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Supplemental Information for:

Lysine Acetylation is a Widespread Protein Modification for Diverse Proteins in Arabidopsis Xia Wu, Man-Ho Oh, Eliezer M. Schwarz, Clayton T. Larue, Mayandi Sivaguru, Brian S.

Imai, Peter M. Yau, Donald R. Ort, and Steven C. Huber

Experimental Procedures

Plant materials

Arabidopsis ecotype Ws-2 was used in the study. Majority of tissues were harvested

from the plants that were grown in soil under the conditions of 22° C, 150 μmol m-2 s-1,

12 h photoperiod with 60% humidity. In some experiments, seedlings were grown on

agar plates containing half-strength MS media, pH 5.7, with 0.7% agar and 0.5% sucrose.

Protein direct extraction.

Direct extraction buffer (DEB) contained 100 mM Tris pH 8.0, 1.5 M 2-

mercaptoethanol, 4% SDS, 15% glycerol, 5 mM NaF, 1 mM Na3VO4, 1 mM AEBSF, 2

mM EDTA and 0.005% bromophenol blue. Arabidopsis frozen tissue powders were

quickly mixed with DEB at a 1:3 ratio (g ml-1) by vortexing for 30 seconds. The mixture

was heated at 95° C for 5 minutes in a heat block, and vortexed again for 30 seconds. The

crude extracts were collected as the supernatants after centrifuging the mixture at 16,000g

for 15 min at room temperature. Protein concentration was measured with the Bradford

assay (Bio-Rad).

Antibodies

Generic anti-lysine acetylation antibodies were obtained from Cell signaling

(#9681S) and Immunechem (#ICP0380) and were used for LysAc peptide affinity

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enrichment. Immunechem antibodies (#ICP0380) was also used for the immunoblotting.

Sequence- and modification-specific antibodies were produced and affinity purified

against the following peptides: anti-eEF-1A-acK306, KNVAVacKDLKRG; and anti-

eEF-1A-acK227, QINEPacKRPSDK, by GenScript and were used to confirm specific

lysine acetylation sites on eEF-1A. Generic anti-lysine trimethylation antibodies were

obtained from Immunechem (#ICP0601). Sequence-specific antibodies against LHCb

were obtained from Agrisera (#AS01 004-10) and custom anti-EF1α antibodies were

produced and affinity purified against the peptide IDRRSGKEIEKEPK and were

produced by GenScript. Blots were blocked with 2% fish gelatin (Sigma-Aldrich) and

Alexa Fluor 680 (Invitrogen) or IRDye 800 (Rockland) was used as the secondary

antibodies. Images were viewed using a Li-Cor Odyssey and analyzed using Odyssey

software.

2-DE

The direct extraction samples were cleaned up with phenol-Tris (pH 8.0) for 3 times.

The proteins were precipitated by 0.1M ammonium acetate in methanol overnight at -

20°C. The pellets were washed 3 times with ice-cold 0.1M ammonium acetate in

methanol and one time of ice-cold 100% ethanol before dissolving in IEF sample buffer

(7 M urea, 2 M thiourea, 4% Chaps, 2% IPG buffer, 65 mM DTT and 0.002% (v/v)

bromophenol blue). We avoided heating with urea and limited the dissolving time to

minimize the in vitro carbamylation on lysine residues (McCarthy et al., 2003). Protein

(250 μg) was loaded on IEF strips (pH 3-10, 13cm; GE Healthcare) using the in-gel

rehydration protocol for 36 kVh at 20° C. After the IEF, the strips were equilibrated in

fresh DTT equilibration buffer (50 mM Tris, pH 8.8, 30% glycerol, 2% SDS, 6 M Urea,

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0.002% bromophenol blue and 65 mM DTT) for 25 min and subsequently in fresh IAA

equilibration buffer (50 mM Tris, pH 8.8, 30% glycerol, 2% SDS, 6 M Urea, 0.002%

bromophenol blue and 135 mM iodoacetamide) for 25min. The equilibrated strips were

loaded on traditional SDS-PAGE gels for the second dimension.

Green Native Gel Electrophoresis

Arabidopsis shoots tissue was ground in a Dounce homogenizer with 1ml

homogenization buffer containing 50 mM Tris, pH 7.4, 10 mM MgCl2, and 5 mM KCl.

The homogenate was filtered through four layers of Miracloth® and samples were

normalized for chlorophyll content before centrifugation at 6,000g for 10 min.

Supernatants were aspirated and thylakoid pellets were solubilized in homogenization

buffer containing 30% glycerol, 1% decylmaltoside and 1% n-octylglucoside at a 20:1

ratio of detergent to chlorophyll (w/w). Insoluble debris was removed by centrifugation

at 16,000g for 10 min. Solubilized thylakoid samples were loaded for first dimension

native green gel analysis according to Allen and Staehelin (Allen and Staehelin, 1991).

