University of Groningen Nature-inspired molecules containing multiple electrophilic positions Dockerty, Paul Jacques

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University of Groningen

Nature-inspired molecules containing multiple electrophilic positions

Dockerty, Paul Jacques

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Publication date:

2018

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Dockerty, P. J. (2018). Nature-inspired molecules containing multiple electrophilic positions: Synthesis and

application as activity-based probes and inhibitors. University of Groningen.

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Chapter 3 Enolcarbamate Probes Label the Aldehyde

Dehydrogenases RALDH1 and ALDH3A1

Chemical probes derived from electrophilic natural product can provide insight in

the reactivity and biological activity of secondary metabolites. The scaffolds of

natural products are often unique and biologically active, making them attractive

leads for probe development. The bicyclic enolcarbamate scaffold found in the

Brabantamide A is particularly interesting in that it contains two electrophilic

centers and that only a few protein targets have been identified. To study which

proteins react with this scaffold, we synthesized a panel of direct and two-step

probes featuring an enolcarbamate. Protein profiling experiments enabled us to

characterize the targets in A549 cells. Rather than the expected serine hydrolases,

our probes strongly labeled retinoic acid aldehyde dehydrogenase 1 and aldehyde

dehydrogenase 3A1 by reacting with a cysteine residue. Both proteins are

upregulated in various cancer cell lines and our probes may therefore find use in

studying these enzymes and provide a lead for the synthesis of inhibitors.

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58 3.1 Introduction

Chemical proteomics using molecular probes has become an important

method to address questions in biologically relevant settings.

1-4

It

contributed to the development of selective inhibitors based on electrophiles

that react with specific amino acids,

5

_{it enabled the identification of drug}

off-targets and it became one of the main tools to investigate the bio-activity of

natural products. The majority of the probes used in these studies, be it

rationally designed, derived from natural products or identified by

fragment screening,

6

_{contain an electrophilic trap that attaches the probe}

covalently to its targets.

5

Either hyper-reactive cysteine, serine, threonine,

tyrosine or lysine residues react with the probe, depending on the type of

electrophile that has been installed. Therefore this so-called warhead

determines together with scaffold of the inhibitor which proteins get

labeled. Specificity within a protein class can be generated by tuning the

scaffold and/or the reactivity of the warhead,

7

but targeting completely

different sub-sets of proteins often requires larger structural changes. This

essentially necessitates the development of novel electrophilic scaffolds that

are suitable leads for probes. Although novel reactive groups can be

obtained by rational design,

8

often secondary metabolites serve as

inspiration. The reactive groups in natural products are structurally divers

and have proven to be biologically active, making them ideal starting points

for probe synthesis. Added advantage of employing natural products for

probe synthesis is that the resulting probes further the understanding of the

biological activity of the respective natural product. Various electrophilic

natural products, such as acivicin,

9

_duocarmycin,

10,11

_{tetrahydrolipstatin}

12,13

and artemisin

14

_{have been successfully converted into probes.}

The bicyclic enolcarbamate scaffold found in Brabantamide A also forms an

attractive lead (Figure 1). Brabantamide A and derivatives thereof showed

inhibition of the growth of bacteria, fungi and oomycetes.

15-17

_{In vitro assays}

revealed that this compound class irreversibly inhibits the mammalian

serine hydrolase lipoprotein-associated phospholipase A2 (PLA2G7).

18

Furthermore, Brabantamide A has been shown to activate phospholipase D

in oomycetes

15

_{and we recently demonstrated in our studies towards the}

antibacterial activity of bicyclic enolcarbamates that several derivatives

block Bocillin-FL labeling of the penicillin-binding proteins in B. subtilis.

19

Two electrophilic centers within the scaffold can react with proteins, namely

the carbamate and the Michael acceptor, and these are thought to be at the

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basis of the biological activities. Mass spectrometry experiments on PLA2G7

indicate that the carbamate can function as an acylating agent.

18

Acylation is

presumably facilitated by the relative low pKa of the enol-leaving group and

the nitrogen being at a bridge-head position of the 5,5-fused ring system,

which enhance the reactivity of the carbamate considerably. Furthermore,

nucleophilic residues may also react with the α,β-unsaturated amide. This

second putative electrophilic center of the enolcarbamate scaffold could

undergo 1,4-conjugate addition. Subsequent retro-Michael reaction and

concomitant extrusion of CO

2

could lead to protein alkylation.

Identification of the proteins that are modified by bicyclic enolcarbamate

thus far relied on in vitro inhibition assays and competitive protein profiling.

We reasoned that chemical probes PJD120 and PJD224 and the recently

reported alkyne containing derivatives PJD2d and PJD4b

19

_{would enable us}

to identify the targets in a less-biased fashion. We here report the results of

activity-based protein profiling experiments with these probes in lysates

from adenocarcinomic human alveolar epithelial cells (A549 cells) and

demonstrate, for the first time to the best of our knowledge, that the

enolcarbamates react with enzymes featuring a nucleophilic cysteine

residues. We found that retinal dehydrogenase 1 (RALDH1) and dimeric

NADP-preferring aldehyde dehydrogenase (ALDH3A1) are the main

targets of bicyclic enolcarbamate probes in A549 cells. These enzymes are

considered to be biomarkers for cancer stem cells and confer cellular

resistance towards cancer chemotherapy. The enolcarbamate probes may

therefore provide an attractive starting point for the development of novel

RALDH inhibitors and diagnostic tools to detect cancer stem cells.

3.2 Results and discussion

3.2.1 Synthesis and in vitro labeling

With the aim to identify the proteins that covalently bind to bicyclic

enolcarbamates, we equipped the scaffold with a tag/bioorthogonal handle

that enables visualization and identification of the probe-protein adducts by

chemical proteomics. The in house available enolcarbamate derivatives

PJD2d and PJD4b could directly serve as probes for profiling experiments.

19

To complement these two-step probes, we exploited the alkyne moiety in

PJD2d and PJD4b for the synthesis of fluorophore-containing probes. By

performing a copper (I)-catalyzed cycloaddition reaction (“click chemistry”)

(5)

60

Figure 1. Structure of the enolcarbamate scaffold and the probes that have been

developed based on this electrophile.

of a BODIPY-azide to PJD2d and PJD4b, we obtained direct probes PJD224

and PJD120, which differ in the positioning of the tag (Figure 1).

We anticipated that the obtained probes would label PLA2G7. A549 cells

express PLA2G7, albeit low levels, and we therefore assessed the reactivity

of our probes in lysates from cell line. Incubating A549 lysate with

increasing concentrations of PJD120 and PJD224 and subsequent in-gel

detection of the fluorescent probe adducts revealed that PJD120 labels a

large number of proteins (Figure 2B). Enolcarbamate PJD224, however,

appeared to be more selective. Low concentrations (1 µM) of this probe

resulted in labeling of two proteins with an approximate molecular weight

of 50 kDa and 55 kDa respectively (Figure 2A). Of these, the 50 kDa protein

is most prominently labeled. The intensity of fluorescent signal at 55 kDa

increased significantly, when the probe concentration was increased to 10 or

100 µM. At these concentrations, several other proteins also start to be

labeled by PJD224. Denaturation of the proteins with heat and SDS (Figure

2A) prior to labeling leads to loss of fluorescent signal, thereby confirming

that PJD224 interacts with correctly folded proteins.

Subsequently, labeling of proteins by the two-step probes was studied. A549

cell lysate was incubated with the probes, after which the probe adducts

were visualized by performing click chemistry using a BODIPY-azide. For

PJD4b, which is the parent compound of PJD120, we again observed a

complex labeling profile (Figure 2B). Incorporation of a tag at the

proline-derived head group is apparently not tolerated. Treating lysate with PJD2d

on the other hand resulted in rather selective labeling. An intense

fluorescent signal is detected around 55 kDa and a less intense band is

1,4-addition N O O H N O PJD2d PJD4b N O O O H N O N O O H N O PJD224 N N N N B N F F N O O O H N O PJD120 N N N N B N F F N O O H N O reactive carbamate R

A

B

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observed at 50 kDa. These fluorescent bands are not due to non-specific

labeling by the fluorophore, because they are not present when PJD2d is

omitted from the reaction mixture (Figure 2B).

