Supporting Information

 
Discovery of Interacting Proteins of ABA Receptor PYL5 Via Covalent Chemical
Capture

Qizhen Zheng, Liang Zhang, Qian Zhang, Zhengyuan Pang, Yang Sun, Zheng Yin and
Zhiyong Lou
   

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Contents

1.  Instruments ............................................................................................................................ 3 
2.  General materials and methods .......................................................................................... 3 
3.  Plasmids construction and site mutation of PYLs ............................................................ 3 
4.  Natural amino acids proteins expression and purification .............................................. 3 
5.  PYR1/PYL5-pBpa expression and purification ................................................................. 4 
6.  ESI-MS on wild type and pBpa-containing recombinant proteins ................................. 5 
7.  HAB1 phosphatase activity assay ...................................................................................... 5 
8.  PYLs-pBpa-HAB1 photo-crosslinking assay ..................................................................... 5 
9.  PYL5-pBpa-RAD23C/CSN1/CAP1 photo-crosslinking assay ........................................ 5 
10.  Sample preparation of Arabidopsis total proteins ......................................................... 6 
11.  Covalent chemical capture assay in TP ......................................................................... 6 
12.  Photo-crosslinking products in-gel trypsin digestion and HPLC-MS/MS analysis .. 7 
13.  Proteins identification and label-free quantification ...................................................... 8 
14.  Statistical analysis ............................................................................................................. 9 
15.  Binding sites identification of PYL5 and interacting partners ..................................... 9 
16.  Yeast two-hybrid assay ..................................................................................................... 9 
17.  Bimolecular fluorescence complementation (BiFC) assay ....................................... 10 
18.  Protein sequence used in this work .............................................................................. 10 
19.  Supplementary figure ...................................................................................................... 13 
20.  Supplementary table ....................................................................................................... 16 
21.  MS2 of crosslinked peptides .......................................................................................... 21 
22.  MS1 of purified PYLs and PYLs-pBpa mutants .......................................................... 30 

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1. Instruments

Protein mass spectrum was collected by an Ultimate 3000 HPLC coupled with
a Q-Exactive Mass Spectrometer (Thermo Fisher Scientific). Photo-
crosslinking experiments were performed by a CL-1000L Ultraviolet Crosslinker
(UVP) with a fixed wave length of 365 nm. The images of both gel and western
blot were all taken by Azure C400 (Azure Biosystems).

2. General materials and methods

pEVOL-pBpa was a gift from Peter Schultz Lab (Addgene plasmid # 31190) in
order to express pBpa corresponding tRNA and aminoacyl-tRNA synthetase.1
E. coli strain DH5α was used for plasmid amplification and BL21(DE3) was
used for protein expression. E. coli cells were growing on Luria-Bertani (LB)
agar solid or broth medium. The unnatural amino acid pBpa was purchased
from adamas. The antibiotic of chloramphenicol, kanamycin and ampicillin were
purchased from solarbio. The mouse anti-His6, anti-Flag antibodies and HRP-
conjugated goat anti-mouse antibodies were purchased from TransGen
Biotech. All antibodies diluted with blocking buffer in a ratio of 1:4000 when use.
All protein concentration was detected by using bicinchoninic acid (BCA)
protein determination method.

3. Plasmids construction and site mutation of PYLs

pET28a plasmids inserted with PYR1, PYL5, RAD23C, CSN1 gene, pET22b
plasmid inserted with CAP1 gene and pGEX-6p-1 plasmids inserted with
PYR1-Flag, PYL5-Flag, HAB1 gene were constructed for corresponding
protein expression in E. coli cells. PYR1/PYL5-TAG gene and the mutants
gene of RAD23C, CSN1 or CAP1 were performed from corresponding
plasmids by site-directed mutations with a mutation kit.

4. Natural amino acids proteins expression and purification

pET28a, pET22b or pGEX-6p-1 inserted with protein gene was transformed

into E. coli BL21(DE3) cells. Cells were grown at 37°C in 800 mL fresh LB

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 medium which containing corresponding antibiotic for about 4 h until OD600
reached 0.5, at which point the temperature was lowered to 16°C. Cells were
induced by 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) before growing
overnight at 16°C. The cells were harvested by centrifugation at 4600 rpm for
15 min, discarding supernatant and the remaining cells lysed by sonication for
30 min in suitable buffer. Buffer 1 (20 mM Tris-HCl, 150 mM NaCl, pH 8.0) was
for PYR1, PYL5, RAD23C and CSN1. Buffer 2 (PBS, pH 7.4) was for CAP1,
CAP1-N, and CAP1-C. Buffer 3 (50 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl2,
5% glycerol, pH 8.0) was for HAB1. The cell lysates were centrifuged at 12000
rpm for 45 min. After that, recombinational proteins in supernatant were purified
by Ni-NTA (Qiagin) affinity column (for His6 tag) or Glutathione-Sepharose
beads (for GST tag). HAB1-GST was eluted by buffer 4 (20 mM Tris-HCl, 10
mM reduced glutathione, pH 8.0). Proteins have no GST tag was cut by
prescission protease overnight at 4°C. All purified proteins were concentrated
and stored at -80°C.

5. PYR1/PYL5-pBpa expression and purification

pET28a-PYR1/PYL5-TAG (or pGEX-6p-1-PYL5-TAG-Flag) and pEVOL-pBpa

were co-transformed into E. coli BL21(DE3) cells. Cells were grown at 37°C in
800 mL fresh LB medium which containing 50 μg/mL kanamycin(100 μg/mL
ampicillin for pGEX-6p-1-PYL5-TAG-Flag) and 25 μg/mL chloramphenical for
about 4 h until OD600 reached 0.5, at which point the 0.2 mM pBpa was added
and the temperature was lowered to 25°C. Cells were induced by 0.2% w/v L-
arabinose and 1 mM IPTG before growing overnight at 25°C. The cells were
harvested by centrifugation at 4600 rpm for 15 min, discarding supernatant and
the remaining cells lysed by sonication for 30 min in buffer 1 (20 mM Tris-HCl,
150 mM NaCl, pH 8.0). The cell lysates were centrifuged at 12000 rpm for 45
min. After that, recombinational proteins in supernatant were purified by Ni-NTA
(Qiagin) affinity column (for His6 tag) or Glutathione-Sepharose beads (for GST
tag). GST tag was cut by prescission protease at 4°C for overnight. Proteins
were further purified by Hitrap Q and Superdex-75 column (GE Healthcare).