Gel image was recorded before transferring to PVDF membrane for immunoblotting. For

second dimension of protein components analysis, individual bands were excised from

green gels. Bands were denatured in gel slices by incubation at 80° C in buffer

containing 100mM Tris pH 6.8, 2% SDS, 5 % β-mercaptoethanol for 10 min. Gel slices

were then physically loaded above the stacking gel of standard SDS-PAGE gels for

electrophoresis. Finally, the gel was transferred to PVDF for immunoblotting.

LC-MS analysis

Frozen tissues were extracted in the buffer containing only competitive protease

inhibitors (100 mM MOPS, pH7.5, 10 mM DTT, 5 mM EDTA, 5 mM caproic acid, 1

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mM benzamidine, 2 mM DPDS, and 2 μM leupeptin). The extracts were cleaned up and

the buffer was changed to 50 mM NH4HCO3, using spin columns (microspin 6, Bio-Rad;

Zeba Desalt spin column 5ml, Thermo Scientific). Then the proteins were denatured by

heating at 95° C for 5min. After further reduction with10 mM DTT and subsequent

alkylation by 50 mM iodoacetamide, the extracts were digested overnight with trypsin in

a 1:20 ratio (w/w) (Promega). The digestion was quenched by addition of 0.1% TFA to

pH 3.0. The digested peptides were purified with C18 SPE column (Discoverysciences)

and dried in speedvac. The dried peptides were either dissolved in 0.1% formic acid, 5%

acetonitrile for LC-MS anlaysis or reconstituted with immunoprecipitation buffer for

enrichment of lysine aceylated peptides as described by Choudhary (Choudhary et al.,

2009). Besides in solution digestion analysis, specific interested spots of 1-D or 2-D gels

were cut out for in gel digestion analysis. Trypsin or Glu-C (Roche) proteases were used

as described by Shevchenko (Shevchenko et al., 2007).

LC-MS/MS analysis was performed on a Waters Q-Tof API-US Quad-ToF mass

spectrometer with a nanoAcquity UPLC system. The columns used were Waters

nanoAcquity UPLC (75 micron X 150 mm 3 micron Atlantis dC18) and Atlantis dC18 5

micron Nanoease trap columns. A linear gradient of 1% to 60% acetonitrile in 0.1%

formic acid over 60 minutes was used to elute peptides from the columns. MS/MS data

were collected using the Data Directed Analysis (DDA) method in MassLynx to fragment

the top four ions in each survey scan. ProteinLynx (Waters) was used to process the

mass spectral data into peak list files (PKL) for analysis by Mascot (Matrix Science)

and/or Phenyx (Genebio). Database searches were performed against the NCBI non-

redundant database limited to Arabidopsis thaliana. Cysteine carbamidomethylation was

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selected as a fixed modification and lysine acetylation, N-terminal acetylation and

methionine oxidation were selected as variable modifications in an error tolerant search

with a mass tolerance of 0.5 Da for parent and fragment ions. Each identified lysine

modification peptide was confirmed manually and assembled into Table 1, Table S2, and

Supplementary Figure S9.

Immunolabeling of LysAc in leaf cells

Fully expanded leaves were dissected out and fixed with 4% paraformaldehyde

(Electron Microscopy Sciences) and dehydrated in graded ethanol series. The leaf

samples were embedded in paraffin after processed in an automatic tissue processor (ASP

300, Leica) as the samples were bathed at 0, 80, 95 and 100% (2x) ethanol followed by

2x xylene and then 3x in 100% paraffin wax. Leaf sections of 5 μm were cut using a

microtome (RM 2265, Leica), floated on a water bath and let adhere to the surface of

positively charged slides. The sections were dewaxed in Histoclear (Electron Microscopy

Sciences) and rehydrated in graded ethanol in PBS. Additional incubation of 0.5% Triton

X-100 and 100% methanol were performed to increase the permeability of the tissues.

Sections were then blocked by IT signal Fx (Invitrogen), before incubating in a cocktail

of anti-LysAc mouse monoclonal antibody (Cell Signaling) for overnight, diluted at

1:100 in PBS with signal Fx. Control slides without adding anti-LysAc was kept in

blocking solution overnight. The sections were probed with Alexa 488 Goat anti-Mouse

secondary antibody (Invitrogen) and counter stained with either a membrane dye

(10µg/mL) FM143 (Invitrogen) or a nuclear dye (100µg/mL in water) Hoechst 33342

(Invitrogen) afterwards. Finally the sections were mounted in Prolong Gold (Invitrogen),

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an anti-fade mounting media under a #1.5 cover glass, cured for 24h, sealed and stored at

4º C until analysis.