Figure 2. Protein profiling with probes PJD2d and PJD224 (A) or 4b and PJD120 (B) on

A549 cell lysate. Cell lysate was incubated with increasing concentrations of the probes. For two-step probes PJD2d the labeled proteins were clicked to BODIPY-N3 using CuSO4.5H2O (100 µM), sodium ascorbate (3 mM) and TABTA (500 µM) prior to being separated by 12% SDS-PAGE gel. Modified proteins were detected by in gel fluorescence scanning using the Cy2 settings.

3.2.2 Target identification and validation

Based on the labeling pattern, we reasoned that both PJD2d and PJD224

target the same proteins, albeit with different efficiencies. Competition

experiments between PJD2d and PJD224 confirmed this. Labeling of both

proteins by PJD224 can be blocked with an excess of PJD2d (Figure 3). Since

conjugating biotin-azide to PJD2d after labeling should enable

0 + Bodipy-N3 + + + + 100 100 10 1 PJD2d [μM] Δ PJD224 [μM] 1 10 100 100Δ

A

100 10 1 0.1 PJD120 [μM] + 100 + 10 Coomassie Bodipy-N3 PJD4b [μM] + 100 + 10

B

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62 straightforward enrichment of the targets, we decided to use PJD2d to

identify which proteins reacted with the enolcarbamate probes. We first

assessed if using biotin-azide as reporter group affected the outcome of the

experiment. Visualization of the biotinylated proteins by

streptavidin-horseradish peroxidase yielded similar results as obtained for

BODIPY-azide (Figure 3).

Figure 3. Validation of the probes. (A) Competition experiment between PJD2d and PJD224. Cell lysate was treated with PJD2d (100 µM) before being reacted with either 1

µM (left panel) or 100 µM (right panel) PJD224. Probe PJD2d blocks labeling of the two main targets of PJD224 (ALDH1A1 and ALDH3A1, vide infra). (B) Protein profiling with probe PJD2d using Biotin-N3 as a reporter group results in an identical labeling pattern as with BODIPY-N3 confirming the specificity of PJD2d’s labeling. Modified proteins were detected by either in-gel fluorescence scanning using the Cy2 settings (A) or chemiluminescence (B).

For the identification of the proteins, we therefore incubated cell lysate with

PJD2d, functionalized the tagged proteins with Biotin-azide and

subsequently enriched them using affinity purification on streptavidin

beads. The isolated proteins were separated on a SDS-PAGE. Following

mass-spectrometry compatible silver staining and trypsin in-gel digestion,

the tryptic peptides were analyzed by LC-MS/MS (see appendix for the list

of proteins). Amongst the 15 proteins identified by MS (with more than 20%

protein coverage), retinal dehydrogenase 1 (RALDH1, also known as

aldehyde dehydrogenase 1A1, ALDH1A1) was retrieved with the best

coverage (72%) and the highest number of unique peptides (34 unique

peptides) (see appendix for the list of peptides). The molecular weight of

RALDH 1 (~55 kDa) corresponded with the fluorescent signal in the

gel-kDa 75 25 -+ + Biotin-N₃ (100 μM) PJD2d (100 μM) + 1 + -1 -100 + PJD224 [μM] PJD2d (100 μM) kDa 75 25 100 WB: Strp-HRP

A

B

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based analysis for PJD2d and PJD224 (Figure 2). Furthermore, dimeric

NADP-preferring aldehyde dehydrogenase (ALDHIII, also known as

aldehyde dehydrogenase 3A1 (ALDH3A1)) was also identified as an

interacting protein (coverage 25 %, 10 unique peptides). The size of this

protein matches with that of the protein that is preferentially labeled by

PJD224. To verify the interaction with our probes (Figure 4A and 4B), we

incubated recombinantly expressed purified RALDH1 and ALDH3A1 with

PJD224. Both enzymes were labeled efficiently with PJD224, while PJD120,

which did not label these proteins in cell lysates, only weakly labeled

RALDH1 (Figure 4A). To determine if PJD2d indeed modified one of the

cysteine residues in the active site of ALDH1A1, we digested the protein

using chymotrypsin and analyzed the peptides by LC-MS/MS. We observed

two peaks leading to two set of different peptide fragments that contain

probe-bound cysteine residues (Cys302 and Cys303), which indicates that

the probe modify one of the two Cys within the active site of the protein

(Appendix). Cys303 is the nucleophilic active-site residue but Cys302, even

if noncatalytic, has also been reported to be modified by compound such as

duocarmycin.

20

RALDH1 and ALDH3A1 belong to the aldehyde dehydrogenase

superfamily. The active site of enzymes from this protein family contains a

catalytic cysteine that is essential for activity. Electrophiles found in natural

products such as the diynes in falcarinol and the 4-chloroisoxazole of

acivicin have been reported to covalently modify this conserved hyper

reactive cysteine residue.

9,10,21

_{Moreover, the cyclopropane in duocarmycin}

has been shown to react with another cysteine residue in the proximity of

the active site of RALDH1. We therefore hypothesized that one of these

cysteine residue also could react with the electrophilic centers of the

enolcarbamate warhead.

To confirm the mass spectrometry results we performed competition

experiments with the broad-spectrum alkylating agent iodoacetamide (IAA)

and the ALDH inhibitor disulfiram (DSF). Both compounds inhibit ALDHs

by reacting with the catalytic cysteine residue in the active site. These

reagents are commonly used to verify the binding mode of ALDHs

inhibitors and probes and to determine the selectivity of novel fluorogenic

ALDHs substrates.

9,10,22-24

Addition of an excess of these compounds to the

purified proteins or lysates blocked labeling by PJD2d and PJD224 (Figure

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64 4, panel C and D), suggesting that the probes also react with cysteine

residues.

Figure 4. Validation of the targets of PJD2d and PJD224 on purified RALDH1 and

recombinant ALDH3A1 (A, B) and on A549 cells lysates (C). (A) Purified protein was incubated with increasing concentrations of PJD224. As a control, purified protein was denatured prior to labelling with the highest concentration of PJD224. (B, C) Purified protein or cell lysate was incubated in the presence or absence of disulfiram (DSF) or iodoacetamide (IAA) for 30 minutes before the indicated amount of probe was added (competition between PJD2d and disulfiram was not successful as the latter is a good ligand for copper preventing the click reaction to occur). For all the gels, the proteins were separated on a 12% SDS-PAGE gel and visualized by in gel fluorescence scanning using the Cy2 settings. Black triangle: ALDH3A1 labeled in lysate. Black triangle*: recombinantly expressed human ALDH3A1, which contains a his-tag. Red triangle: RALDH1.

3.2.3. In situ labeling of ALDHs and selectivity study

Having confirmed that PJD2d and PJD224 label RALDH1 and ALDH3A1 in

vitro in an activity-based fashion, we aimed to study the efficacy of our

probes in situ. A549 cells were cultured overnight in the presence of the

probes. The labeling profile of cells treated with PJD2d is comparable to that

of cell lysates (Figure 5A) For PJD224, the profile is rather different.

Incubating cells with 10 µM of PJD224 led to labeling of RALDH1 and

ALDH3A1. Compared to cell lysates, the intensity of labeling of RALDH1 is

significantly stronger (Figure 5B).

Competitor (1 mM) -IAA + - IAA+ DSF+ Competitor [mM] 0 IAA10 DSF10 PJD224 (1 μM) RALDH1 PJD224 (100 μM) PJD2d (100 μM) PJD224 [mM] RALDH1 0 0.01 0.1 1 1 Δ + + + + + PJD224 [μM] rhALDH3A1 * 0.1 1 10 10 1 lysate Δ + + + + -IAA 1 0 DSF1 rhALDH3A1 - - - - + IAA + - DSF+ *

A

B

C

PJD120 [mM] RALDH1 0.01 0.1 1 1 Δ + + + +

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Figure 5. In situ labeling versus lysates labeling. A549 cells were grown on a T75 culture

flask until 75-85% confluency before adding the indicated amount of probe PJD2d (A) or

PJD224 (B). The cells were cultured for another 17 h (A, B), after which the cells were

lysed and labeled proteins visualized. Lysate labeling was performed in the same way as described for Figure 2. Red triangles indicate RALDH1 and black triangles indicate ALDH3A1.

This dehydrogenase is most likely more active in its original environment

and the activity rapidly decreases after cell lysis.