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 The purified protein was concentrated and stored at -80°C.

6. ESI-MS on wild type and pBpa-containing recombinant proteins

The mass spectrum of different recombinant proteins was acquired on the

Ultimate 3000 HPLC coupled with Q-Exactive Orbitrap mass spectrometer
system (Thermo Fisher Scientific). Purified proteins diluted in buffer (20 mM
Tris-HCl, pH 8.0) to a final concentration of 0.5 mg/mL. 1 μL diluted sample was
loaded onto a C4 (Beijing yuanbaoshan Chrom-Tech Co. Ltd) analytical column
and eluted with a 7.5 min gradient elution method.

7. HAB1 phosphatase activity assay

The phosphatase activity of HAB1 was measured by a Serine/Threonine

phosphatase assay system kit (Promega). In a 50 μL reaction system, 10 μL
5× reaction buffer (250 mM imidazole, 25 mM MgCl2, 1 mM EGTA, and 0.5
mg/mL BSA, pH 7.2) and certain volume ddH2O were added firstly, followed by
a final concentration of 6 μM PYLs (or specific concentration of PYL5 for dose-
response assays), 10 μM ABA and 0.3 μM HAB1, and finally, the peptide
substrate (supplied in kit). The reaction mixture was incubated at 30°C for 20
min and stopped by adding 50 μL molybdate dye. Another 15 min incubation at
room temperature was needed before absorbance measure at 630 nm. The
reaction without HAB1 was set as negative control. The data present the means
± standard deviation from three independent experiment results.

8. PYLs-pBpa-HAB1 photo-crosslinking assay

Purified PYLs-pBpa with or without ABA were incubated with HAB1 at 10 μM

(final concentrations) for 10 min, followed by UV irradiation (365 nm) for 20 min
at a distance of 15 cm at 0°C, and no UV treated samples as control. The photo-
crosslinking products were separated by 12% SDS-PAGE, and stained with
coomassie brilliant blue.

9. PYL5-pBpa-RAD23C/CSN1/CAP1 photo-crosslinking assay

10 μM PYL5-T187/C191pBpa-Flag and 10 μM His6-tagged putative interaction

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 proteins were incubated with or without 100 μM ABA for 30 min, followed by UV
irradiation (365 nm) for 30 min at a distance of 15 cm at 0°C, and no UV treated
samples as control. The photo-crosslinking products were separated by 12%
SDS-PAGE, and detected with anti-His6 and anti-Flag antibody.

10. Sample preparation of Arabidopsis total proteins

Intact Arabidopsis thaliana (col-0) growing in soil for four weeks were treated
with 100 μM ABA for 1 h, after that, washed by deionized water. Dry Arabidopsis
was fully grinded to powder in liquid nitrogen and extracted Arabidopsis total
proteins (hereinafter referred to as TP) with extraction buffer (50 mM Tris-HCl,
150 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 0.1% NP-40, 5% glycerol, 1 mM
PMSF, 50 μM MG132, 1 × cocktail, pH 7.5). TP sample was put on ice for 20
min after adding 100 μL extraction buffer in each 500 μL Arabidopsis powder,
following centrifuged at 17000 g for 10 min. Supernatant was centrifuged for
another 10 min and then transferred into a new tube. TP concentration was
detected by BCA method.

11. Covalent chemical capture assay in TP

Freshly prepared TP was diluted to 5 mg/mL with extraction buffer and

incubated with purified PYL5-T187pBpa-His6 (30 μM) in the presence of ABA
(300 μM) to a final volume of 650 μL for 30 min at 0°C. Samples were treated
with UV irradiation (365 nm) for 15 min at a distance of 15 cm at 0°C and no
UV treated samples as control. All UV treated and non-UV treated samples
were centrifuged at 12000 rpm for 1 min at 4°C to remove precipitation before
added to an eppendorf tube which containing 50 μL Ni-NTA beads, replenishing
600 μL buffer 4 (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) to each samples and
incubated for 1 h with rotary mixer at 4°C. After incubation, samples were
centrifuged at 2500 rpm for 2 min at 4°C and discarding supernatant. Ni-NTA
beads on the bottom of the centrifuge tube were resuspended and washed with
1 mL wash buffer (50 mM Tris-HCl, 150 mM NaCl, 0.2% SDS, 10 mM Imidazole,
10% glycerol, pH 7.5) for four times and washed once with 1mL chilled buffer 4

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 (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) to dilute SDS. In each time, beads
were centrifuged at 2500 rpm for 2 min at 4°C, then discarding supernatant.
After that, beads were resuspended in 200 μL elution buffer (50 mM Tris-HCl,
150 mM NaCl, 200 mM Imidazole, pH 7.5) and mixed for 1min by inversion and
then put on 0°C for 5 min followed by centrifuged at 2500 rpm for 2 min at 4°C.
The photo-crosslinked products in supernatant was pipetted off and transferred
into a clean centrifuge tube. This elution procedure was repeated two times.
Three times of eluent were combined and centrifuged at 12000 rpm for 1 min
at 4°C to remove the residue beads. The supernatant was added to a 10K
ultrafiltration device (Millipore) and centrifuged at 10000 g for 15 min at 4°C.
The protein solutions in ultrafiltration device were exchanged buffer with 500
μL buffer 4 (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) for three times and then
concentrated to a final volume of 40 μL. Concentrated sample was transferred
into another new centrifuge tube respectively followed by adding 10 μL 5 ×
loading buffer (SDS) to each samples, put them on 95°C metal bath for 5 min
and then separated by 12% SDS-PAGE. The gel was stained with coomassie
brilliant blue. Three biological replicates of UV-treated and non-UV treated
assay were processed to ensure the veracity and repeatability of experiments
results.

