Marc D. Driessen 1 , Sarah Mues 2
,
Antje Vennemann 3 , Bryan Hellack 4 , Anne Bannuscher 1 ,
Vishalini Vimalakanthan 1,6 , Christian Riebeling 1 , Rainer Ossig 2 , Martin Wiemann 3 , Jürgen
Schnekenburger 2 , Thomas A. J. Kuhlbusch 4,5 , Bernhard Renard 6 , Andreas Luch 1 and Andrea
Haase 1*
1 German Federal Institute for Risk Assessment (BfR), Department of Chemicals and Product
Safety, Berlin, Germany,
2 Biomedical Technology Center, Westfälische Wilhelms-University, Münster, Germany,
3 IBE R&D gGmbH, Institute for Lung Health, Münster, Germany,
4 Institute of Energy and Environmental Technology (IUTA) e.V., Air Quality & Sustainable
Nanotechnology, Duisburg, Germany,
5 Center for Nanointegration CENIDE, University of Duisburg-Essen, Duisburg, Germany,
6 Robert-Koch-Institut (RKI), Junior Research Group Bioinformatics, Berlin, Germany.
*Corresponding author
1

Figure S1: Kinetics of protein carbonylation in NRK-52E cells for different SiO
2
NPs
NRK52E cells were incubated with 10 µg/mL (3.4 µg/cm 2 ) for the indicated time period (0, 2h, 5h, 8h).
Carbonylation was detected in the cellular lysates after coupling the modified proteins with dinitrophenyl-hydrazine (DNPH) using a specific DNP antibody in immunoblots. Normalization was performed with tubulin signals. All experiments were performed in at least three independent biological repeats. A typical result for each NP is depicted here.
2

Figure S2: Immunoblots of all positive NPs for protein carbonylation in NRK-52E cells.
NRK-52E cells were incubated with the respective NP in the indicated concentration (0 to up to 100
 g/ml (34 µg/cm 2 ), in case of strong cytotoxicity the concentration was adjusted accordingly) for 6h.
Carbonylation was detected in the cellular lysates after coupling the modified proteins with dinitrophenyl-hydrazine (DNPH) using a specific DNP antibody in immunoblots. Normalization was performed with tubulin signals. All experiments were performed in at least three independent biological repeats. A typical result for each NP is depicted here.
3

kD:
97
68
43
29
ZrO
2
Acryl
0 S 5 10 25 50 kD:
97
68
43
ZrO
2
PEG
0 S 5 10 25 50
29 kD:
97
68
43
ZrO
2
Amino
0 S 5 10 25
29
µg/mL
An -DNP
An -Tub kD:
97
68
43
ZrO
2
TODS
0 S 5 10 25 50 kD:
97
68
SiO
2
PEG
0 S 5 10 25 50 kD:
97
68
Ag 200 PVP
0 S 5 10 25 µg/mL
An -DNP
43
29
43
29
29
An -Tub kD:
100
MWCNT NM -400
0 5 15 25 35 50 kD:
100
MWCNT NM -401
0 5 15 25 35 50 kD:
100
50
MWCNT NM -402
0 5 15 25 35 50
µg/mL
50
37
37
25
An -DNP
25
50
37
25
An -Tub
AlOOH kD: 0 5 10 25 50 100
µg/mL
97
68
An -DNP
43
29
An -Tub
Figure S3: Immunoblots of all negative NPs for protein carbonylation in NRK-52E cells.
NRK-52E cells were incubated with the respective NP in the indicated concentration (0 to up to 100
 g/ml (34 µg/cm 2 )) for 6h. Carbonylation was detected in the cellular lysates after coupling the modified proteins with dinitrophenyl-hydrazine (DNPH) using a specific DNP antibody in immunoblots.
Normalization was performed with tubulin signals. All experiments were performed in at least three independent biological repeats. A typical result for each NP is depicted here.
4

Figure S4: 2D immunoblots for all positive NPs
NRK-52E cells were incubated with the respective NP at 10
 g/ml (3.4 µg/cm 2 ). Carbonylated proteins were labeled by using dinitrophenyl-hydrazine (DNPH) after separation in first dimension. Labeled proteins were detected in 2D immunoblots using an anti-DNP antibody. All experiments were performed in at least three independent biological repeats. A typical result for each NP is depicted here.
5