10

_{Analysis of the cells after}

overnight incubation revealed that PJD224 affects the cell viability and we

therefore also cultured cells for only 2 hours with the probe. Fortunately, the

cells do not show any phenotypic differences and labeling is more intense

than after overnight incubation. Under these conditions, PJD224 can be used

to profile the activity of RALDH1 and ALDH3A1 in situ (Figure 6A). To test

the selectivity of the probe over closely related proteins of RALDH1, we

transiently transfected HEK293T cells with RALDH1 and the homologs

RALDH2 (ALDH1A2) and RALDH3 (ALDH1A3). Of these proteins, only

RALDH1 was efficiently labeled by PJD2d and PJD224, indicating that the

probes are selective (Figure 6B).

Both RALDH1 and ALDH3A1 are involved in variety of biological processes

including detoxification, retinoic acid signaling and protection against

oxidative stress.

25,26

_{Furthermore, they are also overexpressed in several}

types of cancer. RALDH1 is reported to be a reliable marker in solid tumors

(breast, liver, lung, ovary and others), which is especially useful when

healthy tissue displays normal expression.

10 100 in situ lysate PJD2d [μM] PJD224 [μM] 10 100 lysate in situ

A

B

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66

Figure 6. In situ labeling in A549 cells (A) and transfected HEK293T cells (B). (A) A549

cells were cultured with the indicated amount of PJD224 for 2 h, after which the cells were lysed and labeled proteins visualized. (B) HEK293T cells transfected with Flag-tagged RALDH1, RALDH2 or RALDH3 were incubated with PJD224 or PJD2d for 2 h, after which the cells were lysed and labeled proteins visualized. Western-blot was performed to visualize the FLAG-tagged enzymes (RALDH1, RALDH2 and RALDH3) and confirm the successful transfection. (A). Black triangle: endogenously expressed protein that is labeled in HEK293T cells. Red triangle: RALDH1.

RALDH1 was recently investigated as a potential therapeutic target and

diagnostic marker. The expression of this aldehyde dehydrogenase is

generally related with worse prognosis in cancer

27

_{and plays a role in cancer}

drug

resistance

via

inactivation

of

cyclophosphamide

and

oxaphosphorines.

28

_{ALDH3A1 also displays cell protection from drugs and}

participates extensively to detoxification and cell proliferation. The ability to

label RALDH1 and ALDH3A1 in situ could therefore provide a new

opportunity to study their activity and could potentially lead to new

diagnostic tools and inhibitors.

3.3 Conclusion

In conclusion, we characterized two novel targets of the enolcarbamate

scaffold and here demonstrate that this electrophile reacts with a

heterogenous set of proteins. Besides the previously reported serine

hydrolases and transpeptidases, also proteins from the aldehyde

dehydrogenase family react with this scaffold. Enolcarbamates modify the

hyper reactive cysteine in RALDH1 and ALDH3A1 and probes based on

this scaffold therefore provide a new means to study these enzymes both on

lysates and in situ. Finally, to further expand the chemical toolbox for

unrelated protein profiling the targets of other electrophiles and scaffolds

containing several electrophiles forms an attractive approach.

PJD224 [μM] 0 1 10 -+ Probe [μM] AF-647-N3 + + 10 1 PJD2d + 10+ 1 + +10 1 + 10+ 1

mock RALDH1 RALDH2 RALDH3 mock RALDH1 RALDH2 RALDH3

1 0.1 0.1 1 0.1 1 0.1 1 - - - -PJD224 IB: Flag fluorescence

A

B

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3.4 Experimental section

3.4.1. General bio-procedures

Proteins. Recombinant human ALDH3A1 protein was obtained from Abcam as 50 µg at 0.5

mg/mL and immediately aliquoted and stored at -80 °C. Wild-type ALDH1A1 and the C302S and C302A mutants were obtained from the Sieber Lab.

Vectors. For the preparation of the different constructs, full-length human cDNA was

purchased from Bioscource and cloned into the mammalian expression vector pcDNA3.1, containing genes for ampicilin and neomycin resistance. hALDH1a1, hALDH1a2 and hALDH1a3 were cloned into pcDNA3.1. A FLAG-linker was cloned into the vector at the C-terminus of hALDH1a1, hALDH1a2 and hALDH1a3. All plasmides were grown in XL-10 Z-competent cells and prepped (Maxi Prep, Qiagen). The sequences were confirmed by sequence analysis at the Leiden Genome Technology Centre.

Cell Culture conditions. A549 cell line was grown in t75 culture flasks in DMEM supplemented

with 10% FBS (Fetal Bovine Serum), 1% L-Glutamine, and 1% Pen/Strep in an incubator at 37 °C

and 5% CO2 humidified air. At about 70-90% confluency cells were detached from the flask by

Trypsin/EDTA treatment, pelleted and either reseeded in 4 mL complete cell culture medium per t75 flask or washed two times with PBS and stored at -80 °C before preparing cell lysates. HEK293T cells were grown in DMEM with stable glutamine and phenolred (PAA) with 10%

New Born Calf serum, penicilin and streptomycin at 37 °C and 7% CO2. Cells were passaged

every 2-3 days by resuspending in medium and seeding them to appropriate confluence.

Probes, reagents and material. Stock solutions of the enol-cyclocarbamate probe and

BODIPY-N3 were prepared in DMSO and stored at -20 °C. Stock solutions of TABTA ligand was

prepared in water and stored at -20 °C. Stock solutions of CuSO4.5H2O and sodium ascorbate

were prepared fresh in water before each experiment. Sample buffer (SB) 4× contains 200 mM Tris-HCl (pH 6.8), 400 mM dithiothreitol (DTT), 8% sodium dodecyl sulfate (SDS), 0.4% bromophenol blue and 40% glycerol. Stock solutions of iodoacetamide and disulfiram (purchased from Sigma) were prepared in DMSO and stored at -20 °C.

SDS-PAGE, western blot and pull-down analysis. Laemmli type SDS-PAGE was performed

according to standard literature procedures.29_{Gels were prepared using acrylamide-bis}

ready-to-use solution 40% (37.5:1) (Merck Millipore) and separated on a Mini-PROTEAN Tetra cell (Bio-Rad). Fluorescence scanning of SDS-PAGE gels was performed on a typhoon 9500 FLA model (GE Healthcare) using the CY2 settings for BODIPY (blue laser excitation at 488 nm and emission filter at 520 nm). Coomassie staining was carried out with colloidal CBB G250 staining according to the manufactures protocol (Roti-Blue, Carl Roth). Silver staining was carried out using typical mass-spectrometry compatible protocol with a 0.1% silver nitrate aqueous solution and 0.04% formaldehyde in a 2% sodium carbonate aqueous solution as developing agent.30

For western blot analysis, proteins were transferred to a PVDF membrane (GE Healthcare) subsequent to separation by gel electrophoresis using a Mini Trans-Blot system for wet blotting (Bio-Rad) or a Trans-Blot Turbo system (Bio-Rad). Transfer was performed according to the manufacturer’s protocol using Tobin buffer without methanol. Biotinylated proteins were

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68

stained using streptavidin-HRP and FLAG-tagged enzymes were stained using monoclonal mouse anti-FLAG as primary antibody (F3165, Sigma-Aldrich) and polyclonal goat-anti-mouse HRP as secondary antibody (sc2005, Santa Cruz Biotechnology). Subsequent visualisation with Clarity Western ECL substrate on a ChemiDoc XRS (Bio-Rad) was performed according to manufacturer’s protocol (Bio-Rad). BioRad precision plus protein standards dual color was used as molecular weight marker. Pull-down were performed using Dynabeads® MyOne™ Streptavidin C1 and DynaMag™-2 Magnet (ThermoFisher Scientific).

3.4.2. SDS-PAGE labeling and western blot with A549 lysates

A549 cells were lysed using a NP40 lysis buffer [0.5 % NP40, Tris·HCl (10 mM), NaCl (150 mM),

MgCl2 (5 mM), pH 7.4] during 10 min over ice, the lysates were then centrifugated during 10

min at 10,000 rpm and the supernatant was collected and submitted to a Bradford protein assay to assess the protein concentration before subsequent dilution to 1 mg/mL. One t75 flask usually provided 200-250 µL at 1 mg/mL of protein.

Two-steps labeling: 1 µL of enol-cyclocarbamate PJD2d, PJD4b or DMSO was incubated one

hour at 37 °C with 19 µL of A549 lysates (1 mg/mL). BODIPY-N3 or Biotin-N3 was then clicked

using the following conditions: BODIPY-N3 or Biotin-N3 (100 µM), CuSO4.5H2O (100 µM),

TABTA (500 µM) and sodium ascorbate (3 mM) for 2 hours. SB (with DTT) was then added and the proteins resolved on a 12% SDS-PAGE. Proteins were visualized by in-gel fluorescent

scanning on a Typhoon scanner when BODIPY-N3 was used as a reporter molecule. For the

Biotin labeling experiments, the proteins were then transferred on a PVDF membrane and the proteins were visualized by chemiluminescence.