12. Photo-crosslinking products in-gel trypsin digestion and HPLC-MS/MS

analysis

Crosslinking products in each lane in gel were divided into three parts and the
highly abundant protein of PYL5-T187pBpa as an individual part. Each part of
protein gel was cut into granules before put into a clean centrifuge tube
respectively, 500 μL 50% acetonitrile in 50 mM NH4HCO3 was added, and the
solution was placed at 37°C until the gel color faded. The blue solution was
discarded and enough acetonitrile was added to make gel dehydration. The dry
gel granules were reduced with 5 mM DTT and alkylated with 11 mM IAA
followed by adding enough acetonitrile to make gel dehydration again. The dry
gel granules were added moderate trypsin working solution (10 ng/μL trypsin

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 in 50 mM NH4HCO3) and incubated at 37°C for 16 h. Peptides in solution and
gel were extracted three times with 0.1% TFA in 50% acetonitrile aqueous
solution for 1 h. Extracts were then combined and dried by vacuum centrifugal.
Dry tryptic peptides were dissolved in 20 μL 0.1% TFA in water and analyzed
by HPLC-MS/MS.
For HPLC-MS/MS analysis, the peptides were separated and identified by a 2
h gradient elution at a flow rate 0.30 µL/min with a Thermo-Dionex Ultimate
3000 HPLC and a Thermo Scientific Q Exactive mass spectrometer combining
system. The analytical column was a home-made fused silica capillary column
(75 µm ID, 150 mm length; Upchurch, Oak Harbor, WA) packed with C18 resin
(300 Å, 5 µm, Varian, Lexington, MA). Mobile phase A consisted of 0.1% formic
acid, and mobile phase B consisted of 80% acetonitrile and 0.1% formic acid.
The Q-Exactive mass spectrometer was operated in the data-dependent
acquisition mode using Xcalibur 2.1.2 software and there was a single full-scan
mass spectrum in the orbitrap (300-1800 m/z, 70,000 resolution) followed by
20 data-dependent MS/MS scans at 27% normalized collision energy (HCD).

13. Proteins identification and label-free quantification

Proteins were identified and quantified by running MaxQuant_1.5.5.1 against

the Uniprot Swiss-Prot database of Arabidopsis (download on 23rd, November,
2017). The search criteria were as follows: full tryptic specificity was required;
two missed cleavage was allowed; Oxidation (M), acetyl (protein N-term), and
carbamidomethyl (C) were set as the variable modifications; match between
runs option (0.7 min match time and 20 min alignment time) was needed to
help find the non-identified MS features in other runs; the minimal ratio count
for protein quantification was 1, and all unmodified peptides plus those peptides
that had Oxidation (M), acetyl (protein N-term) or carbamidomethyl (C)
modifications were used for protein quantification; the “iBAQ” option was
enabled to calculate absolute protein abundances; the false discovery rate
(FDR) at the peptide spectrum match level and the protein level was set to 0.01
using a reverse, decoy database. Other parameters for protein identification

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 and quantification were set as software default.

14. Statistical analysis

Three biological replicates data in proteinGroups.txt file from MaxQuant

running results was used for subsequent analysis. Proteins that matched to
reverse, only identified by sites and identified less than two independent
experiments were removed from further analysis. Data were normalized before
unpaired Student’s t-test analyzing. Proteins meeting the ratio of +UV/-UV ≥ 3
and the p-value was ≤ 0.05 were considered to have significant difference.

15. Binding sites identification of PYL5 and interacting partners

Proteins were identified and quantified by running pLink 2.3.5 against the
corresponding protein sequences. The search criteria were as follows:
conventional crosslinking (HCD), cross-linker pBpa-F or pBpa-X [X also
represents F, α cross-linking sites F or X, β cross-linking sites A C D E F G H I
K L M N P Q R S T V W X Y, cross-linker composition C(7)H(4)O(1), cross-
linker mass shift 104.026]; enzyme trypsin_P (or trypsin_P and chymotrypsin
for PYL5-T187pBpa), up to 3 cleavages; peptide mass minimum 400 and
maximum 6,000 Da per chain, peptide length mininum 4 amino acids and
maximum 60 amino acids per chain; precursor mass tolerance 20 ppm,
fragment mass tolerance 20 ppm; variable modifications carbamidomethyl[C]
and oxidation[M], no fixed modifications. Other parameters for binding sites
identification were set as software default. The binding sites suggested by
pLink 2 were in cross_linkes_sites.csv file.

16. Yeast two-hybrid assay

The full length CAP1, CAP1-N (residues 1-230) and CAP1-C (residues 316-
476) gene were cloned into pGADT7-Rec vector (Clontech, CA, USA) and the
PYL5 gene was cloned into the pGBKT7 vector (Clontech, CA, USA). GAL4-
DNA-binding domain (BD) fused with PYL5 was used as prey, and GAL4-
activating domain (AD) fused with CAP1/CAP1-N/CAP1-C were used as baits.
The interaction was determined by growth assay on media lacking
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 His/Ade/Leu/Trp. Dilutions (1, 0.1, 0.01, and 0.001) of saturated cultures were
spotted onto the plates, and photographed after 4 days.

17. Bimolecular fluorescence complementation (BiFC) assay

For BiFC assay, the full-length coding sequence of PYL5 was subcloned into
the binary vector YFPn to generate PYL5-YFPn plasmid, and the coding
sequences of full length CAP1, CAP1-N (residues 1-230), and CAP1-C
(residues 316-476) were subcloned into the binary vector YFPc to generate
CAP1/CAP1-N/CAP1-C-YFPc plasmids. Agrobacterium tumefaciens strain
GV3101 with indicated YFPn or YFPc vectors were incubated, harvested, and
resuspended in infiltration buffer [0.1 mM acetosyringone, 2 mM
Na3PO4·12H2O, 0.5% D-Glucose and 50 mM MES to identical concentrations
(OD600=1.0), pH 5.8]. Equal concentrations and volumes of Agrobacterium
strains were mixed and coinfiltrated into N. benthamiana leaves with a
needleless syringe. After infiltration, plants were placed at 24°C for 48 h before
observation. YFP fluorescence was detected with a Zeiss microscope (LSM710,
Jene, Germany) and analyzed using the ZEN 2009 software. Scale bar = 50 µm.