Figure S5: Identified modified proteins
Depicted is one corresponding duplicate gel image of a untreated control sample, which were run in parallel to the 2D immunoblots shown in Supplementary Figure S4 visualizing all cellular proteins not only the modified ones of the 2D immunoblots. Modified proteins as identified in 2D immunoblots were excised from the duplicate gels and identified by mass spectrometry. All proteins that could be identified are marked in this image with green marks. Yellow labels (roman numbers) refer to proteins, which are identified only on basis of the Mascot score and 1 MS/MS (i.e. likely identified). Protein identities are given in Table S1.
6

Table S1. Proteomic identification assigned to the respective NP treatment. Column 1 refers to the spot number (Figure S5). Column 2 gives additional information. Single means the respective spot could be clearly identified on the duplicate gel by transferring the spot from the 2D blot, multiple means that several spots had to be considered (see Materials and Methods). Columns 3 - 8 list identification parameters. Each of the following columns refers to a NP treatment while x means the spot had similar intensity after NP treatment compared to control, xx and xxx indicating stronger intensities than in control).
7

Identifica tion x x x x x x x x x x x x x x x tbc x x x x x x x x x x x x x x x x x x x x
Spots defining PCA
1st Dimension
5th Dimension
2nd Dimension
2nd Dimension
5th Dimension
2 3
2 3
2 4
2 5
2 6
2 7
2 0
2 1
2 2
14
15
16
17
18
19
2 0
2 8
2 8
2 9
3 0
3 1
6
6
7
4
5
1
2
2
3
10
11
12
13
8
9
S po t num b.
s po t t ype
UniP R O t ID single single single single
E F 2 _ R A T
E F 2 _ R A T
F UB P 2 _ R A T
T E R A _ R A T multiple F UB P 2 _ R A T single H S P 7 C _ R A T multiple multiple single
T C P G _ R A T
LM N A _ R A T
F UB P 1_ R A T multiple H S P 7 C _ R A T multiple S T IP 1_ R A T single single single single
F UB P 1_ R A T
T KT _ R A T
C A LR _ R A T
KP Y M _ R A T single single single single single multiple single single single single multiple single single single multiple single
C H 6 0 _ R A T
T C P E _ R A T
KP Y M _ R A T
N UC B 1_ R A T
P D IA 1_ R A T
T B A 1B _ R A T
T B A 1B _ R A T
T B A 1C _ R A T
C A P 1_ R A T
A T P A _ R A T
P D IA 1_ R A T
V IM E _ R A T
N H R F 1_ R A T
A C T B _ R A T
S E P 11_ R A T
E N O A _ R A T multiple P D IA 6 _ R A T multiple H N R P F _ R A T single multiple single
C A P G _ R A T
P G K1_ R A T
P G K1_ R A T
49,9
51,6
59,7
56,9
53,7
38,8
41,7
49,7
47,1
60,9
59,5
57,8
53,5
56,9
50,1
50,1
48,1
45,7
38,8
44,5
44,5
74,2
70,8
60,6
74,3
67,2
95,2
95,2
74,2
89,3
70,8
62,5
67,2
67,6
48
57,8
M W
[ k D a ] pI
301,7
102,6
491,5
94,0
571,9
67,7
537,7
75,0
108,9
310,9
601,7
33,9
198,2
954,5
153,8
250,5
469,9
143,0
33,8
276,1
318,8
273,8
143,1
137,4
137,0
134,2
1029,3
108,9
116,0
49,0
670,4
106,4
135,4
174,1
98,5
552,1
4,8
5,1
5,7
5,3
6,2
6,2
5
7,2
9,2
5,9
5,5
6,6
5
4,8
4,9
4,9
5
5,3
6,1
8
8
6,4
5,4
6,2
7
7,2
6,4
6,4
6,4
5,1
5,4
6,4
7,2
7,2
4,3
6,6
S c o re
# o f pe pt ide s
S C [ %]
N o t re a t m .
S iO 2
Unm o d.
S iO 2
A m ino
S iO 2
P ho s ph.
S iO 2
N M - 2 0 0
S iO 2
N M - 2 0 3
A g 5 0
P V P
A g 5 0
C it ra t e
A g
N M - 3 0 0
2
9
2
3
1
9
4
3
11
1
4
19
3
12
4
4
1
5
6
8
3
2
4
2
3
16
5
2
2
4
3
5
3
13
12
2
8,9
4
6,2
6,1
6,5
25,2
4
8,1
3,4
22,8
4,1
5,5
11,9
6,5
33,7
13,1
13,9
31,1
3,7
30,7
11
34,9
4,6
6,5
9,4
28,3
3
11,5
40,5
13,1
13,1
25,2
13,7
3,7
20,6
23,7 x x x x x x x x x x x xxx x x x x x x xxx xx xx x x x x x x x x xxx x x x x x x x xxx x x x x x xx xx x x x xx xxx x x x x x x x x xx x x xxx xxx x x x x x x x x x x x x x x x x x x x x x x x x x x x x xx x x xx x x x x x xx x x x x x x x x x x x xx x x xx xx x xx x x x x x x x x x x x xx xx x x x x x x x x
T iO 2
N M - 10 5
Z nO
N M - 111
Z nO
N M - 110
S t irre d x x x x+/ x x x x x x
Z nO
N M - 110
Ult ra s .
x x x x x x x x x x x xx xx x x x x x x x x xx x x x xx x x x x x x x x x x x x x x x x x x x x x x x x x x x x x
8