Direct labeling: 1 µL of enol-cyclocarbamate PJD224, PJD120 or DMSO was incubated one hour at 37 °C with 19 µL of A549 lysates (1 mg/mL). SB (with DTT) was then added and the proteins were resolved on a 12% SDS-PAGE followed by in-gel fluorescent scanning on a Typhoon scanner.

3.4.3. Pull-down experiment with A549 lysates (adapted procedure

from Li et al.)

31

Pull-down. (i) ABP reaction. A549 cells were lysed using NP40 buffer and used immediately.

500 µL of A549 lysate (1 mg/mL) was incubated with 100 µM of PJD2d or DMSO for one hour

at 37 °C followed by click conditions (Biotin-N3 100 µM, CuSO4.5H2O 100 µM, TABTA 500 µM

and sodium ascorbate 3 mM) for 2 hours.

(ii) Denaturation. SDS was added to 1% final concentration and the mixture was vortexed and

boiled at 100 °C for 5 min.

(iii) Chloroform/Methanol precipitation. 750 µL of methanol was addded to the 250 µL protein solution (protein samples were usually splitted in two and combined in the next step). The samples were vortexed vigorously. 200 µL of chloroform were added and the samples were

vortexed vigorously. Add 600 µL of H2O (to get phase separation), vortex the tube vigorously

and centrifuge it (2 min at room temperature, 9,000g). We discarded the upper layer and then add 600 µL of methanol, vortex the mixture at low speed, thoroughly, and centrifuge it (2 min at room temperature, 9,000g) and finally discarded the supernatant by pipetting and air-dry the pellet for exactly 5 min the tube upside-down.

(iv) Proteins were rehydrated in 180 µL of urea buffer (8 M urea/100 mM NH4HCO3) for 15

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(v) Reduction and alkylation. DTT was added to a final concentration of 5 mM and incubated 30 min at 37 °C. Iodoacetamide (IAA) was then added to a final concentration of 15 mM and incubated 30 min in the dark.

(vi) The tubes were centrifuged for 5 min at top speed and the supernatant transferred to a new 2-mL eppendorf.

(vii) Chloroform/Methanol precipitation. (see step iii)

(viii) Dilution with SDS. 25 µL of 2% SDS were added and heated to 70 °C to help protein solubilisation. 900 µL PD buffer (50 mM Tris-HCl (pH 7.5) and 150 mM NaCl) was then added stepwise to slowly dissolve the pellet and the tubes were centrifuged at top speed to remove all insoluble lumps of proteins.

(ix) Pull-down with paramagnetic beads. 50 µL of streptavidin magnetic beads (prewashed with water, PD buffer and 0.05% (wt/vol) SDS PD buffer) were added and incubated for 1 h at room temperature under constant shaking.

(x) The tube were placed on the Dynamag for 3 min and the supernatant was removed. (xi) The beads were then washed once with PD buffer containing 0.1% SDS, twice with PD buffer, once with fresh 4 M urea in 50 mM NH4HCO3 buffer, once with fresh 50 mM Tris-HCl (pH 7.5) and 10 mM NaCl and twice with water.

(xii) Elution from the beads. The beads were boiled with 100 µL of 1× sample buffer containing 10 µM biotin for 5 min at 100 °C.

Samples were run on a 12% SDS-Page and a MS compatible silver stain was performed and a section around 50 kDa excised (around one main band). The silver stained bands were washed three times with milliQ water, reduced using 10 mM DTT in 50 mM ammonium bicarbonate (ABC) at 56 °C, alkylated using 55 mM iodoacetamide at RT in the dark. Gel pieces were washed three times with 50 mM ABC, shrunk using 100% acetonitrile and dried at 37 °C. 100 ng trypsin in 25 µL 50 mM ABC was added to the gel pieces and incubated at RT. After 1 hour additional 50 mM ABC was added to cover the gel pieces with fluid. Digestion was performed overnight at 37 °C. After digestion the gel pieces were acidified by adding 5 µL of formic acid. Peptides were extracted after sonicating the sample for 10 min. The supernatant was pipetted in a clean eppendorf cup. An extra extraction was done by adding 100 µL of acetonitrile and sonicate the sample for 10 min. The supernatants were combined and subsequently dried in a speedvac. The dried samples were dissolved in 15 µL 0.1%FA.

Mass spectrometry. LCMS of the tryptic peptides was performed using an Ultimate 3000 HPLC

system (Thermo Fisher Scientific) coupled online to a Q-Exactive-Plus mass spectrometer with a NanoFlex source (Thermo Fisher Scientific) equipped with a stainless steel emitter. Tryptic digests were loaded onto a 5 mm × 300 µm i.d. trapping micro column packed with PepMAP100 5 µm particles (Dionex) in 0.1% FA at the flow rate of 20 µL/min. After loading and washing for 3 minutes, peptides were back-flush eluted onto a 50 cm × 75 µm i.d. nanocolumn, packed with Acclaim C18 PepMAP100 2 µm particles (Dionex). The following mobile phase gradient was delivered at the flow rate of 300 nL/min: 2–50% of solvent B in 90 min; 50–80 % B in 1 min; 80 % B during 9 min, and back to 2 % B in 1 min and held at 2% A for

17 minutes. Solvent A was 100:0 H2O/acetonitrile (v/v) with 0.1% formic acid and solvent B was

0:100 H2O/acetonitrile (v/v) with 0.1% formic acid. MS data were acquired using a

data-dependent top-10 method dynamically choosing the most abundant not-yet-sequenced precursor ions from the survey scans (300–1650 Th) with a dynamic exclusion of 20 seconds. Sequencing was performed via higher energy collisional dissociation fragmentation with a target value of 1e5 ions determined with predictive automatic gain control. Isolation of

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70

precursors was performed with a window of 1.6. Survey scans were acquired at a resolution of 70,000 at m/z 200. Resolution for HCD spectra was set to 17,500 at m/z 200 with a maximum ion injection time of 110 ms. The normalized collision energy was set at 28. Furthermore, the S-lens RF level was set at 60 and the capillary temperature was set at 250 °C. Precursor ions with single, unassigned, or six and higher charge states were excluded from fragmentation selection

Data processing. The software PEAKS 8.0 (Bioinformatics Solutions Inc., Waterloo, Ontario,

Canada)) was applied to the spectra generated by the Q-exactive plus mass spectrometer to search against a Human Protein database (Uniprot/SwissProt , 20195 entries). Searching for the variable modifications carbamidomethylation, PEG-Biotin and oxidation of methionine was done with a maximum of 5 posttranslational modifications per peptide at a parent mass error tolerance of 10 ppm and a fragment mass tolerance of 0.02 Da. False discovery rate was set at 0.1%.

3.4.4. Labeling of recombinant ALDH3A1

Before each experiment 40 µL of Tris-HCl 20 mM was added to 5 µL (0.5 mg/mL) of protein and this stock solution was used in the following experiment. 9 µL of protein was incubated with DMSO or different concentration of PJD224 during 30 min at 37 °C; for the competition experiment, iodoacetamide or disulfiram was incubated during 15 min at 37 °C prior to incubation with probe PJD224. After incubation, SB (with DTT) was added and the proteins resolved on a 12% SDS-PAGE followed by in-gel fluorescent scanning on a Typhoon scanner.

3.4.5 Labeling of hALDH1A1 and binding site identification (protein

was obtained from the group of Prof. Dr. Stephan A. Sieber – for

cloning, expression and purification see Koch et al.)

20

Proteins were diluted to 30 ng/µL in PBS before each experiment. 9 µL of protein was incubated with DMSO or different concentration of PJD224 or PJD120 during 30 min at 37 °C; for the competition experiment, iodoacetamide was incubated during 30 min or DTT for 15 min at 37 °C prior to incubation with probe PJD224.

After incubation, SB (with DTT) was added and the proteins resolved on a 12% SDS-PAGE followed by in-gel fluorescent scanning on a Typhoon scanner.

To discover the binding site of PJD2d in ALDH1A1 the enzyme was labeled, tryptic digested and the peptide fragments analyzed by ESI tandem mass spectrometry. 5 µL ALDH1A1 (8 mg/mL) was added to 13 µL 10 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl2, pH 7.8. To this was added 2 µL PJD2d (3.6 mM in DMSO). The reaction mixture was incubated for 1 hour at 37 °C and 300 rpm. Binding was confirmed by intact protein measurement.

