: Electron transport
Energy metabolism: Electron transport
Energy metabolism: Electron transport
Energy metabolism: Electron transport
Energy metabolism: Electron transport
Energy metabolism: Electron transport
Energy metabolism: Fermentation
Energy metabolism: Fermentation
Energy metabolism: Other
Energy metabolism: Pentose phosphate pathway
Energy metabolism: Pentose phosphate pathway
Energy metabolism: Pentose phosphate pathway
Energy metabolism: Sugars
Fatty acid and phospholipid metabolism: Biosynthesis
Fatty acid and phospholipid metabolism: Degradation
fliE
fliF
yeeJ
ygbK
ygbL
ygbM
yagI
argF
insB
insA
valS
holC
pepA
pqqL
yddB
tar
talB
7
73
74
75
76
77
78
79
80
81
82
83
84
85
predicted molybdochelatase
Rac prophage region hypothetical protein
IS5 transposase and trans-activator
ECK1366:JW5904:b1371
predicted tail fiber protein
galactitol-specific enzyme IIA component of PTS
IS5 element protein
galactitol-specific enzyme IIA component of PTS
predicted transporter
(alpha)-aspartyl dipeptidase
23S rRNA pseudouridine synthase
IS3 element protein InsF
transposase InsF for insertion sequence IS3A/B/C/D/E/fA
9306
1429172
1430440
1430528
1430763
2177052
2177051
2178383
4231321
4233732
4233944
566361
2172669
9893
1430698
1429460
1430698
1434125
2176732
2178031
2178069
4232952
4233043
4234816
567227
2173535
86
galactitol utilization operon repressor
2173864
2173532
87
galactitol-1-phosphate 5-dehydrogenase
2175010
2173970
88
cyclic diguanylate phosphodiesterase (EAL) domain protein
4279061
4280647
89
regulatory protein SoxS
4280973
4280650
redox-sensitive transcriptional activator SoxR
4281059
4281523
inner membrane transport protein
7959
6529
8238
9191
90
mog
insH
stfR
gatA
insH
gatA
yjbB
pepE
yjbC
insF
soxR
91
92
tal
transaldolase
93
hemN
oxygen-independent coproporphyrinogen III oxidase
3584636
3583263
94
hypothetical protein
3584635
3584751
95
protein YihI
3585334
3584825
Hypothetical proteins: Conserved
Hypothetical proteins: Conserved
Hypothetical proteins: Conserved
Hypothetical proteins: Conserved
Hypothetical proteins: Conserved
Hypothetical proteins: Conserved
Hypothetical proteins: Domain
Hypothetical proteins: Domain
Hypothetical proteins: Domain
Hypothetical proteins: Domain
Hypothetical proteins: Domain
Hypothetical proteins: Domain
Mobile and extrachromosomal element functions: Transposon
functions
Mobile and extrachromosomal element functions: Transposon
functions
Mobile and extrachromosomal element functions: Transposon
functions
Mobile and extrachromosomal element functions: Transposon
functions
Mobile and extrachromosomal element functions: Transposon
functions
Mobile and extrachromosomal element functions: Transposon
functions
Protein fate: Degradation of proteins, peptides, and
glycopeptides
Protein fate: Degradation of proteins, peptides, and
glycopeptides
Protein fate: Degradation of proteins, peptides, and
glycopeptides
Protein fate: Degradation of proteins, peptides, and
glycopeptides
Protein fate: Degradation of proteins, peptides, and
glycopeptides
8
96
maltodextrin phosphorylase
4087943
4090336
4090346
4166335
4166902
1782115
1783109
43188
4092430
4166874
4169016
1783053
1784398
44129
Protein fate: Degradation of proteins, peptides, and
glycopeptides
Protein fate: Other
Protein fate: Protein and peptide secretion and trafficking
Protein fate: Protein and peptide secretion and trafficking
Protein fate: Protein modification and repair
Protein fate: Protein modification and repair
Protein synthesis: Ribosomal proteins: synthesis and modification
44180
45466
Protein synthesis: Ribosomal proteins: synthesis and modification
transcriptional regulator
288386
287628
Protein synthesis: Ribosomal proteins: synthesis and modification
ornithine carbamoyltransferase
conserved hypothetical protein
insertion element IS1 2/3 protein InsB
inosine-guanosine kinase
inner membrane protein YbaL
289529
289629
290376
499349
502462
288525
289862