18. Protein sequence used in this work

The pBpa incorporating sites are colored in red. The positions for site-directed
mutations are colored in blue.
PYR1-His MW 22711.44 Da
1 MPSELTPEER SELKNSIAEF HTYQLDPGSC SSLHAQRIHA PPELVWSIVR
51 RFDKPQTYKH FIKSCSVEQN FEMRVGCTRD VIVISGLPAN TSTERLDILD
101 DERRVTGFSI IGGEHRLTNY KSVTTVHRFE KENRIWTVVL ESYVVDMPEG
151 NSEDDTRMFA DTVVKLNLQK LATVAEAMAR NSGDGSGSQV TLEHHHHHH

PYL5-His MW 24824.84 Da
MGSSHHHHHHSSGLVPRGSH
1 MRSPVQLQHG SDATNGFHTL QPHDQTDGPI KRVCLTRGMH VPEHVAMHHT
51 HDVGPDQCCS SVVQMIHAPP ESVWALVRRF DNPKVYKNFI RQCRIVQGDG
101 LHVGDLREVM VVSGLPAVSS TERLEILDEE RHVISFSVVG GDHRLKNYRS
151 VTTLHASDDE GTVVVESYIV DVPPGNTEEE TLSFVDTIVR CNLQSLARST
201 NRQ

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 PYL5-Flag MW 24067.94 Da
GPLGS
1 MRSPVQLQHG SDATNGFHTL QPHDQTDGPI KRVCLTRGMH VPEHVAMHHT
51 HDVGPDQCCS SVVQMIHAPP ESVWALVRRF DNPKVYKNFI RQCRIVQGDG
101 LHVGDLREVM VVSGLPAVSS TERLEILDEE RHVISFSVVG GDHRLKNYRS
151 VTTLHASDDE GTVVVESYIV DVPPGNTEEE TLSFVDTIVR CNLQSLARST
201 NRQDYKDDDDK

HAB1(180-511) MW 37233.65 Da
GPLGS
180 SVYELDCIPLW GTVSIQGNRS
201 EMEDAFAVSP HFLKLPIKML MGDHEGMSPS LTHLTGHFFG VYDGHGGHKV
251 ADYCRDRLHF ALAEEIERIK DELCKRNTGE GRQVQWDKVF TSCFLTVDGE
301 IEGKIGRAVV GSSDKVLEAV ASETVGSTAV VALVCSSHIV VSNCGDSRAV
351 LFRGKEAMPL SVDHKPDRED EYARIENAGG KVIQWQGARV FGVLAMSRSI
401 GDRYLKPYVI PEPEVTFMPR SREDECLILA SDGLWDVMNN QEVCEIARRR
451 ILMWHKKNGA PPLAERGKGI DPACQAAADY LSMLALQKGS KDNISIIVID
501 LKAQRKFKTR T

RAD23C-His MW 45572.31 Da
ME
1 MKIFVKTLKG THFEIEVKPE DSVVDVKKNI ESVQGADVYP AAKQMLIHQG
51 KVLKDETTIE ENKVAENSFI VIMMNKSKPA SAAASSASAG TSQAKSIPPS
101 TSQPSISPQT PASVSAPVAP APTRPPPPAP TPTPAPVAAT ETVTTPIPEP
151 VPATISSSTP APDSAPVGSQ GDVYGQAASN LAAGSNLEST IQQILDMGGG
201 TWDRETVVLA LRAAFNNPER AVEYLYTGIP EQAEVPPVAR PPASAGQPAN
251 PPAQTQQPAA APASGPNANP LDLFPQGLPN VGGNPGAGTL DFLRNSQQFQ
301 ALRAMVQANP QVLQPMLQEL GKQNPNLMRL IQDHQADFLRL INEPVEGGGE
351 SGNLLGQMAA GMPQPQAIQV THEEREAIER LEAMGFERAL VLEVFFACNK
401 NEELAANYLL DHMHEFEELE HHHHHH

CSN1-His MW 51904.31 Da
ME
1 MERDEEASGP MMEMCTNGGE ETSNRRPIIS GEPLDIEAYA ALYKGRTKIM
51 RLLFIANHCG GNHALQFDAL RMAYDEIKKG ENTQLFREVV NKIGNRLGEK
101 YGMDLAWCEA VDRRAEQKKV KLENELSSYR TNLIKESIRM GYNDFGDFYY
151 ACGMLGDAFK NYIRTRDYCT TTKHIIHMCM NAILVSIEMG QFTHVTSYVN
201 KAEQNPETLE PMVNAKLRCA SGLAHLELKK YKLAARKFLD VNPELGNSYN
251 EVIAPQDIAT YGGLCALASF DRSELKQKVI DNINFRNFLE LVPDVRELIN
301 DFYSSRYASC LEYLASLKSN LLLDIHLHDH VDTLYDQIRK KALIQYTLPF
351 VSVDLSRMAD AFKTSVSGLE KELEALITDN QIQARIDSHN KILYARHADQ
401 RNATFQKVLQ MGNEFDRDVR AMLLRANLLK HEYHARSARK LLEHHHHHH

CAP1-His MW 52034.95 Da
1 MEEDLIKRLE AAVTRLEGIS SNGGGVVSLS RGGDFSSAAG IDIASSDPSI
51 LAYEDLISQC VGRALTAAEK IGGPVLDVTK IVAEAFASQK ELLVRIKQTQ
101 KPDLAGLAGF LKPLNDVTMK ANAMTEGKRS DFFNHLKAAC DSLSALAWIA
151 FTGKDCGMSM PIAHVEESWQ MAEFYNNKVL VEYRNKDADH VEWAKALKEL
201 YLPGLREYVK SHYPLGPVWN ASGKPASAPA KGPPGAPAPP PAPLFSAESS
251 KPSSSSNQKQ GMSAVFQQLS SGAVTSGLRK VTDDMKTKNR ADRSGAVSAV
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 301 EKETRTSKPA FSKTGPPKME LQMGRKWAVE NQIGKKDLVI SECDSKQSVY
351 IYGCKDSVLQ IQGKVNNITI DKCTKVGVVF TDVVAAFEIV NCNNVEVQCQ
401 GSAPTVSVDN TTGCQLYLNK DSLETAITTA KSSEINVMVP GATPDGDWVE
451 HALPQQYNHV FTEGKFETTP VSHSGALEHH HHHH

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19. Supplementary figure

Figure S1

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Figure S1. The protein mass of PYLs and PYLs-pBpa

14
 
Figure S2

Figure S2. The inhibition of Wild-type PYL5 and PYL5-T187pBpa to HAB1 in

the absence of ABA. The concentrations of each PYLs were set with molar ratios
to HAB1 as 1:1, 10:1, and 20:1, respectively. Reaction without HAB1 was set as
negative control and reaction without inhibitor was set as positive control. Each
reaction was repeated three times and error bars represent standard deviations.
The details of the experiments are described in above supplementary information.

Figure S3

Figure S3. The dose-response inhibition of wild-type PYL5 and PYL5-

T187pBpa to HAB1 when ABA presence. The molar ratios of PYLs to HAB1 as
2:1, 4:1, 6:1, 8:1, 10:1, 15:1 and 20:1, respectively. The details of the
experiments are described in above supplementary information.