80 µL urea buffer (8 M urea, 100 mM NH4HCO3) was added, followed by 1 µL DTT (1 mM) to

reduce disulfide bonds. The samples were shaken for 15 minutes at 65 °C at 650 rpm and then allowed to cool down to room temperature for at least 5 minutes. Then 6 µL iodoacetamide (0.5 M) was added to alkylate the free thiols and incubated for 30 minutes at room temperature in the dark. After alkylation 900 µL 100 mM Tris.HCl, 10 mM CaCl2, pH 8.0 and 1 µL chymotrypsin (0.5 µg/µL) was added followed by overnight digestion at 37 °C and 950 rpm. The digestion was stopped by addition of 50 µL formic acid. The samples were then desalted with stage tips (Empore disk C-18, 47 mM, Agilent Technologies).

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The peptides were analyzed on a MultiMate 3000 nano HPLC system coupled to a Orbitrap FusionTM TribidTM mass spectrometer. Samples were injected onto an in-house packed precolumn (100µm x 15 mm; Reposil-Pur C18-AQ 3 µm, Dr. Maisch, Germany) and eluted via a homemade analytical nano-HPLC column (15 cm x 50 µm; Reposil-Pur C18-AQ 3 µm). The gradient was run from 0% to 30% solvent B (5/95/0.1 water/acetonitrile/formic acid) in 60 min. A tip of 5 µm was drawn at the end of the nano-HPLC which acted as the electrospray needle of the MS source. Precursors were measured in the Orbitrap in a scan range from 400 to 1500 m/z. Precursors were isolated in the quadrupole and fragmentation was performed using higher-energy collisional dissociation (HCD). Resulting fragments were measured and assignment of the measured fragments was done manually.

3.4.6. In situ experiments (adapted procedure from Wirth et al)

10

Labeling in A549 cells. Cells were grown to 75-85% confluence in t75 cell culture flasks. Probes PJD2d or PJD224 were added in the medium with a final DMSO concentration of 0.1 %. Cells

were incubated for 2 h or 17 h with various concentrations of probes at 37 °C and 5 % CO2.

Subsequently, the cells were washed two times with PBS and the pellets were lysed using a

NP40 lysis buffer [0.5 % NP40, Tris·HCl (10 mM), NaCl (150 mM), MgCl2 (5 mM), pH 7.4]

during 10 min over ice, the lysates were then spin down during 10 min at 10,000 rpm and the supernatant was collected and submitted to a Bradford protein assay to assess the protein

concentration before subsequent dilution to 1 mg/mL.

For cells treated with PJD2d, the following click conditions were applied to 20 µL of lysates:

BODIPY-N3 100 µM, CuSO4.5H2O 100 µM, TABTA 500 µM and sodium ascorbate 3 mM for 2

hours and SB (with DTT) was then added and the proteins resolved on a 12% SDS-PAGE followed by in-gel fluorescent scanning on a Typhoon scanner.

For cells treated with PJD224, SB (with DTT) was added to 20 µL of lysates and the proteins resolved on a 12% SDS-PAGE followed by in-gel fluorescent scanning on a Typhoon scanner.

Labeling in transfected HEK cells. One day prior to transfection 4*105 cells were seeded in a

6-wells plate. Cells were transfected by addition of a 3:1 mixture of polyethyleneimine (3.6 µg) and plasmid DNA (1.2 µg) in 12 µL serum free medium per well. The medium was refreshed after 24 hours and after 72 hours the medium was removed and 2 mL serum free medium was added. To the medium 2 µL probe (stock in DMSO) was added and mixed carefully. The cells

were then incubated for 1 hour at 37 °C and 7% CO2. The medium was then removed and the

cells harvested by suspending them in two times 2 mL PBS. The suspension was centrifuged for 5 minutes at 1200 rpm. The PBS was then removed and the cells lysed by adding 150 µL 0.5% type NP40 lysis buffer (0.5% type NP40 (Sigma-Aldrich), Tris.HCl (10 mM), NaCl (150 mM),

MgCl2 (5 mM), pH 7.4). The protein concentration was determined with Quick Start Bradford

assay (Bio-Rad). The protein fractions were diluted to a total protein concentration of 1 mg/mL. For the samples treated with the direct probe (PJD224) 30 µL lysate was taken. To this 10 µL standard 4× SDS-PAGE sample buffer was added and the samples were denaturated at 100°C for 5 minutes. To of the samples (40 µL) treated with DMSO or the two-step probe (PJD2d) 5 µL

of a freshly prepared “click” mixture containing 9 mM CuSO4 (2.5 µL/sample, 18 mM in H2O),

45 mM sodium ascorbate (1.5 µL/sample, 150 mM in H2O), 1.8 mM THPTA (0.5 µL/sample, 18

mM in DMSO) and 9 µM Alexa Fluor 647 azide (0.5 µL/sample, 90 µM in DMSO from Thermo Fischer Scientific). The samples were incubated for 1 hour at 37 °C and then 15 µL standard 4×

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72

SDS-PAGE sample buffer was added. The samples were denaturated at 100°C for 5 minutes. All samples were loaded on SDS-PAGE gel (10% acrylamide). The gels were visualized with a ChemiDoc XRS (Bio-Rad) using Cy2, Cy3 and Cy5 settings.

3.4.7. Synthetic procedure

General remarks. TLC analysis was performed on Merck silica gel 60/Kieselguhr F254, 0.25

mm. Compounds were visualized using KMnO4 stain (K2CO3 (40 g), KMnO4 (6 g), H2O (600

mL) and 10% NaOH (5 mL)). Flash chromatography was performed using SiliCycle silica gel

type SiliaFlash P60 (230 – 400 mesh) as obtained from Screening Devices. 1_{H- and}13_C-NMR

spectra were recorded on a Varian AMX400 or a Varian 400-MR (400 and 100.59 MHz,

respectively) using CDCl3. Chemical shift values are reported in ppm with the solvent

resonance as the internal standard (CDCl3: δ 7.26 for 1H, δ 77.06 for 13C). Data are reported as

follows: chemical shifts (δ), multiplicity (s = singlet, d = doublet, dd = double doublet, ddd = double double doublet, td = triple doublet, t = triplet, q = quartet, b = broad, m = multiplet), coupling constants J (Hz), and integral. High-resolution mass spectra (HRMS) were recorded on a Thermo Scientific LTQ Orbitrap XL.

PJD224: Enol cyclocarbamate PJD224 was

prepared by mixing 2d (26 mg, 0.12 mmol, 1 eq)

and BODIPY-N3 (40 mg, 0.12 mmol, 1 eq) in 1.7

mL (tBuOH:MeOH:H2O; 1:1:1) and subsequently

adding 300 µL of 80 mM CuSO4.5H2O (in water)

and 450 µL of 80 mM sodium ascorbate (in water). The reaction was stirred overnight. After TLC showed complete consumption of starting

material, ethyl acetate was added and the crude mixture washed with H2O. The organic layer

was dried over Na2SO4, concentrated under reduced pressure and purified by silica gel flash

column chromatography using ethyl acetate : pentane (8 : 2) as eluent to yield PJD224 (49.6 mg,

73% yield) as an orange oil. Rf [silica, ethyl acetate : pentane (8 : 2)] = 0.18. 1_{H NMR (400 MHz,}

CDCl3): δ = 7.15 (broad s, 1H), 6.05 (s, 2H), 5.18 (broad s, 1H), 4.47 (t, J = 8.0 Hz, 1H), 4.40 (s, 2H),

3.65 (dt, J = 11.4, 7.8 Hz, 1H), 3.30 (ddd, J = 11.7, 8.8, 4.2 Hz, 1H), 2.92 (t, J = 8.3 Hz, 2H), 2.50 (s,

6H), 2.39 (s, 6H), 2.29 - 1.91 (m, 5H), 1.91 - 1.62 (m, 5H), 1.50 (s, 2H). 13_{C NMR (101 MHz,}

CDCl3): δ = 155.8, 155.3, 154.0, 145.6, 140.2, 131.3, 121.7, 67.1, 63.2, 46.0, 31.4, 31.2, 29.9, 29.7, 28.0,

27.0, 26.4, 14.4. IR νmax/cm−1_{: 3346, 2992, 1712, 1671, 1612, 1592, 1198, 1055, 994 cm}−1_{. HRMS:}

