Chemistry & Biology, Volume 22

Supplemental Information

Probes to Monitor Activity of the Paracaspase MALT1

Janna Hachmann, Laura E. Edgington-Mitchell, Marcin Poreba, Laura E. Sanman, Marcin Drag, Matthew Bogyo, and Guy S. Salvesen

Supplemental Information

Supplemental Figures

Figure S1 (related to Figure 1)

LCMS of biotin-LVSR-AOMK

TIC of +Q1: from Sample 54 (biotin-LVSR-AOMK pure) of 1208_Laura.wiff (Turbo Spray) Max. 6.2e8 cps.

1 2 3 4 5 6 7 8 9 10 11 12

Time, min

0.0

1.0e8

2.0e8

3.0e8

4.0e8

5.0e8

6.0e8

Inte

nsity, cps

5.35

12.55

2.45

Channel 1 from Sample 54 (biotin-LVSR-AOMK pure) of 1208_Laura.wiff Max. 0.9 %.

0 1 2 3 4 5 6 7 8 9 10 11 12

Time, min

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

Inte

nsity, %

+Q1: 5.259 to 5.359 min from Sample 54 (biotin-LVSR-AOMK pure) of 1208_Laura.wiff (Turbo Spray) Max. 4.3e6 cps.

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800

m/z, amu

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

4.3e6

Inte

nsity, cps

846.4

714.4423.4

133.1

507.4

408.2

312.1

100.9 1692.5864.8439.31270.0295.5 488.8 696.6620.5 1292.7 1441.7930.8719.6 1533.51073.3 1169.5

Acq. File: 1208_Laura.wiff Sample Name: biotin-LVSR-AOMK pure

Sample Number: N/A

Figure S2 (related to Figure 1)

LCMS of Cy5-LVSR-AOMK

TIC of +Q1: from Sample 50 (Cy5 LVSR AOMK final) of 1208_Laura.wiff (Turbo Spray) Max. 2.3e8 cps.

1 2 3 4 5 6 7 8 9 10 11 12

Time, min

0.0

5.0e7

1.0e8

1.5e8

2.0e8

2.3e8

Inte

nsity, cps

5.81

12.53

9.516.36

9.071.11 2.711.706.992.95 4.66 8.685.48 11.637.16

Channel 1 from Sample 50 (Cy5 LVSR AOMK final) of 1208_Laura.wiff Max. 4.6 %.

0 1 2 3 4 5 6 7 8 9 10 11 12

Time, min

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

Inte

nsity, %

5.78

+Q1: 5.659 to 5.860 min from Sample 50 (Cy5 LVSR AOMK final) of 1208_Laura.wiff (Turbo Spray) Max. 2.3e6 cps.

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800

m/z, amu

2.0e5

4.0e5

6.0e5

8.0e5

1.0e6

1.2e6

1.4e6

1.6e6

1.8e6

2.0e6

2.2e6

Inte

nsity, cps

630.0

1259.2

640.8 1679.1124.3 1574.5839.81126.6

1270.4149.3 371.1 433.1 614.4279.2 1287.0 1519.3686.5 958.6 983.5 1712.0

Acq. File: 1208_Laura.wiff Sample Name: Cy5 LVSR AOMK final

Sample Number: N/A

Figure S3 (related to Figure 2)

The 60 kDa species contains mostly the C-terminal fragment of MALT1 including the caspase-like domain and the Ig3 domain. After mass spectrometry analysis, the frequency with which the different peptides were detected was determined and the amino acids covered by these peptides were calculated. The frequency with which each amino acid was observed in the different peptides was graphed versus the position in full-length MALT1 to determine which part of the protein made up the 60 kDa species. For example, amino acid 41 was discovered in 2 different peptides (encompassing either amino acids 40-52 or 41-52) with a total occurrence of 16. The frequency for this amino acid was thus graphed as 16. DD, death domain; Ig, immunoglobulin-like domain.

0 10 20 30 40 50 60 70 80

Ig1 Ig2DD caspase-like Ig31 824

Freq

uenc

y of

am

ino

acid

det

ectio

n

Figure S4 (related to Figure 3)

MALT1 gets cleaved upon overexpression. MALT1-WT or C464A containing an N- or C-terminal Flag-tag, respectively, were overexpressed in the presence or absence of Bcl10 in HEK-293A cells. Cell lysis was followed by SDS-PAGE and Western blot analysis with the indicated antibodies. Note that when Bcl10 is expressed in combination with MALT1-WT (lanes 1 and 2) a smaller Bcl10 cleavage product below the main species is visible. The size difference between overexpressed (lanes 1-4) and endogenous (lanes 5-8) Bcl10 is explained by the Flag-tag on the Bcl10 construct.