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20. Supplementary table

Table S1
Protein Protein names Gene Mol. p- Ratio
IDs names weight value
[kDa]
P93004 Aquaporin PIP2-7 PIP2-7 29.74 0.0185 43.32
Q9SVN5 Probable methionine--tRNA ligase 89.85 0.0296 21.31
P22954 Probable mediator of RNA polymerase II MED37D 71.39 0.0490 21.26
transcription subunit 37c
Q84L31 Ubiquitin receptor RAD23c RAD23C 44.25 0.0144 14.91
Q7XZU1 Phosphoinositide phosphatase SAC4 SAC4 94.05 0.0206 11.86
P45432 COP9 signalosome complex subunit 1 CSN1 50.58 0.0143 11.52
Q9LZ66 Sulfite reductase [ferredoxin], chloroplastic SIR 71.95 0.0128 10.55
Q9LV35 Actin-interacting protein 1-2 AIP1-2 66.04 0.0065 8.83
Q9FYR6 Proline--tRNA ligase, OVA6 60.75 0.0355 8.17
chloroplastic/mitochondrial
Q94A28 Aconitate hydratase 3, mitochondrial ACO3 108.48 0.0431 8.07
Q9ZUC1 Quinone oxidoreductase-like protein 40.99 0.0098 7.89
At1g23740, chloroplastic
Q9C9W5 Glycerate dehydrogenase HPR, HPR 42.25 0.0064 7.73
peroxisomal
Q9FXH1 Pentatricopeptide repeat-containing protein DYW7 100.81 0.0204 7.21
Q9C7X0 Disease resistance protein ADR2 ADR2 113.98 0.0317 6.68
Q94K85 Cathepsin B-like protease 3 CATHB3 39.42 0.0232 6.53
Q9LFW1 UDP-arabinopyranose mutase 2 RGP2 40.89 0.0051 6.32
Q8H156 GTP-binding nuclear protein Ran-3 RAN3 25.08 0.0075 6.26
Q93VP3 Eukaryotic translation initiation factor 5A-2 ELF5A-2 17.14 0.0003 5.91
Q9M8D3 Probable 153.95 0.0136 5.78
phosphoribosylformylglycinamidine
synthase, chloroplastic/mitochondrial
Q8S3U9 Exocyst complex component SEC5A SEC5A 121.90 0.0124 5.61
Q9SAR5 Ankyrin repeat domain-containing protein 2 AKR2 36.99 0.0232 5.36
Q9LDZ0 Heat shock 70 kDa protein 10, HSP70- 72.99 0.0003 5.15
mitochondrial 10
Q8GUM2 Heat shock 70 kDa protein 9, mitochondrial HSP70-9 73.07 0.0283 4.76

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 Q94AM1 Organellar oligopeptidase A, OOP 88.76 0.0020 4.57
chloroplastic/mitochondrial
Q9FLT2 Inositol polyphosphate multikinase beta IPK2b 33.49 0.0210 4.51
P46645 Aspartate aminotransferase, cytoplasmic ASP2 44.27 0.0283 4.45
isozyme 1
Q9M876 Tyrosine--tRNA ligase F16B3.29 56.62 0.0258 4.42
Q42029 Oxygen-evolving enhancer protein 2-1, PSBP1 28.10 0.0023 4.21
chloroplastic
Q9SI75 Elongation factor G, chloroplastic CPEFG 86.06 0.0045 4.11
Q8H0S9 Puromycin-sensitive aminopeptidase MPA1 99.12 0.0081 4.07
Q9FKK7 Xylose isomerase XYLA 53.72 0.0206 3.90
P93736 Valine--tRNA ligase VALRS 125.92 0.0316 3.85
Q5XF33 Magnesium-chelatase subunit ChlI-2, CHLI2 46.10 0.0115 3.78
chloroplastic
Q9FPS3 Ubiquitin carboxyl-terminal hydrolase 24 UBP24 60.44 0.0124 3.76
O65902 Cyclase-associated protein 1 CAP1 50.97 0.0287 3.72
P93028 Ubiquitin-activating enzyme E1 1 UBA1 120.25 0.0329 3.72
Q9CAJ0 Protein phosphatase 2C 16 HAB1 55.74 0.0139 3.54
Q42290 Probable mitochondrial-processing 59.16 0.0205 3.50
peptidase subunit beta
Q8LGH4 Cullin-4 CUL4 91.47 0.0254 3.48
Q9LVJ1 Subtilisin-like protease SBT1.4 At3g1406 81.82 0.0211 3.40
7
Q9LPW0 Glyceraldehyde-3-phosphate GAPA2 42.85 0.0419 3.24
dehydrogenase GAPA2, chloroplastic
Q9LRA7 Probable alpha,alpha-trehalose-phosphate TPS9 98.50 0.0275 3.23
synthase [UDP-forming] 9
Q9SYI0 Protein translocase subunit SECA1, SECA1 115.18 0.0023 3.19
chloroplastic
F4ICX9 TSK-associating protein 1 TSA1 84.38 0.0307 3.16
P11832 Nitrate reductase [NADH] 1 NIA1 103.04 0.0352 3.16
Q41188 Cold shock protein 2 CSP2 19.15 0.0010 3.13
Q56Z59 Patellin-3 PATL3 56.10 0.0137 3.11
P93032 Isocitrate dehydrogenase [NAD] regulatory IDH2 39.59 0.0278 3.11
subunit 2, mitochondrial
Q9SF85 Adenosine kinase 1 ADK1 37.84 0.0069 3.03

Table S1. Putative PYL5 interaction partners.