(ESI+) Calculated mass [M+H-HF]+_C29H36BFN7O3 = 560.29567 found: 560.29423; Calculated

mass [M+Na]+_C₂₉_H₃₆_BF₂_N₇_O₃_{Na = 602.28384, found: 602.28182.}

PJD120: Enol cyclo carbamate PJD120 was prepared by mixing 4b (14 mg, 34.6 nmol, 1 eq) and BODIPY-N3 (12.5 mg, 35 nmol, 1.01 eq) in 500 µL (tBuOH:MeOH:H2O;

1:1:1) and subsequently adding 175 µL of 40 mM CuSO4.5H2O (in water) and 262 µL of 40 mM

sodium ascorbate (in water). The reaction was stirred overnight. After TLC showed complete consumption of starting material,, ethyl acetate was added and the crude mixture washed with

N O O O H N O N N N N B N F F N O O H N O N N N N B N F F

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H2O. The organic layer was dried over Na2SO4, concentrated under reduced pressure and

purified by silica gel flash column chromatography using methanol: DCM (1: 9) as eluent to

yield PJD120 (26 mg, 98% yield) as an orange oil. Rf [silica, methanol: DCM (1: 9)] = 0.20. 1_H

NMR (400 MHz, CDCl3): δ = 6.54 (t, J = 5.2 Hz, 1H), 6.05 (s, 2H), 5.15 (s, 1H), 4.82 - 4.54 (m, 2H), 4.47 (s, 1H), 4.39 (s, 2H), 3.87 (s, 1H), 3.42 - 3.21 (m, 3H), 3.05 - 2.83 (m, 1H), 2.50 (s, 6H), 2.38 (s, 6H), 2.10 - 1.93 (m, 4H), 1.85 - 1.60 (m, 6H), 1.60 - 1.47 (m, 5H), 1.26 (s, 15H), 0.91 - 0.83 (m, 3H). 13_{C NMR (101 MHz, CDCl}₃_):_δ_{= 163.2, 155.9, 154.1, 153.6, 145.5, 140.1, 131.3, 121.7, 100.2, 79.9,} 77.3, 62.7, 61.8, 52.7, 50.1, 39.7, 37.9, 31.9, 31.2, 30.1, 29.6, 29.6, 29.6, 29.5, 29.3, 29.3, 28.0, 26.9, 22.7, 16.5, 14.4, 14.1. IR νmax/cm−1_{: 3303, 2956, 2812, 1892, 1699, 1612, 1534, 1393, 1192, 1012, 992}

cm−1. HRMS: (ESI+) Calculated mass [M+H-HF]+_C₄₁_H₆₀_BFN₇_O₄_{= 744.47839 found: 744.47807.}

BODIPY-N3 was synthesized following a procedure from Verdoes et al. and the NMR datas

were in accordance with literature.32

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Hoogendoorn, S.; McGuire, R.; Florea, B. I.; Meeuwenoord, N.; van den Elst, H.; van der Marel, G. A.; Brouwer, J.; Di Marzo, V.; Overkleeft, H. S.; van der Stelt, M. Angew. Chem. Int. Ed. 2013, 52 (46), 12081.

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(21) Heydenreuter, W.; Kunold, E.; Sieber, S. A. Chem. Commun. 2015, 51, 15784. (22) Adam, G. C.; Sorensen, E. J.; Cravatt, B. F. Nat. Biotechnol. 2002, 20 (8), 805. (23) Yagishita, A.; Ueno, T.; Esumi, H.; Saya, H.; Kaneko, K.; Tsuchihara, K.; Urano, Y. Bioconjugate Chem. 2017, 28 (2), 302.

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3.6 Appendix

Protein list identified with probe PJD2d on A549 lysates after pull-down

experiment.

Protein List Protein

Group ProteinID Accession -10lgP Coverage(%) #Peptides #Unique PTM Mass DescriptionAvg.

1 6 P04264|K2C1_HUMAN 387.69 77 122 93 Y 66039 Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 PE=1 SV=6

2 2 P35527|K1C9_HUMAN 377.69 90 114 110 Y 62064 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 PE=1 SV=3

4 1 P35908|K22E_HUMAN 341.57 90 87 61 Y 65433 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens GN=KRT2 PE=1 SV=2

5 10 P02538|K2C6A_HUMAN 295.14 71 77 3 Y 60045 Keratin, type II cytoskeletal 6A OS=Homo sapiens GN=KRT6A PE=1 SV=3

6 8 P04259|K2C6B_HUMAN 293.51 71 78 4 Y 60067 Keratin, type II cytoskeletal 6B OS=Homo sapiens GN=KRT6B PE=1 SV=5

16 24 P00352|AL1A1_HUMAN 267.98 72 35 34 Y 54862Retinal dehydrogenase 1 OS=Homo sapiens GN=ALDH1A1 PE=1 SV=2

11 30 O95678|K2C75_HUMAN 239.13 50 48 16 Y 59560 Keratin, type II cytoskeletal 75 OS=Homo sapiens GN=KRT75 PE=1 SV=2

30 45 P14618|KPYM_HUMAN 230.63 60 30 30 Y 57937 Pyruvate kinase PKM OS=Homo sapiens GN=PKM PE=1 SV=4

20 20 P02768|ALBU_HUMAN 217.56 45 30 30 Y 69367 Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2

18 5 Q04695|K1C17_HUMAN 214.79 57 33 10 Y 48106 Keratin, type I cytoskeletal 17 OS=Homo sapiens GN=KRT17 PE=1 SV=2

35 12910 P10809|CH60_HUMAN 208.85 40 20 20 Y 61055 60 kDa heat shock protein, mitochondrial OS=Homo sapiens GN=HSPD1 PE=1 SV₌₂

29 12 P15924|DESP_HUMAN 204.58 16 49 48 Y 331774 Desmoplakin OS=Homo sapiens GN=DSP PE=1 SV=3

19 66 Q9NSB2|KRT84_HUMAN 198.06 54 33 24 Y 64842 Keratin, type II cuticular Hb4 OS=Homo sapiens GN=KRT84 PE=2 SV=2

34 14 P14923|PLAK_HUMAN 197.21 31 24 24 Y 81745 Junction plakoglobin OS=Homo sapiens GN=JUP PE=1 SV=3

36 16 Q02413|DSG1_HUMAN 187.19 18 14 14 Y 113748 Desmoglein-1 OS=Homo sapiens GN=DSG1 PE=1 SV=2

39 56 P29401|TKT_HUMAN 166.81 30 17 17 Y 67878 Transketolase OS=Homo sapiens GN=TKT PE=1 SV=3

23 31 Q7Z794|K2C1B_HUMAN 162.77 17 15 6 Y 61901 Keratin, type II cytoskeletal 1b OS=Homo sapiens GN=KRT77 PE=2 SV=3

38 75 P05165|PCCA_HUMAN 155.61 27 19 18 Y 80059 Propionyl-CoA carboxylase alpha chain, mitochondrial OS=Homo sapiens GN=PCC_{A PE=1 SV=4}

24 100 Q86Y46|K2C73_HUMAN 145.07 15 15 1 Y 58923 Keratin, type II cytoskeletal 73 OS=Homo sapiens GN=KRT73 PE=1 SV=1

45 18 P60709|ACTB_HUMAN 142.95 28 11 11 Y 41737 Actin, cytoplasmic 1 OS=Homo sapiens GN=ACTB PE=1 SV=1

45 19 P63261|ACTG_HUMAN 142.95 28 11 11 Y 41793 Actin, cytoplasmic 2 OS=Homo sapiens GN=ACTG1 PE=1 SV=1

41 37 P78386|KRT85_HUMAN 142.67 24 15 12 Y 55802 Keratin, type II cuticular Hb5 OS=Homo sapiens GN=KRT85 PE=1 SV=1

28 17 Q8N1N4|K2C78_HUMAN 142.14 16 12 4 Y 56866 Keratin, type II cytoskeletal 78 OS=Homo sapiens GN=KRT78 PE=2 SV=2

40 88 P30838|AL3A1_HUMAN 136.08 25 11 10 Y 50395Aldehyde dehydrogenase, dimeric NADP-preferring OS=Homo sapiens GN=ALDH3_{A1 PE=1 SV=3}

42 123 P81605|DCD_HUMAN 133.87 72 10 9 Y 11284 Dermcidin OS=Homo sapiens GN=DCD PE=1 SV=2