Confocal laser scanning microscopy and image analysis

Samples were imaged using either a 40x C-apochromat 1.2 NA water immersion

objective or a 63x plan apochromat 1.4 NA DIC oil objective in a Zeiss LSM 710 laser

scanning confocal microscope (Carl Zeiss). The images were obtained using a 405 UV

diode laser (420-455 emission for Hoechst), 488 Ar laser for LysAc (505-555 emission),

chlorophyll autofluorescence (660-740 emission) and 561 DPSS laser for FM143 (566-

616 emission) obtained in a sequential manner. Laser power, gain and offset were kept

constant across the samples and scanned in a high resolution format of 1024x1024 pixels

with 2 frames averaging. Multiple 2D image stacks has been taken along the Z axis if

necessary. Raw data of Z stacks were deconvolved using a blind deconvolution algorithm

under 10 constrained iterations and medium noise setting (Autoquant version X 2.2,

Mediacybernatics). Single optical plane of individual channels of LysAc, nuclei stain,

membrane stain and chlorophyll autofluorescence were recorded (with or without zoom),

and all optical planes were displayed as a gallery and 3D volume rendered using the

Imaris suite software (Bitplane). Differential interference contrast (DIC) images in the

same 3D stacks for a specific optical plane were picked up using either the Imaris

program or the Zen 2009 software (Carl Zeiss). The Imaris software was also used to

create 3D animations showing all optical planes and saved at 10 frames per second avi

format movie with 5% default compression. All final images were organized and

assembled in Photoshop software (Adobe Systems) and the levels were adjusted in all

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images at the same time (entire panel) to display the best signal to noise ratio range. The

images were representative of three independent experiments.

Recombinant protein purification and protein interaction analysis

Recombinant calmodulin proteins (CaM) were expressed in BL21 (DE3) cells

(Novagen) and purified with phenyl-Sepharose affinity chromatography (GE Healthcare).

Protein interactions were monitored with Octet QK (Forte-Bio). Biotinylated eEF-1A

peptides were immobilized on SA sensors (Forte-Bio) and the association of CaM

analytes were tested in the binding buffer of 50mM MOPS (pH7.5), 0.5% NP-40 and

100μM CaCl2. The equilibrium constant (KD) was calculated based on 1:1 model in Octet

Origin software (Forte-Bio).

LITERATURE CITED Allen KD, Staehelin LA (1991) Resolution of 16 to 20 chlorophyII-protein complexes

using a low lonic strength native green gel system. Analytical Biochemistry 194: 214-222

Choudhary C, Kumar C, Gnad F, Nielsen ML, Rehman M, Walther TC, Olsen JV, Mann M (2009) Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325: 834-840

McCarthy J, Hopwood F, Oxley D, Laver M, Castagna A, Righetti PG, Williams K, Herbert B (2003) Carbamylation of proteins in 2-D electrophoresis--myth or reality? J Proteome Res 2: 239-242

Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M (2007) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protocols 1: 2856-2860

Table S1 Functional examples of LysAc candidate sites

Implication of the LysAc site Position Protein AT number Score Peptide

Interfere with the inter salt bridge formation of Rubisco holoenzyme K252 Ribulose bisphosphate

carboxylase large chain AtCg00490, Rbcl 73 GHYLNATAGTCEE(ox)MI(ac)KR

Within ATP binding domain, control ATP synthase catalytic activity K178 ATP synthase CF1 beta

subunit, chloroplastic AtCg00480 51 IGLFGGAGVG(ac)KTVLI(ox)MELINNIAK

Inhibit translation, inhibit CaM binding K306 Eukaryotic elongation factor 1A

At5g60390, At1g07940, At1g07930, At1g07920 36 NVAV(ac)KDLK

Play a role in LHCb trimer assoiciation K37 Chlorophyll a/b binding protein (LHCP AB 180)

At1g29910, At1g29920,At1g29930 44 R(ac)KTVAKPKGPSGSPWYGSD

Alternative splicing K41 Chlorophyll a-b binding protein CP26, LHCB5 At4g10340 49 LFS(ac)KKKPAPAKSKAVSE

Tissue Go Function Location Position Protein AT number Mr(expt) Mr(calc) Delta MC Score Peptide