17
 
Table S2
Peptide 1 Peptide 2
No. PYR1-T162pBpa HAB1
1 MFADXVVK Y(21)L(41)KPYVIPEPEVTFMPR
2 MFADXVVK SIG(7)DRY(2)L(43)K(15)PYVIPEPEVTFMPR
3 MFADXVVK IENAGGK
4 MFADXVVK ILMWHKKNGAPPLAER

PYL5-T187pBpa (Trypsin) HAB1

1 SVTTLHASDDEGTVVVESYIVD Y(15)LKPYVIPEPEVTFMPR
VPPGNTEEETLSFVDXIVR
2 SVTTLHASDDEGTVVVESYIVD S(7)IGDRYLKPYVIPEPEVTFMPR
VPPGNTEEETLSFVDXIVR

PYL5-T187pBpa (Trypsin and HAB1

Chymotrypsin)
1 VDXIVR SIGDRYL(22)K(3)PY
2 VDXIVR YLKPY
3 VDXIVR IENAGGK
4 IVDVPPGNTEEETLSFVDXIVR SIGDRYL(11)K(2)PY
5 IVDVPPGNTEEETLSFVDXIVR SIGDRYLKPYVIPEPEVTF
6 SVTTLHASDDEGTVVVESYIVD SIGDRYL(10)KPY
VPPGNTEEETLSFVDXIVR

PYL5-C191pBpa HAB1
1 XNLQSLAR Y(18)L(23)K(3)PYVIPEPEVTFMPR
2 XNLQSLAR SIGD(2)RY(34)L(130)K(9)PYVIPEPEVTFMPR
3 XNLQSLAR AQRK
4 XNLQSLAR DRLHFALAEEIER
5 XNLQSLAR RILMWHK
6 XNLQSLAR ILMWH(2)K
7 XNLQSLAR KNGAPPLAER
8 XNLQSLAR DELCKR
9 XNLQSLAR IKDELCK

PYL5-T187pBpa RAD23C
1 VDXIVR LEAMGF

PYL5-C191pBpa RAD23C
1 XNLQSLAR LINEP(4)VEGGGES(2)G(2)NLLG(2)Q(2)MAAG
18
 
 MPQPQAIQVTHEER
2 XNLQSLAR AMVQANPQVL(14)Q(37)P(2)ML(7)Q(8)E(58)L
(6)G(28)K
3 XNLQSLAR Q(2)NP(5)N(5)L(45)MR
4 XNLQSLAR NSQ(2)Q(8)F(11)Q(8)A(7)L(3)R
5 XNLQSLAR VLKDETTIEE(3)NK
6 XNLQSLAR LIQ(3)DHQADFLR
7 XNLQSLAR VAENSFIVIMMNK
8 XNLQSLAR QM(2)LIHQGK
9 XNLQSLAR LEAMGFER
10 XNLQSLAR EMKI(2)FVK
11 XNLQSLAR ALVLEVFFACNK

PYL5-T187pBpa CSN1
1 VDXIVR AMLLR
2 VDXIVR AEQKK
3 VDXIVR ELEALITDNQIQAR
4 VDXIVR CEAVDR
5 VDXIVR LELVPDVRELINDFY
6 VDXIVR NATF
7 VDXIVR ELINDFYSSR
8 VDXIVR TNLIK
9 IVDVPPGNTEEETLSFVDXIVR AM(2)LLR

PYL5-C191pBpa CSN1
1 XNLQSLAR NAT(14)F(212)Q(5)K
2 XNLQSLAR NATF(2)QKVLQMGNEFDR
3 XNLQSLAR HADQ(26)R(22)N(7)A(3)TF(3)Q(4)K
4 XNLQSLAR VLQ(2)MGNEFDR
5 XNLQSLAR VLQMG(2)N(2)E(3)F(2)D(3)RDVR
6 XNLQSLAR AMLLR(9)A(2)NL(4)LK
7 XNLQSLAR A(23)MLLR
8 XNLQSLAR LENELSSYRTNLIK
9 XNLQSLAR MAYDEIK

PYL5-T187pBpa CAP1
1 VDXIVR TDVV(2)AAF
2 VDXIVR VTDDMKTK
3 VDXIVR TSKPAF
4 VDXIVR IGGPVLDVTK
19
 
 5 VDXIVR AACDSLSALAW
6 VDXIVR SDFFNHLK
7 VDXIVRCNLQSLAR VGVVF
8 VDXIVRCNLQSLAR VLVEYRNK

PYL5-C191pBpa CAP1
1 XNLQSLAR DCGM(6)SMPIAHVEESWQMAEFYNNK
2 XNLQSLAR QG(2)M(5)S(5)AV(2)FQQLSSGAVTSGLR
3 XNLQSLAR KVTDDMK
4 XNLQSLAR VTD(3)DM(4)K
5 XNLQSLAR VTDDM(2)KTK
6 XNLQSLAR M(4)ELQMGR
7 XNLQSLAR ETRTSKPAFSK
8 XNLQSLAR ADRSGAVSAVEK
9 XNLQSLAR GPPGAPAPPPAPLFSAESSKPSSSSNQK
10 XNLQSLAR VLVEYRNK
11 XNLQSLAR DLVISECDSK
12 XNLQSLAR EEDLIK
13 XNLQSLAR TSKPAFSK
Table S2. Crosslinked peptides and binding sites suggested by pLink 2. X
represents the photo-crosslinking amino acid pBpa. The crosslinking site based on
the MS/MS spectra is colored in red. The numbers in brackets represent spectral
counts.

20
 
21. MS2 of crosslinked peptides

PYR1-T162pBpa and HAB1

 
21
 
PYR1-T187pBpa and HAB1

 
PYR1-C191pBpa and HAB1

22
 
  
PYR1-T187pBpa and RAD23C

23
 
PYR1-C191pBpa and RAD23C

24
 
PYR1-T187pBpa and CSN1

25
 
PYR1-C191pBpa and CSN1

26
 
PYR1-T187pBpa and CAP1

 
PYR1-C191pBpa and CAP1

27
 
 28
 
 29
 
22. MS1 of purified PYLs and PYLs-pBpa mutants

PYR1-His
PYR1-wt-His #357-379 RT: 3.00-3.12 AV: 23 NL: 3.08E5
T: FTMS + p ESI Full ms [500.0000-2500.0000]
842.12887
z=27 874.44117
100 812.08929
784.15512 z=28 z=26
z=29
95
758.05050
z=30
90

85

80 733.56522
z=31
75 909.45844
z=25
70 947.31035
710.76692
z=32 z=24
65 764.05293
z=?
60
Relative Abundance

55
988.45472
50 z=23

45
1033.29350
40 z=?