51 147 Q8NHM5|KDM2B_HUMAN 130.21 5 4 4 Y 152614 Lysine-specific demethylase 2B OS=Homo sapiens GN=KDM2B PE=1 SV=1

50 83 P11142|HSP7C_HUMAN 129.35 14 9 4 N 70898 Heat shock cognate 71 kDa protein OS=Homo sapiens GN=HSPA8 PE=1 SV=1

49 369 O60701|UGDH_HUMAN 126.81 24 10 10 Y 55024 UDP-glucose 6-dehydrogenase OS=Homo sapiens GN=UGDH PE=1 SV=1

48 73 P04406|G3P_HUMAN 125.68 31 9 8 Y 36053 Glyceraldehyde-3-phosphate dehydrogenase OS=Homo sapiens GN=GAPDH PE=1_SV=3

67 317 Q86YZ3|HORN_HUMAN 122.11 3 5 5 N 282389 Hornerin OS=Homo sapiens GN=HRNR PE=1 SV=2

54 36 P07355|ANXA2_HUMAN 120.22 21 8 8 Y 38604 Annexin A2 OS=Homo sapiens GN=ANXA2 PE=1 SV=2

52 3197 P30101|PDIA3_HUMAN 118.66 23 9 9 Y 56782 Protein disulfide-isomerase A3 OS=Homo sapiens GN=PDIA3 PE=1 SV=4

56 65 P06576|ATPB_HUMAN 112.58 10 4 4 Y 56560 ATP synthase subunit beta, mitochondrial OS=Homo sapiens GN=ATP5B PE=1 SV₌₃

58 3085 P06744|G6PI_HUMAN 109.98 15 7 7 Y 63147 Glucose-6-phosphate isomerase OS=Homo sapiens GN=GPI PE=1 SV=4

57 2442 P07237|PDIA1_HUMAN 106.81 16 8 8 Y 57116 Protein disulfide-isomerase OS=Homo sapiens GN=P4HB PE=1 SV=3

72 86 P13798|ACPH_HUMAN 105.94 10 7 7 N 81225 Acylamino-acid-releasing enzyme OS=Homo sapiens GN=APEH PE=1 SV=4

90 61 Q08554|DSC1_HUMAN 103.79 4 3 3 Y 99987 Desmocollin-1 OS=Homo sapiens GN=DSC1 PE=1 SV=2

86 105 Q5D862|FILA2_HUMAN 101.79 2 4 4 N 248072 Filaggrin-2 OS=Homo sapiens GN=FLG2 PE=1 SV=1

53 12913 P11413|G6PD_HUMAN 101.72 10 6 6 Y 59257 Glucose-6-phosphate 1-dehydrogenase OS=Homo sapiens GN=G6PD PE=1 SV=4

89 138 Q13835|PKP1_HUMAN 100.25 5 4 3 N 82861 Plakophilin-1 OS=Homo sapiens GN=PKP1 PE=1 SV=2

70 29 P05089|ARGI1_HUMAN 99.69 13 5 5 Y 34735 Arginase-1 OS=Homo sapiens GN=ARG1 PE=1 SV=2

71 12911 Q16881|TRXR1_HUMAN 99.67 11 5 5 Y 70906 Thioredoxin reductase 1, cytoplasmic OS=Homo sapiens GN=TXNRD1 PE=1 SV=3

73 115 P01040|CYTA_HUMAN 99.09 66 5 5 Y 11006 Cystatin-A OS=Homo sapiens GN=CSTA PE=1 SV=1

55 76 P07900|HS90A_HUMAN 98.08 6 5 1 N 84660 Heat shock protein HSP 90-alpha OS=Homo sapiens GN=HSP90AA1 PE=1 SV=5

62 91 P08238|HS90B_HUMAN 96.00 6 5 2 N 83264 Heat shock protein HSP 90-beta OS=Homo sapiens GN=HSP90AB1 PE=1 SV=4

88 62 P06733|ENOA_HUMAN 95.39 15 5 5 Y 47169 Alpha-enolase OS=Homo sapiens GN=ENO1 PE=1 SV=2

31 38 Q6KB66|K2C80_HUMAN 95.30 9 5 3 N 50525 Keratin, type II cytoskeletal 80 OS=Homo sapiens GN=KRT80 PE=1 SV=2

64 27 P29508|SPB3_HUMAN 94.96 11 5 5 N 44565 Serpin B3 OS=Homo sapiens GN=SERPINB3 PE=1 SV=2

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76 ALDH1A1 peptide list measured from pull-down experiment with PJD2d

on A549 lysates (72% protein coverage).

total 72 peptides

P00352|AL1A1_HUMAN - ALDH1A1

| Protein Coverage | Supporting Peptides | Protein Coverage:

Supporting Peptides:

Peptide Uniq -10lgP Mass Length ppm m/z z RT Fraction Scan Source File #Spec Start End PTM

K.IGPALSC(+57.02)GN TVVVKPAEQTPLTALHVA

SLIK.E Y 84.71 3183.7688 31 3.1 796.9520 4 54.82 6 17057 20161213dOCKERTHY_SILVERdIRECT.raw 11 180 210 Carbamidomethylation K.GYFVQPTVFSNVTDE

MR.I Y 71.61 1988.9302 17 3.2 995.4755 2 58.42 6 18451 20161213dOCKERTHY_SILVERdIRECT.raw 3 379 395 K.KFPVFNPATEEELC(+

57.02)QVEEGDKEDVD

K.A Y 71.22 3051.4019 26 2.5 763.8597 4 49.16 6 14840 20161213dOCKERTHY_SILVERdIRECT.raw 8 37 62 Carbamidomethylation R.IAKEEIFGPVQQIMK.F Y 67.42 1729.9436 15 1.5 577.6560 3 49.76 6 15076 20161213dOCKERTHY_SILVERdIRECT.raw 3 396 410

K.LYSNAYLNDLAGC(+5

7.02)IK.T Y 66.78 1713.8396 15 4.1 857.9306 2 54.55 6 16950 20161213dOCKERTHY_SILVERdIRECT.raw 4 114 128 Carbamidomethylation R.ANNTFYGLSAGVFTK. D Y 65.14 1588.7886 15 6.4 795.4067 2 53.92 6 16705 20161213dOCKERTHY_SILVERdIRECT.raw 7 421 435 K.RANNTFYGLSAGVFTK .D Y 64.45 1744.8896 16 2.6 582.6387 3 47.41 6 14153 20161213dOCKERTHY_SILVERdIRECT.raw 3 420 435 R.TIPIDGNFFTYTR.H Y 59.80 1543.7671 13 3.6 772.8936 2 56.81 6 17828 20161213dOCKERTHY_SILVERdIRECT.raw 9 144 156 K.GYFVQPTVFSNVTDE

M(+15.99)R.I Y 58.84 2004.9250 17 4.6 1003.4744 2 54.14 6 16793 20161213dOCKERTHY_SILVERdIRECT.raw 6 379 395 Oxidation (M) R.ELGEYGFHEYTEVK.T Y 56.64 1699.7729 14 2.9 567.5999 3 41.82 6 11955 20161213dOCKERTHY_SILVERdIRECT.raw 5 477 490 K.FPVFNPATEEELC(+57

.02)QVEEGDKEDVDK.A Y 56.61 2923.3069 25 4.5 975.4473 3 53.92 5 18283 20161213dOCKERTHY_SILVEREXTRACT1.raw 1 38 62 Carbamidomethylation R.IAKEEIFGPVQQIM(+ 15.99)K.F Y 56.33 1745.9386 15 4.5 582.9894 3 42.80 5 13936 20161213dOCKERTHY_SILVEREXTRACT1.raw 9 396 410 Oxidation (M) R.IFVEESIYDEFVR.R Y 56.12 1644.8035 13 2.2 823.4108 2 61.45 6 19619 20161213dOCKERTHY_SILVERdIRECT.raw 5 309 321 R.IFVEESIYDEFVRR.S Y 55.82 1800.9045 14 2.7 601.3104 3 56.07 6 17539 20161213dOCKERTHY_SILVERdIRECT.raw 7 309 322 K.IFINNEWHDSVSGK.K Y 54.64 1644.7896 14 2.9 549.2720 3 40.62 5 13065 20161213dOCKERTHY_SILVEREXTRACT1.raw 6 23 36 R.YC(+57.02)AGWADK