Identifica tion x x x x x x x x x x x x x x x x x x x x x x x x x x x x tbc x x x x x x
Spots defining PCA
4th Dimension
1st Dimension
1st Dimension
3rd Dimension
5th Dimension
2nd Dimension
4 9
5 0
5 1
5 2
4 7
4 8
4 8
4 4
4 5
4 5
4 6
4 1
4 2
4 3
5 5
5 5
5 6
5 7
5 2
5 3
5 4
5 8
3 8
3 9
4 0
4 1
3 6
3 7
3 7
3 2
3 3
3 3
3 4
3 4
3 5
S po t num b.
s po t t ype
UniP R O t ID single single single multiple multiple multiple
IF 4 A 2 _ R A T
A C T B _ R A T
A C T G _ R A T
A C T B _ R A T
A C T G _ R A T
D H S O _ R A T multiple multiple multiple
C S N 4 _ R A T
A R G I1_ R A T
T H IC _ R A T multiple single
A N X A 1_ R A T
H N R D L_ R A T multiple multiple
G 3 P _ R A T
A N X A 1_ R A T multiple single single
A K1A 1_ R A T
A LD R _ R A T
C A Z A 2 _ R A T single multiple multiple single
T X N L1_ R A T
G B B 1_ R A T
G B B 2 _ R A T
A N X A 3 _ R A T single G B LP _ R A T multiple A R P C 2 _ R A T multiple G B LP _ R A T single multiple single single
P N P H _ R A T
T H T M _ R A T
G D IR 1_ R A T
P G A M 1_ R A T single single single single single single single multiple
P G A M 2 _ R A T
P G A M 1_ R A T
P S A 6 _ R A T
G S T M 2 _ R A T
R A N _ R A T
T P IS _ R A T
R A N _ R A T
A P T _ R A T
35,1
34,4
35,1
32,3
32,9
23,4
28,8
36,5
35,8
32,9
32,2
37,3
37,3
36,3
28,2
28,8
27,4
25,7
24,4
26,8
24,4
19,5
46,3
35
41,1
38,8
35,3
35,8
38,8
46,4
41,7
41,8
41,7
41,8
38,2
M W
[ k D a ] pI
6,5
5,9
5,1
6,7
7,6
6,8
7,1
4,8
5,6
5,6
6
6,8
6,3
5,6
6,9
7
6,9
7
8,8
6,7
6,3
6,2
7
9,1
8,1
7
5,6
6,8
6,9
5,3
5,3
5,3
5,3
5,3
7,1
40,4
32,7
14,2
15,6
10,8
50,5
42,5
16,3
23,4
18,9
15,6
5
5
33
16,6
46,9
25,2
67,9
17,6
45
37,5
35,6
23,7
5
13,5
32,4
5,4
43
4
7,1
34,4
31,7
53,9
40
31,1
S c o re
# o f pe pt ide s
S C [ %]
N o t re a t m .
S iO 2
Unm o d.
S iO 2
A m ino
S iO 2
P ho s ph.
S iO 2
N M - 2 0 0
501,7
310,5
171,0
137,7
129,2
439,2
381,0
151,6
362,4
151,2
205,2
73,2
73,2
561,6
182,8
345,0
230,0
570,1
212,4
340,2
365,1
247,0
91,1
589,1
63,3
530,3
86,5
265,2
464,1
138,6
650,6
410,0
976,1
849,0
304,2
2
10
7
18
14
7
9
6
3
3
8
7
4
2
10
2
2
5
7
3
7
8
14
3
3
7
5
4
5
9
8
2
2
10
1 x x x x x x x x x x x x x x x x x x xx xx x x x x x xx xx xxx x x x x xx xx x x x xx x x x x x x x x xx x x x x x x x x x x x x x x x x x x x x x x x x x x x x xx x x x x x x xx
S iO 2
N M - 2 0 3
A g 5 0
P V P
A g 5 0
C it ra t e
A g
N M - 3 0 0
T iO 2
N M - 10 5 xx x x xx xx x x x x x x x x x
Z nO
N M - 111 x x x x
Z nO
N M - 110
S t irre d xx x x x x x x x x x x x x x x x x x x x xxx xxx x x x x x x xx x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x xx x x x x x x x x x x x x x x x
Z nO
N M - 110
Ult ra s .
x x x x x x x x x x x x x x x x x x x
9