35 880.45069
z=? 1082.49762
30 z=21

25

20
802.66455 866.80091 961.21476
z=? 833.61368 903.04121
15 z=1 z=? z=? 1073.29072
z=? 979.54137 1022.18065 z=?
z=? z=?
10 916.58028 1043.38694
995.86903 1095.30319
z=? z=? z=?
z=?
5

0
700 750 800 850 900 950 1000 1050 1100
m/z

PYR1-H60pBpa-His
PYR1-H60-PBPF-His #367-394 RT: 3.08-3.23 AV: 28 NL: 2.45E5
T: FTMS + p ESI Full ms [500.0000-2500.0000]
816.16209
761.81793 z=28
100 z=30
788.05334
95 z=29

90 846.35247
z=27
85

80 737.34081 878.86619
z=31 z=26
75

70

913.89990
65
z=25
714.23639
60 z=32 952.06266
z=24
Relative Abundance

55
993.41280
z=?
50

45

40
884.71702 1038.52197
z=? z=?
35
1087.92775
30 z=?
939.77062
25 z=?
780.66166
20 839.31775 907.12975
z=1
802.44596 z=? 871.24694 z=?
15 z=? z=? 980.51621
919.82152 z=? 1030.33722 1098.35855
10 z=? 1045.61967 z=?
z=? 958.18394
z=?
z=? 1003.59100
z=? 1060.26449
5
z=?

0
700 750 800 850 900 950 1000 1050 1100
m/z

30
 
PYR1-F159pBpa-His
PYR1-F159-PBPF-His #371-385 RT: 3.09-3.18 AV: 15 NL: 8.45E4
T: FTMS + p ESI Full ms [500.0000-2500.0000]
787.70855
z=29
100
846.02009
z=? 878.55752
95 815.73334 z=?
z=28
90
761.48542
z=30
85

80 913.66006 951.56158
z=25 z=24
992.89098
75 737.01817 z=23
z=31
70

65

60
Relative Abundance

55 713.95478
z=32 1038.02285
50 z=22 1087.35570
z=21
45

40

35 1045.04308
z=?
777.95723 809.37504
30 z=? z=?
25 986.75027
870.44205 z=? 1029.60118 1094.59914
z=1 z=? z=?
20 885.29335
826.68362 z=?
15 z=1
927.42589
965.11457 1012.81362 1114.04325
10 z=?
z=? z=? 1062.19206 z=?
z=?
5

0
700 750 800 850 900 950 1000 1050 1100
m/z

PYR1-T162pBpa-His
PYR1-T162-PBPF-His #358-375 RT: 3.01-3.11 AV: 18 NL: 3.29E5
T: FTMS + p ESI Full ms [500.0000-2500.0000]
763.01889
z=30 789.32967
100 z=29
817.44844
738.43839
z=28 847.68625
95 z=31
z=27
90

85
880.28972
80 z=26

75
915.38133
z=25
70 715.42436
z=32
65

60
953.47983
Relative Abundance

z=24
55

50 693.74530
z=33 994.93528
45 z=?

40 1040.06863
z=22
35

30
1089.54802
25 924.34176 z=21
776.12140
z=?
20 z=? 810.26839
z=1 988.62727
z=? 1004.93513
15
947.26967 z=?
833.39841 z=? 1051.24771
10 z=? 867.44099 906.05476 z=?
z=? 1100.92686
z=? 972.45357 1028.97062 1080.16909 z=?
5 z=? z=? z=?

0
700 750 800 850 900 950 1000 1050 1100
m/z

31
 
PYR1-L166pBpa-His
PYR1-L166-PBPF-His #376-394 RT: 3.12-3.23 AV: 19 NL: 1.43E5
T: FTMS + p ESI Full ms [500.0000-2500.0000]
788.88038
z=29
100
762.61792 816.98337
95 z=30 z=28 847.27803
z=27
90 738.08211
z=31
85

80
879.82782
75 z=26

70 715.01777
z=32 915.02061
z=25
65
953.10308
z=?
60
Relative Abundance

55

50

45

40 994.45596
z=23
776.40091
35 1039.56842
z=?
z=22
30
923.24843 1089.02322
25 809.83829 z=?
z=?
z=?
20 1096.17028
z=?
15 831.67478
z=? 1030.59120
944.92919 z=? 1078.30627
857.39237 891.13137 z=? z=?
10 z=? 971.62280 1002.19026
z=? 1047.52743 1115.66710
z=? z=? z=?
z=?
5

0
700 750 800 850 900 950 1000 1050 1100
m/z

PYR1-T173pBpa-His
PYR1-T173-PBPF-His #337-396 RT: 2.86-3.24 AV: 60 NL: 1.08E5
T: FTMS + p ESI Full ms [500.0000-2500.0000]
817.44795
z=28
100 789.32934
763.05215 z=29
95 738.47036 z=30
z=31
90

85 847.72360
z=27
80
880.25125
75 z=?
715.36237
z=32
70

915.38087
65
z=25
60
Relative Abundance

55 953.52263
z=24
50

45 909.21409
z=? 994.89125
40 z=?

35 1040.15944
z=22
30

1089.54776
25
z=21
802.72667
20 z=?
1082.23218
799.98775 1002.59140 z=?
z=? z=?
15 832.42077 864.47688
z=1 z=?
10 936.59786 977.01326
z=? z=? 1021.51497 1050.48340
5 z=? z=? 1100.35677
z=?
0
700 750 800 850 900 950 1000 1050 1100
m/z

32
 
PYL5-His
PYL5-wt-His #359-388 RT: 3.03-3.20 AV: 30 NL: 8.34E4
T: FTMS + p ESI Full ms [500.0000-2500.0000]
749.31764 772.60881
z=33 z=32
100
727.25053
z=34 824.04916
95
797.46601 z=30
z=?
90 852.49836
z=?
85

80

75
706.50070
z=?
70

65 882.83711 915.49868
z=? z=27
60
Relative Abundance

55

754.59190 950.70953
50
z=?
z=?
45 732.54561
711.64388 z=? 778.20433 803.24220
z=1 z=32 z=?
40 830.11689 988.73680
z=30 858.70588 z=25
35 789.36488 z=29
889.34136 922.16669
z=? z=27
847.57601 z=?
30 817.44646
z=? z=? 869.93727
z=1 957.51805
25 z=? 995.89616
970.96277 z=?
20
z=?
15 945.14112 983.89068
z=? z=?
899.05578
10
z=?