IQGR.T Y 52.82 1423.6666 12 1.3 475.5634 3 35.17 6 9336 20161213dOCKERTHY_SILVERdIRECT.raw 6 132 143 Carbamidomethylation K.LEC(+57.02)GGGPW GNK.G Y 52.54 1173.5237 11 0.4 587.7693 2 32.74 6 8416 20161213dOCKERTHY_SILVERdIRECT.raw 4 368 378 Carbamidomethylation K.YILGNPLTPGVTQGPQ IDKEQYDK.I Y 50.26 2673.3650 24 2.3 892.1310 3 49.22 5 16426 20161213dOCKERTHY_SILVEREXTRACT1.raw 6 330 353 K.VAFTGSTEVGK.L Y 49.52 1094.5608 11 1.0 548.2882 2 32.42 6 8295 20161213dOCKERTHY_SILVERdIRECT.raw 3 242 252 R.QAFQIGSPWR.T Y 49.36 1188.6040 10 4.2 595.3118 2 48.82 5 16295 20161213dOCKERTHY_SILVEREXTRACT1.raw 8 69 78 R.LLLATMESMNGGK.L Y 48.72 1363.6840 13 0.4 682.8495 2 48.64 6 14637 20161213dOCKERTHY_SILVERdIRECT.raw 1 101 113 R.HEPIGVC(+57.02)G QIIPWNFPLVM(+15.99

)LIWK.I Y 46.50 2762.4441 23 2.0 921.8239 3 75.00 5 26638 20161213dOCKERTHY_SILVEREXTRACT1.raw 1 157 179 Carbamidomethylation;Oxidation (M) K.EEIFGPVQQIM(+15.9 9)K.F Y 45.48 1433.7224 12 3.1 717.8707 2 46.19 5 15271 20161213dOCKERTHY_SILVEREXTRACT1.raw 1 399 410 Oxidation (M) R.LLLATM(+15.99)ESM (+15.99)NGGK.L Y 44.23 1395.6738 13 2.9 698.8462 2 34.91 5 10785 20161213dOCKERTHY_SILVEREXTRACT1.raw 2 101 113 Oxidation (M) K.EEIFGPVQQIMK.F Y 41.58 1417.7275 12 4.2 709.8740 2 54.27 6 16842 20161213dOCKERTHY_SILVERdIRECT.raw 1 399 410 R.VTLELGGK.S N 39.58 815.4752 8 6.2 408.7474 2 35.66 6 9524 20161213dOCKERTHY_SILVERdIRECT.raw 7 266 273 K.LADLIERDR.L Y 39.45 1099.5985 9 3.0 550.8082 2 32.69 5 9944 20161213dOCKERTHY_SILVEREXTRACT1.raw 9 92 100 K.ILDLIESGKK.E Y 38.56 1114.6597 10 2.8 558.3387 2 37.51 5 11818 20161213dOCKERTHY_SILVEREXTRACT1.raw 4 354 363 K.EAGFPPGVVNIVPGYG PTAGAAISSHM(+15.99

)DIDKVAFTGSTEVGK.L Y 38.35 4159.0571 42 -2.7 1040.7688 4 57.65 5 19716 20161213dOCKERTHY_SILVEREXTRACT1.raw 4 211 252 Oxidation (M) K.EGAKLEC(+57.02)G GGPWGNK.G Y 35.21 1558.7197 15 0.3 520.5807 3 31.96 6 8125 20161213dOCKERTHY_SILVERdIRECT.raw 1 364 378 Carbamidomethylation K.SLDDVIKR.A Y 35.14 944.5291 8 2.9 315.8512 3 32.09 5 9713 20161213dOCKERTHY_SILVEREXTRACT1.raw 8 413 420 K.FKSLDDVIKR.A Y 33.56 1219.6924 10 2.5 407.5724 3 34.93 6 9239 20161213dOCKERTHY_SILVERdIRECT.raw 2 411 420 K.LADLIER.D Y 32.84 828.4705 7 4.3 415.2443 2 37.26 5 11718 20161213dOCKERTHY_SILVEREXTRACT1.raw 4 92 98 K.SLDDVIK.R Y 32.77 788.4280 7 5.3 395.2234 2 36.53 5 11429 20161213dOCKERTHY_SILVEREXTRACT1.raw 3 413 419 R.YC(+57.02)AGWADK .I Y 32.52 969.4014 8 5.3 485.7106 2 30.61 5 9132 20161213dOCKERTHY_SILVEREXTRACT1.raw 1 132 139 Carbamidomethylation K.LIKEAAGK.S Y 27.78 828.5068 8 3.1 415.2620 2 19.57 5 4988 20161213dOCKERTHY_SILVEREXTRACT1.raw 1 253 260 K.RVTLELGGK.S Y 26.88 971.5764 9 4.0 486.7974 2 30.83 5 9219 20161213dOCKERTHY_SILVEREXTRACT1.raw 3 265 273 R.DRLLLATM(+15.99)E SM(+15.99)NGGK.L Y 26.34 1666.8019 15 4.7 556.6105 3 34.27 5 10551 20161213dOCKERTHY_SILVEREXTRACT1.raw 1 99 113 Oxidation (M) K.EAGFPPGVVNIVPGYG PTAGAAISSHM(+15.99

)DIDK.V Y 23.70 3082.5068 31 3.1 1028.5127 3 55.85 5 19027 20161213dOCKERTHY_SILVEREXTRACT1.raw 1 211 241 Oxidation (M) K.EAGFPPGVVNIVPGYG

PTAGAAISSHMDIDKVA

FTGSTEVGK.L Y 23.06 4143.0620 42 3.2 1036.7761 4 59.73 6 18965 20161213dOCKERTHY_SILVERdIRECT.raw 1 211 252

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LC-MS/MS results. LC–MS/MS binding-site identification in human

ALDH1A1 with probe PJD2d. Displayed are fragmented peptides (y and b

ions) identified by MS/MS sequencing. The modified cysteine residues are

Cys302 or Cys303.

RT:0,00 - 52,00 0 5 10 15 20 25 30 35 40 45 50 Time (min) 0 20 40 60 80 100 Re lat ive A bu nd an ce 0 20 40 60 80 100 Re lat ive A bu nd an ce 0 20 40 60 80 100 Re lat ive A bu nd an ce 20,06_21,39 24,57 28,59 21,50 16,05 _23,06 28,80 26,26 14,86 33,37 33,72 17,77 29,29 34,88 13,44 29,89 35,80 38,33 13,31 39,92 41,3744,48 11,00 50,20 5,92 46,53 1,53 3,90 7,51 24,22 21,02 19,87 23,68 28,74 18,25 24,91 33,55 13,18 29,10 35,18 38,33 39,67 44,00 10,57 45,30 1,662,59 6,21 9,45 47,44 51,58 24,27 21,10 27,40 32,63 NL: 1,41E10 Base Peak m/z= 160,00-2000,00 MS L2173118a NL: 9,19E9 m/z= 570,43-571,43 MS L2173118a NL: 3,02E9 TIC F: FTMS + c NSI d Full ms2 570,9317@hcd32,00 [110,0000-1723,0000] MS L2173118a

L2173118a #7621RT:21,10AV:1NL:1,96E8

F:FTMS + c NSI d Full ms2 570,9317@hcd32,00 [110,0000-1723,0000] 200 300 400 500 600 700 800 900 1000 1100 1200 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Re lat ive A bu nd an ce 664,38 593,34 777,46 614,27 522,30 133,04 301,13 843,32 386,21 934,33 429,19 307,64 227,11 503,24 683,29 178,23 _246,13 _314,14 906,33 486,21 791,33 757,27 870,35 1086,16 1182,731239,89

L2173118a #9473RT:24,27AV:1NL:5,59E8

F:FTMS + c NSI d Full ms2 570,9317@hcd32,00 [110,0000-1723,0000] 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Re lat ive A bu nd an ce 664,37640 680,25513 593,33911 140,07036 295,12701 266,12424 777,45947 195,11259 _340,17249 451,20410 522,30322 611,23596 793,34015 226,10577 499,29575 745,85388 423,20892 807,65948 913,56165959,80249 b1 b2 b3 b4 b5 b6 b7 b8 b9 b10 b11 b12 y12 y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 y1

b1 b2 b3 b4 b5 b6 b7 b8 b9 b10 b11 b12 y12 y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 y1

y8 b6 y7 b5 y6 y5 y4 b2 b2 b3 b4 y4 y5 y6 y7 b5 t=21,1 t=24.27

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