289873
500653
500786
97
98
99
100
101
102
malQ
rpsG
fusA
4-alpha-glucanotransferase
ribosomal protein S7
translation elongation factor G
protein FixB
protein FixC
protein FixB
103
gidA
glucose inhibited division protein A
104
105
106
107
108
109
argF
110
glycerol-3-phosphate regulon repressor
4079810
4080568
111
112
113
114
115
gspL
gspK
dapD
4082148
4082337
4176494
4177492
185947
4080550
4083563
4175331
4176509
185123
116
117
118
119
120
glnD
nifJ
abgR
ydaL
ydaM
188650
1442498
1406455
1407693
1409509
185978
1438974
1407363
1408256
1408277
Regulatory functions: Other
Regulatory functions: Other
Regulatory functions: Other
Regulatory functions: Protein interactions
Regulatory functions: Protein interactions
121
122
yehA
yehB
transcriptional regulatory protein RtcR
protein RtcB
general secretion pathway protein L
general secretion pathway protein K
2,3,4,5-tetrahydropyridine-2,6-dicarboxylate Nsuccinyltransferase
protein-P-II uridylyltransferase
pyruvate:ferredoxin (flavodoxin) oxidoreductase
predicted DNA-binding transcriptional regulator
conserved hypothetical protein
predicted diguanylate cyclase, GGDEF domain signalling
protein
predicted fimbrial-like adhesin protein
predicted outer membrane protein
Protein synthesis: Translation factors
Protein synthesis: Translation factors
Protein synthesis: tRNA aminoacylation
Protein synthesis: tRNA and rRNA base modification
Purines, pyrimidines, nucleosides, and nucleotides: Nucleotide
and nucleoside interconversions
Purines, pyrimidines, nucleosides, and nucleotides: Salvage of
nucleosides and nucleotides
Regulatory functions: DNA interactions
Regulatory functions: DNA interactions
Regulatory functions: DNA interactions
Regulatory functions: DNA interactions
Regulatory functions: Other
2191748
2194244
2190714
2191764
Regulatory functions: Small molecule interactions
Transcription: Degradation of RNA
9
123
124
125
126
yiaM
yiaL
yiaK
glmU
predicted transporter
conserved hypothetical protein
2,3-diketo-L-gulonate dehydrogenase, NADH-dependent
fused N-acetyl glucosamine-1-phosphate uridyltransferase
and glucosamine-1-phosphate acetyl transferase
L-glutamine:D-fructose-6-phosphate aminotransferase
3896087
3896672
3897682
3721481
3895614
3896205
3896684
3722851
3723013
3724842
439401
437539
440325
440567
440773
439426
440325
442221
Transport and binding proteins: Anions
Transport and binding proteins: Anions
Transport and binding proteins: Anions
acrB
msrA
1-deoxyxylulose-5-phosphate synthase, thiamine-requiring,
FAD-requiring
geranyltranstransferase
exonuclease VII small subunit
sulfurtransferase required for thiamine and 4-thiouridine
biosynthesis
multidrug efflux system protein
methionine sulfoxide reductase A
Transcription: DNA-dependent RNA polymerase
Transcription: Other
Transcription: Transcription factors
Transport and binding proteins: Amino acids, peptides and
amines
Transport and binding proteins: Amino acids, peptides and
amines
Transport and binding proteins: Anions
127
glmS
128
dxs
129
130
131
ispA
xseB
thiI
132
133
483627
4446856
480478
4446218
134
hda
ATPase regulatory factor involved in DnaA inactivation
2617477
2616731
135
uraA
uracil transporter
2618816
2617527
136
upp
uracil phosphoribosyltransferase
2619528
2618902
137
pgm
phosphoglucomutase
713980
715620
138
ybfP
lipoprotein, putative
715834
716328
139
ybfG
hypothetical protein
716779
716369
140
napF
2306831
2306337
141
malP
ferredoxin-type protein, predicted role in electron transfer
to periplasmic nitrate reductase (NapA)
maltodextrin phosphorylase
4087943
4090336
142
malQ
4-alpha-glucanotransferase
4090346
4092430
Transport and binding proteins: Anions
Transport and binding proteins: Carbohydrates, organic alcohols,
and acids
Transport and binding proteins: Carbohydrates, organic alcohols,
and acids
Transport and binding proteins: Carbohydrates, organic alcohols,
and acids
Transport and binding proteins: Carbohydrates, organic alcohols,