0
700 720 740 760 780 800 820 840 860 880 900 920 940 960 980 1000
m/z

PYL5-N176pBpa-His
PYL5-N176pBpa-His_190918194401 #537-784 RT: 3.61-5.03 AV: 248 NL: 1.85E4
T: FTMS + p ESI Full ms [150.0000-2000.0000]
955.85873
100
994.05249
95
920.49308 1035.43034
90

85
887.61967
80
1080.36005
75
857.11725
70
828.57860
65
1129.46486
60 801.94535
Relative Abundance

55

50
1183.20416
45

40
863.28865 894.08643 962.74358
35 842.19799 926.93755 1001.72863
905.54878
814.88973 1042.80777
30 974.60502
941.25649 1088.18823
1095.04542
25 1018.66452
1057.44168
1137.61014
20 1175.92182
1112.89829 1154.13117 1212.21548
15

10

0
800 820 840 860 880 900 920 940 960 980 1000 1020 1040 1060 1080 1100 1120 1140 1160 1180 1200
m/z

33
 
PYL5-S183pBpa-His
PYL5-S183pBpa-His_190918210523 #374-680 RT: 2.90-4.57 AV: 307 NL: 4.56E4
T: FTMS + p ESI Full ms [150.0000-2000.0000]
759.58969 783.38983
100
808.59524
95 835.48154

90
737.33707
85
777.79379

80 754.25536 864.32557

75 802.85151
732.07075 829.51349
70 716.24179
858.11574
65
895.08756
60
691.45628
Relative Abundance

55 888.72974

50
854.70988
677.66263
45
685.37574
40 704.51992
725.10992
35
786.69540
845.79356
30 745.48921
764.94470
811.91905
25 868.04298
795.13189
20
898.79972
873.82355
15

10

0
680 690 700 710 720 730 740 750 760 770 780 790 800 810 820 830 840 850 860 870 880 890 900
m/z

PYL5-T187pBpa-His
PYL5-T187pBpa-His #411-488 RT: 3.03-3.44 AV: 78 NL: 7.94E4
T: FTMS + p ESI Full ms [150.0000-2000.0000]
777.35619
100

95
753.82986 802.43246
90 829.04667

85
731.68879
80

75
710.81249 857.63497
70
691.03981
65
750.14571

60 888.33572
Relative Abundance

55
672.44426
50
853.76629
45
736.80683 808.14392
40 715.87004
654.80175
720.59861 759.25666
35 782.76218
835.01350 863.84107
30
696.01413
25 677.28083
764.48967 871.76430 894.62473
659.43254 815.56405 842.07343
794.33764
20 883.42501

15

10

0
660 680 700 720 740 760 780 800 820 840 860 880 900
m/z

34
 
PYL5-C191pBpa-His
PYL5-C191-PBPF-His #358-387 RT: 3.04-3.20 AV: 30 NL: 3.46E4
T: FTMS + p ESI Full ms [500.0000-2500.0000]
802.34007
100

95 777.29862

90
731.54739
753.89553 829.05192
85

80

75 710.72928 857.46534

70
888.12603
65 758.22207 788.42084

60
863.67509
Relative Abundance

55 724.67103 920.90628
956.36632 994.49190
834.98421
50
815.45624
950.45984
736.92813 894.55793
45
846.09559
40 875.20363
906.33962 1001.50185
35 937.72463
978.28527
30 963.71364

25

20

15

10

0
700 720 740 760 780 800 820 840 860 880 900 920 940 960 980 1000
m/z

PYL5-Flag
6P-PYL5-wt #362-386 RT: 3.06-3.20 AV: 25 NL: 1.09E5
T: FTMS + p ESI Full ms [500.0000-2500.0000]
860.53917
803.23712
z=28
100 z=30
830.93446
z=29
95 777.32691
753.12938 z=31
z=32
90

85

80 730.33786 870.72914
z=? z=?
75 892.41044
z=27
70
926.69537
z=26
65

60
Relative Abundance

708.85796
z=? 963.68404
55 z=?

50

45
1003.92025
40 z=1

35 688.69010
z=? 1047.43984
z=23 1094.81854
30 z=1
912.57744 1147.04840
z=? z=?
25
669.56107
20 z=2

15
1104.54606 1156.87835
651.49150 944.64276 z=?
z=? 981.45517 z=?
10 z=? z=? 1028.52071
1081.54215
z=?
5 z=?

0
650 700 750 800 850 900 950 1000 1050 1100 1150
m/z

35
 
PYL5-T187pBpa-Flag
6P-PYL5-T187-PBPF #360-382 RT: 3.05-3.20 AV: 23 NL: 4.66E4
T: FTMS + p ESI Full ms [500.0000-2500.0000]
777.39166
z=?
100

95

90 750.65408
z=?
870.59881
85 z=25
806.22122
z=? 837.11323
80 z=?
906.91402
75 z=?

70
946.21382
z=23
65
725.59891
60 z=?
Relative Abundance

55

50

45 989.27054
702.25883
z=22
40 z=? 1036.23440
z=21
35 1087.99659
684.20412 z=?
z=2
30
853.68739 1078.88942
890.28964 926.69270
25 z=1 z=1 z=?
z=? 1145.15251
20 964.14816 z=?
659.81818
z=? 1006.15718
z=? z=? 1101.97914
15 z=?
1048.93653
z=? 1176.59711
10 1120.99025
z=? z=?
5

0
650 700 750 800 850 900 950 1000 1050 1100 1150
m/z

PYL5-C191pBpa-Flag
6P-PYL5-C191-PBPF #370-425 RT: 3.13-3.47 AV: 56 NL: 2.54E4
T: FTMS + p ESI Full ms [500.0000-2500.0000]
757.75633
z=32
100
808.23915
95 782.13694 z=1
z=31 835.97123
90 z=?

85
865.79233
80 z=28

75 880.36444
z=26
70 897.82059
z=?
65 734.82612
z=?
60
Relative Abundance

713.21210
55 z=? 932.39064
z=26 969.64669
50 z=25

45
692.97824 1010.00477
40 z=1 z=?
906.87277
35 684.20356 1053.74472
z=? 1101.82749
z=2 941.71190 z=?
30 z=1 z=1
980.82719
z=1 1154.01014
25 673.70118 1023.51580 1070.12390 z=?
z=?
z=? z=?
20

15 645.28335
z=?
10

0
650 700 750 800 850 900 950 1000 1050 1100 1150
m/z

Reference
1. Chin, J. W.; Martin, A. B.; King, D. S.; Wang, L.; Schultz, P. G., Addition of a
photocrosslinking amino acid to the genetic code of Escherichia coli. Proc Natl
Acad Sci U S A 2002, 99 (17), 11020-4.

36