Identifica tion x x x x x x x x x x x tbc x x x x x x x x x x x x x x x x x x x x x x x
Spots defining PCA
2nd Dimension
1st Dimension
3rd Dimension
7 7
7 7
7 7
7 7
7 5
7 6
7 6
7 1
7 2
7 3
7 4
7 0
7 0
7 1
8 1
8 2
8 3
8 4
7 8
7 9
8 0
8 5
6 7
6 8
6 8
6 9
6 5
6 5
6 6
5 9
6 0
6 1
6 2
6 3
6 4
S po t num b.
s po t t ype
UniP R O t ID single single
M L12 B _ R A T
E Z R I_ R A T multiple multiple
D P Y L2 _ R A T
P D IA 3 _ R A T single T C P B _ R A T multiple H N R H 1_ R A T multiple multiple single
T B A 1C _ R A T
T B B 5 _ R A T
A LD H 2 _ R A T single multiple multiple single multiple multiple multiple
E N O A _ R A T
E N O A _ R A T
O A T _ R A T
E N O A _ R A T
A C T A _ R A T
A C T B _ R A T
G 3 P _ R A T multiple A N X A 2 _ R A T multiple G R P 7 5 _ R A T multiple H S P 7 C _ R A T single V IM E _ R A T single multiple multiple
C A Z A 1_ R A T
A T P B _ R A T
E Z R I_ R A T single single single single single single single
A N X A 1_ R A T
G 3 P _ R A T
R LA 0 _ R A T
A N X A 2 _ R A T
F UB P 2 _ R A T
N D US 1_ R A T
D C 1I2 _ R A T single single single multiple
D C 1I2 _ R A T
G R P 7 8 _ R A T
E F T U_ R A T
C H 6 0 _ R A T multiple T B A 1B _ R A T
32,9
56,3
69,3
38,8
35,8
34,2
38,7
42
41,7
35,8
38,7
73,8
70,8
53,7
74,2
79,4
71,1
71,1
72,3
49,5
60,9
50,1
49,9
49,6
56,5
47,1
47,1
48,3
47,1
19,8
69,3
62,2
56,6
57,4
49,2
M W
[ k D a ] pI
7
8,1
5,9
7,6
5,4
5,2
5,8
7,6
6
5,4
5,1
5,2
5,3
8,1
5,1
5,1
7,2
5,9
6,4
5,6
5,1
4,9
6,2
6,2
6,5
6,2
5
4,8
6,6
4,8
5,8
6
5,9
6
5,7
75,3
969,2
597,9
107,0
93,8
148,0
84,2
78,0
104,7
140,3
84,2
500,4
426,4
998,4
119,5
266,9
138,1
71,4
484,2
144,8
925,6
55,9
132,0
190,4
699,9
175,7
848,4
107,2
169,6
127,2
149,4
87,3
1068,1
417,6
251,5
4
2
2
2
2
17
13
7
22
2
9
1
2
4
3
16
2
8
3
6
3
2
2
3
4
13
3
5
13
6
4
2
19
3
3
9,8
52,9
19,6
11,4
7,4
10,7
7,8
4,2
7,2
17,4
7,4
13,4
13
49,8
5,2
16,7
10
34,7
7,2
11,1
7,5
6,4
9,8
13,5
32,9
15,4
38,9
5,5
9,2
18,6
5,3
5,9
36,8
17,8
18,3
S c o re
# o f pe pt ide s
S C [ %]
N o t re a t m .
S iO 2
Unm o d.
S iO 2
A m ino
S iO 2
P ho s ph.
S iO 2
N M - 2 0 0 x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x xx x x x x x x x x x x x xxx xxx x x x x x x xx x x xxx xx x x x x x x x x x xxx xxx x x x x x x x x x x x x x x x x xx xx x x x x x x x xx x x x x x x
S iO 2
N M - 2 0 3
A g 5 0
P V P
A g 5 0
C it ra t e
A g
N M - 3 0 0
T iO 2
N M - 10 5
Z nO
N M - 111
Z nO
N M - 110
S t irre d x x xx x x x x x x x x xx xxx x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x xx xxx x x xxx x xx x x x x x x
Z nO
N M - 110
Ult ra s .
x x xx x x x x x x x x x x x x x x x x x
10