and acids
Transport and binding proteins: Carbohydrates, organic alcohols,
and acids
Transport and binding proteins: Cations and iron carrying
compounds
Transport and binding proteins: Cations and iron carrying
compounds
Transport and binding proteins: Cations and iron carrying
compounds
Transport and binding proteins: Cations and iron carrying
compounds
Transport and binding proteins: Nucleosides, purines and
pyrimidines
10
143
cysM
cysteine synthase B
2545029
2544118
144
145
146
147
148
149
150
151
152
153
154
155
156
pstS
pstC
pstA
pstB
phoU
thiL
pgpA
yajO
yajL
panE
gntT
gntY
gntX
3725156
3726283
3727242
3728315
3729103
434858
435813
437359
442865
443739
4093857
4094792
4095534
3726196
3727242
3728132
3729088
3729828
435835
436331
436385
442275
442828
4092541
4094217
4094851
157
158
159
160
bioH
yhgA
yhgG
feoB
phosphate transporter subunit
phosphate transporter subunit
phosphate transporter subunit
phosphate transporter subunit
DNA-binding transcriptional regulator
thiamin-monophosphate kinase
phosphatidylglycerophosphatase A
predicted oxidoreductase, NAD(P)-binding
conserved hypothetical protein
2-dehydropantoate reductase, NADPH-specific
gluconate transporter, high-affinity GNT I system
predicted gluconate transport associated protein
gluconate periplasmic binding protein with
phosphoribosyltransferase domain, GNT I system
carboxylesterase of pimeloyl-CoA synthesis
predicted transposase
predicted DNA-binding transcriptional regulator
fused ferrous iron transporter, protein B
Transport and binding proteins: Nucleosides, purines and
pyrimidines
Transport and binding proteins: Other
Transport and binding proteins: Porins
Transport and binding proteins: Unknown substrate
Unclassified: Role category not yet assigned
Unclassified: Role category not yet assigned
Unclassified: Role category not yet assigned
Unclassified: Role category not yet assigned
Unclassified: Role category not yet assigned
Unclassified: Role category not yet assigned
unknown
unknown
unknown
Unknown function: Enzymes of unknown specificity
4095572
4097249
4097688
4100009
4096342
4096371
4097452
4097688
Unknown function: General
Unknown function: General
Unknown function: General
Unknown function: General
11
Western blot analysis
Biological replicate 1
A
Control ptsA
malQ
RseP
rffE
dxs
M
g2
Biological replicate 2
B
Total density
Density Measurements of Glycosylated AcrA using
Lectin Probed Western Blots
160000
140000
120000
100000
80000
60000
40000
20000
0
Control (empty vector) PtsA
MalQ
RseP
RffE
g1
Dxs
Genes
Biological replicate 1
C
Control ptsA
malQ RseP
rffE
dxs
Biological replicate 2
Density Measurements of Glycosylated AcrA
using Anti-his C-term Probed Western Blots
D
500000
Total Density
400000
300000
200000
100000
0
Control
ptsA
malQ
RseP
rffE
dxs
Genes
12
AcrA Glycosylation efficiencies estimated using
Anti-his C-term Probed Western Blots
Glycosylation efficiency %
E
50.0
45.0
40.0
35.0
30.0
25.0
20.0
15.0
10.0
5.0
0.0
Control
ptsA
malQ
RseP
rffE
dxs
Genes
FIGURE S3. AcrA protein produced in engineered cells was purified by his-tag chromatography and
probed by western blotting using methods described previously (Pandhal et al. 2011a) A) Lectin
peroxidase blot (specific for the glycan present on AcrA). The top band is di-glycosylated protein, the
bottom band is mono-glycosylated protein, B) Quantifications from lectin peroxidase western blot
analysis based on pixel size and intensity using ImageQuant™ IQTL Software (GE Healthcare)
(Pandhal et al. 2011a). C) Anti-his C-term antibody (Life Technologies) blot. The top band is diglycosylated protein, the middle band is mono-glycosylated protein and the bottom band is
aglycosylated protein. D) Quantifications from anti-his C-term western blot analysis based on pixel
size and intensity using ImageQuant™ IQTL Software. E) Glycosylation efficiency calculations based
on anti-his C term western blots (Pandhal et al. 2011a).