_-MALT1

_-Bcl10

_-`-tubulin

–– + Flag-MALT1-C464A

MALT1-C464A-FlagBcl10

++

–+ +

–

+–

–

kDa

97 -

31 -

97 -

_-Flag

–+ – Flag-MALT1-WT

MALT1-WT-Flag–+–

––

116 -

116 -

–– +

+–

–– –

–

––

––+ –

–+–

––

Supplemental Experimental Procedures

Synthesis of Cy5-LVSR-AOMK and biotin-LVSR-AOMK

All reagents were purchased from commercial suppliers and used without further

purification. All solvents were HPLC grade. Reactions were analyzed by LC-MS using an

API 150EX single-quadropole mass spectrometer (Applied Biosystems). Reverse HPLC

was conducted with an AKTA explorer 100 (Amersham Pharmacia Biotech) using a C18

column.

The method for the synthesis of Cy5-LVSR-AOMK and biotin-LVSR-AOMK was adapted

from a previously described literature procedure (Edgington et al., 2012; Edgington et

al., 2013). Boc-Leu-Val-Ser(OtBu)-Arg(Pbf)-OH was prepared using standard solid

phase peptide synthesis on 2-chlorotrityl resin. This acid was converted to a

chloromethyl ketone (CMK) using the previously described method.

Isobutylchloroformate (25 µL, 0.19 mmol, 1.1 equiv.) was added dropwise to a solution

of Boc-Leu-Val-Ser(OtBu)-Arg(Pbf)-OH (150 mg, 0.17 mmol) and N-methylmorpholine

(23 µL, 0.21 mmol, 1.2 equiv.) in dry THF (1 mL) at -78°C, and the reaction mixture was

stirred for 1 h under argon atmosphere. Diazomethane was prepared from diazald (0.21

g, 0.93 mmol, 5.4 equiv.) and added dropwise to the reaction mixture at 0°C. After 30

min, the reaction was allowed to warm to room temperature, and the reaction mixture

was stirred for 3 h. A 1:1 solution of hydrochloric acid and acetic acid (500 µL) was then

added dropwise while stirring at 0°C. The reaction mixture was diluted with EtOAc, and

the organic phase was washed with water, saturated NaHCO3, and brine. The organic

layer was dried with MgSO4, filtered, and concentrated using a rotary evaporator,

followed by HPLC purification. The resulting CMK was a white powder (10.1 mg, 0.011

mmol, 6.7%). Boc-Leu-Val-Ser(OtBu)-Arg(Pbf)-CMK (10.1 mg, 0.011 mmol) was then

dissolved in 1 mL dry DMF and 2,6-dimethylbenzoic acid (2.5 mg, 0.017 mmol, 1.5

equiv.) was added along with KF (12.7 mg, 0.22 mmol, 20 equiv.). The reaction mixture

was stirred overnight under argon atmosphere at room temperature. The DMF was

removed in vacuo and 25% TFA in DCM was added for 45 min to remove the protecting

groups followed by concentration of the reaction mixture under reduced pressure. The

deprotected intermediate was then purified by HPLC to yield a white powder (2.7 mg,

0.004 mmol, 39%), which was divided into two aliquots. To one aliquot, Cy5-OSu was

coupled by published methods (Edgington et al., 2012; Edgington et al., 2013) followed

by HPLC purification to yield the compound Cy5-LVSR-AOMK (LE40) (1.7 mg, 0.001

mmol, 63%), a blue powder. To the other aliquot, Biotin-OSu was added according to the

same method and purified by HPLC to yield the compound biotin-LVSR-AOMK (LE42) (1

mg, 0.001 mmol, 54%), a white powder.

Mass spectrometry analysis

Mass spectrometry analysis of proteins extracted from SDS-PAGE gels has been

described elsewhere (Sacchetti et al., 2013). The analysis was performed by the

proteomics core facility at Sanford-Burnham Medical Research Institute. Briefly, the

coomassie-stained protein bands were excised from the gel and subjected to in-gel

trypsin digestion using mass spectrometry grade trypsin (Promega). The digested

samples were analyzed by 1D-LC/MS/MS using an LTQ linear ion trap mass

spectrometer (Thermo Scientific). MS/MS spectra were searched against the Human IPI

database using Sorcerer Enterprise with SEQUEST (SageN) software. The minimum

trans-proteomic pipeline (TPP) probability score for proteins to 0.95 was set to assure a

very low error rate (much less than FDR 1%) with reasonably good sensitivity.

Supplemental References

Edgington, L.E., van Raam, B.J., Verdoes, M., Wierschem, C., Salvesen, G.S., and Bogyo, M. (2012). An optimized activity-based probe for the study of caspase-6 activation. Chem Biol 19, 340-352.

Edgington, L.E., Verdoes, M., Ortega, A., Withana, N.P., Lee, J., Syed, S., Bachmann, M.H., Blum, G., and Bogyo, M. (2013). Functional imaging of legumain in cancer using a new quenched activity-based probe. J Am Chem Soc 135, 174-182.

Sacchetti, C., Motamedchaboki, K., Magrini, A., Palmieri, G., Mattei, M., Bernardini, S., Rosato, N., Bottini, N., and Bottini, M. (2013). Surface polyethylene glycol conformation influences the protein corona of polyethylene glycol-modified single-walled carbon nanotubes: potential implications on biological performance. ACS nano 7, 1974-1989.