Identifica tion tbc tbc tbc x x tbc tbc tbc x tbc tbc tbc tbc tbc tbc tbc tbc tbc tbc tbc tbc x x x x x tbc x x x x x x x
Spots defining PCA
3rd Dimension
3rd Dimension
1st Dimension
5th Dimension
5th Dimension
2nd Dimension
3rd Dimension
4th Dimension
3rd Dimension
S po t num b.
s po t t ype
UniP R O t ID multiple multiple single single single multiple multiple multiple multiple single single single single
T B A 1C _ R A T
T B B 5 _ R A T
H S P 7 4 _ R A T
KP Y M _ R A T
T C P A _ R A T
A LD O A _ R A T
P R S 7 _ R A T
N H R F 1_ R A T
A C Y 1A _ R A T
S H 3 G 1_ R A T
KH D R 2 _ R A T
ID H C _ R A T
A C O T 2 _ R A T single single single multiple multiple multiple single multiple multiple single single single single multiple multiple single single multiple
S H LB 1_ R A T
S H LB 1_ R A T
A LD R _ R A T
R B B P 7 _ R A T
P G K1_ R A T multiple C A P R 1_ R A T multiple D LD H _ R A T multiple S E P T 2 _ R A T
E F 2 _ R A T
A L9 A 1_ R A T
R UV B 1_ R A T
D LD H _ R A T
D H E 3 _ R A T
A R P 3 _ R A T
P D IA 6 _ R A T
R O A 2 _ R A T
P S B 7 _ R A T
E N P L_ R A T
A C T A _ R A T
A C T B _ R A T
D C 1I2 _ R A T
10 4
10 4
10 4
10 1
10 2
10 3
9 8
9 9
10 0
9 5
9 6
9 6
9 7
110
111
112
10 5
10 6
10 7
10 8
10 9
9 2
9 3
9 4
9 4
9 0
9 0
9 1
8 7
8 8
8 9
8 5
8 5
8 6
37,5
29,9
92,7
42
41,7
71,1
95,2
53,6
50,2
54
61,4
47,3
48,1
40,8
40,8
35,8
47,8
44,5
78,1
54
41,6
48,5
38,8
45,8
41,5
38,8
46,7
49,7
49,9
49,6
94
57,8
60,3
39,3
M W
[ k D a ] pI
5,2
5,3
5,1
9
8,1
4,7
8,1
5,6
5
6,4
6,6
6
8
5,1
8
6,1
5,8
5,8
6,3
4,9
8
5,4
6
6,5
7,7
5,6
5,7
6
6,6
5,9
8,3
5
4,8
5,1
S c o re
# o f pe pt ide s
S C [ %]
N o t re a t m .
S iO 2
Unm o d.
S iO 2
A m ino
S iO 2
P ho s ph.
S iO 2
N M - 2 0 0
118,6
82,6
63,8
48,1
105,3
41,2
121,0
71,6
46,0
47,0
77,1
42,0
469,9
24,4
24,4
29,7
50,5
106,8
44,9
89,5
464,3
283,6
53,7
378,9
61,6
24,8
184,2
112,5
167,7
218,0
183,8
110,8
201,1
181,3
2
2
1
2
2
2
1
1
8
3
1
1
1
1
2
9
1
1
1
1
2
4
4
2
1
7
1
7
2
4
5
4
5
4
5,6
5,6
3,8
11,3
5,4
2,5
3,8
2,6
25,2
6,3
3
2,4
2,8
2,8
4,9
38,5
3,6
3,6
2,5
2,1
7,7
20,6
3,9
21,8
5,4
2,3
12,6
13
7,3
12,1
19,8
13,1
9
7,3 x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x xx xxx x x x xx x x x x x xxx xxx x x x x x x x x x x xx x x x x x
S iO 2
N M - 2 0 3
A g 5 0
P V P
A g 5 0
C it ra t e
A g
N M - 3 0 0
T iO 2
N M - 10 5 x x x x x x x x xx x x
Z nO
N M - 111 xx x x
Z nO
N M - 110
S t irre d x x x x x x x x xx xx xx x x x x x x xx x x x x x xx xx xx xx x xx x x x xx xx xx x x x xx x x x x x x x x x x x x x x x x x x x x
Z nO
N M - 110
Ult ra s .
x x x x x x x x
11

12