13
A
1
Biological replicate
2
1
Control
Control
1
2
dxs
Density Measurements of Glycosylated CH2 using
Anti-his C-term Probed Western Blots
B
Total Density
2
ptsA
160000
140000
120000
100000
80000
60000
40000
20000
0
Control
ptsA
dxs
Genes
C
Glycosylation efficiency %
CH2 Glycosylation efficiencies estimated using
Anti-his C-term Probed Western Blots
40.0
35.0
30.0
25.0
20.0
15.0
10.0
5.0
0.0
Control
ptsA
dxs
Genes
FIGURE S4. Human IgG Fc fragment (CH2) produced in engineered cells was purified by his-tag
chromatography and probed during western blotting with the anti-his C-term antibody using methods
described previously (Pandhal et al. 2011a). A) Western blot image. The top band is glycosylated
protein and bottom band is aglycosylated protein. B) Quantifications from western blot analysis based
on pixel size and intensity using ImageQuant™ IQTL Software (Pandhal et al. 2011a). C)
Glycosylation efficiency calculations based on anti-his C term western blot
14
2.5
2
Western 1.5
blot fold
change
1
0.5
0
0
1
2
3
pSRM fold change
FIGURE S5. A correlation of glycosylation efficiency fold changes calculated using western blots
(anti-his C-term antibody) and pSRM. Fold changes were calculated by comparing glycosylation
efficiency in E. coli phenotypes to control cells.
15
Growth Curves
0.9
A
0.8
0.7
O.D. (600nm)
0.6
AcrA pgl Control
0.5
AcrA pgl pts
0.4
AcrA pgl malQ
0.3
AcrA pgl rseP
0.2
AcrA pgl dxs
0.1
AcrA pgl rffE
0
0
50
100
150
200
250
Time (mins)
0.0250
0.0200
0.0150
0.0100
0.0050
0.0000
B
AcrA pgl AcrA pgl pts AcrA pgl
Control
malQ
AcrA pgl AcrA pgl dxsAcrA pgl rffE
rseP
E. coli phenotypes
FIGURE S6. Growth data for E. coli phenotypes. A) Growth curves for E. coli phenotypes where the
grey shaded area indicates the period when protein production is induced. B) Growth rates for the E.
coli phenotypes.
16
Absolute quantitation
Unknown sample
Isotope standard
•
15
14
•
Quantified N
AcrA protein and
AcrA glycoprotein
1
Non-quantified N
AcrA protein and
AcrA glycoprotein
2
SDS-PAGE
separation
Trypsin
3
digestion
pSRM
4
14
N glycopeptide
from engineered
cell
15
N glycopeptide
from standard
AVFDNN*NSTLLPGAFATITSEGFIQK
5
Glycan reporter ions
Peptide b & y ions
Total protein (X) = area under non-modifiable
14
N peptide/ area under non-
15
modifiable N peptide
Glycoprotein (Y) = area under
14
15
N glycopeptide/ area under N glycopeptide
Glycosylation efficiency (Z) = X/Y*100
6
17
FIGURE S7. The workflow for absolute quantitation of both glycosylated and aglycosylated peptides
using pseudo selective reaction monitoring (pSRM). 1. A known quantified amount of 15N labelled
protein consisting of glycopeptides and non-modifiable peptides is prepared using techniques
described in the methods section. This is mixed with an unknown amount of the same mixture
prepared from the engineered E. coli cells. 2. The protein mixture is loaded onto SDS-PAGE gels
where the isotopic and non-isotopic protein is indistinguishable. 3. The protein band at the correct
molecular weight is excised and in-gel digestion with trypsin is performed. 4. Total ion count targeting
glycopeptides m/z 1041.4 (14N-labelled) and 1051.4 (15N-labelled) that co-elute. Non-modifiable
peptides are also targeted in the same run. The inset shows the area under eluting peptide peaks. 5.
Transition ions for peptide m/z 1041.4 confirm the peptide identity (red circles). The reporter ions for
glycans and b and y ions for peptide fragments are labelled. 6. Total protein quantity is estimated from
non-modifiable peptide ratio (14N/15N) (Agarda et al. 2012; Arnott et al. 2002; Lehmann et al. 2008;
Pandhal et al. 2011a; Singh et al. 2012; Zhang et al. 2011).
18
Ionisation efficiency of glycopeptides
1
Dc
2
3
4
19
FIGURE S8. Ionisation efficiency of both normal and heavy labelled glycosylated peptide (m/z 1041.4
and 1051.1, respectively) was tested using a 1:1 mix of protein sample which was digested with
trypsin prior to pseudo selective reaction monitoring (pSRM) on a high capacity ion trap. 1. Total ion
count from pSRM for m/z 1041.4 (dark line) and 1051.2 (light line). The inset shows the area under
m/z 1041.4 and 1051.2 eluting peaks at 34.2 min. 2. Extracted ion chromatogram for transition ion
m/z 407 from peptide m/z 1041.4 (dark line) and extracted ion chromatogram for transition ion m/z
409 from peptide m/z 1051.2 (light line). 3. MS/MS spectrum from pSRM target m/z 1041.4 4. MS/MS
spectrum from pSRM target m/z 1051.2.
20
FIGURE S9. Glycan structure confirmation. A. Glycan structure originally characterised in
Campylobacter jejuni (Young et al. 2002). B. Mass spectrum showing fragmentation ions for
glycopeptide ATFENASKDFNR. Both 2+ and 3+ species of the glycosylated tryptic peptide
are evident (Pandhal et al. 2011b). C. Mass spectrum showing fragmentation ions for
glycopeptide DFNR. 1+ Y ion species show glycan structure as indicated in part A. The
glycan attachment site on asparagine (N) is underlined.
21
References
Agarda NJ, Mahrusa S, Trinidada JC, Lynna A, Burlingamea AL, Wellsa JA. 2012. Global kinetic analysis
of proteolysis via quantitative targeted proteomics. Proceedings of National Acadamy of
Sciences.
Arnott D, Kishiyama A, Luis EA, Ludlum SG, Marsters JC, Jr., Stults JT. 2002. Selective detection of
membrane proteins without antibodies: a mass spectrometric version of the Western blot.
Mol Cell Proteomics 1(2):148-56.
Lehmann U, Wienkoop S, Tschoep H, Weckwerth W. 2008. If the antibody fails--a mass western
approach. Plant J. 55(6):1039-46.
Pandhal J, Noirel J, Ow S, Wright PC. 2011a. Improving N-glycosylation efficiency in Escherichia coli
using shotgun proteomics, metabolic network analysis and selective reaction monitoring.
Biotechnol. Bioeng. 108(4):902-912.
Pandhal J, Ow SY, Noirel J, Wright PC. 2011b. Improving N-glycosylation efficiency in Escherichia coli
using shotgun proteomics, metabolic network analysis, and selective reaction monitoring.
Biotechnol. Bioeng. 108(4):902-912.
Singh P, Batth TS, Juminaga D, Dahl RH, Keasling JD, Adams PD, Petzold CJ. 2012. Application of
targeted proteomics to metabolically engineered Escherichia coli. Proteomics 12(8):1289-99.
Young NM, Brisson JR, Kelly J, Watson DC, Tessier L, Lanthier PH, Jarrell HC, Cadotte N, St Michael F,
Aberg E and others. 2002. Structure of the N-linked glycan present on multiple glycoproteins
in the Gram-negative bacterium, Campylobacter jejuni. J. Biol. Chem. 277(45):42530-9.
Zhang H, Liu Q, Zimmerman LJ, Ham AJ, Slebos RJ, Rahman J, Kikuchi T, Massion PP, Carbone DP,
Billheimer D and others. 2011. Methods for peptide and protein quantitation by liquid
chromatography-multiple reaction monitoring mass spectrometry. Mol Cell Proteomics
10(6):M110 006593.
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