shoot Other biological p chloroplast K252 Ribulose bisphosphate ca AtCg00490, 1949.5456 1949.9088 -0.3632 1 73 GHYLNATAGTCEE(ox)MI(ac)KRshoot Response to abiotchloraplast,K178 ATP synthase CF1 beta suAtCg00480 2485.6756 2485.3978 0.2778 1 51 IGLFGGAGVG(ac)KTVLI(ox)MELINNIAKshoot Response to abiotchloraplast,K178 ATP synthase CF1 beta suAtCg00480 2485.6726 2485.3978 0.2748 1 46 IGLFGGAGVG(ac)KTVLI(ox)MELINNIAKroot Other metabolic p n.a. K257 N-acetyltransferase hookleAt5g67430 1140.492 1140.698 -0.206 1 29 LQV(ac)KGASRLKshoot Other cellular procchloroplast K216 33 kDa oxygen-evolving p At5g66570 2423.2471 2423.2737 -0.0266 1 30 VPFLFTV(ac)KQLDASGKPDSFTGKshoot Other cellular procchloroplast K216 33 kDa oxygen-evolving p At5g66570 2423.5612 2423.2737 0.2875 1 56 VPFLFTV(ac)KQLDASGKPDSFTGKroot Protein metabolismribosome, eK227 Eukaryotic elongation fact At5g60390, 2628.1221 2628.4235 -0.3014 1 91 GPTLLEALDQINEP(ac)KRPSDKPLRroot Protein metabolismribosome, eK79 Eukaryotic elongation fact At5g60390, 1776.8634 1776.9662 -0.1027 1 65 GITIDIALW(ac)KFETTKroot Protein metabolismribosome, eK79 Eukaryotic elongation fact At5g60390, 1776.8710 1776.9662 -0.0951 1 50 GITIDIALW(ac)KFETTKshoot Protein metabolismribosome, eK36 Eukaryotic elongation fact At5g60390, 799.5110 799.4552 0.0559 1 23 LGGID(ac)KRroot Protein metabolismribosome, eK36 Eukaryotic elongation fact At5g60390, 799.4438 799.4552 -0.0113 1 26 LGGID(ac)KRshoot Protein metabolismribosome, eK306 Eukaryotic elongation fact At5g60390, 927.7124 927.5389 0.1735 1 32 NVAV(ac)KDLKshoot Protein metabolismribosome, eK306 Eukaryotic elongation fact At5g60390, 927.5894 927.5389 0.0505 1 36 NVAV(ac)KDLKroot Protein metabolismribosome, eK306 Eukaryotic elongation fact At5g60390, 927.4624 927.5389 -0.0765 1 31 NVAV(ac)KDLKshoot Protein metabolismribosome, eK187 Eukaryotic elongation fact At5g60390, 2735.2684 2735.3265 -0.0581 1 57 VGYNPD(ac)KIPFVPISGFEGDNMIERshoot Protein metabolismribosome, eK187 Eukaryotic elongation fact At5g60390, 2735.3791 2735.3265 0.0526 1 55 VGYNPD(ac)KIPFVPISGFEGDNMIERroot Protein metabolismribosome, eK187 Eukaryotic elongation fact At5g60390, 2735.0734 2735.3265 -0.2531 1 57 VGYNPD(ac)KIPFVPISGFEGDNMIERshoot Protein metabolismribosome, eK227 Eukaryotic elongation fact At5g60390, 2628.3433 2628.4235 -0.0802 1 75 GPTLLEALDQINEP(ac)KRPSDKPLRshoot Protein metabolismribosome, eK227 Eukaryotic elongation fact At5g60390, 2628.9049 2628.4235 0.4814 1 79 GPTLLEALDQINEP(ac)KRPSDKPLRflower n.a. endomemb K873 Putative cell expansion proAt5g49680, 1313.4836 1313.7779 -0.2943 2 28 IV(ac)KSSRVQISR root n.a. endomemb K873 Putative cell expansion proAt5g49680, 1313.5608 1313.7779 -0.2171 2 21 IV(ac)KSSRVQISR shoot Developmental pr endomemb K141 Probable 3-ketoacyl-CoA sAt5g49070 883.0310 883.5239 -0.4929 2 17 KN(ac)KLSPRroot n.a. n.a. K142K146RNA recognition motif (RRAt5g46840 799.4338 799.4552 -0.0214 2 27 (ac)KVGE(ac)KRshoot Response to abiotchloroplast,K92 Ribulose bisphosphate ca At5g38410 2365.5541 2365.1201 0.4340 1 43 (deamination)N(ac)KWIPCVEFELEHGFVYRshoot Response to abiotchloroplast,K92 Ribulose bisphosphate ca At5g38410 2365.5721 2365.1201 0.4520 1 87 (deamination)N(ac)KWIPCVEFELEHGFVYRroot Protein metabolismn.a. K409 DNAJ heat shock N-terminAt5g37380 1059.3596 1059.5560 -0.1964 1 34 ESAD(ac)KLSLRroot Protein metabolismn.a. K409 DNAJ heat shock N-terminAt5g37380 1059.3930 1059.5560 -0.1630 1 22 ESAD(ac)KLSLRshoot Other cellular procendomemb K37 Glutaredoxin-C4 At5g20500 841.4178 841.4657 -0.0479 1 27 (ac)KTISSHKshoot Other cellular procendomemb K37 Glutaredoxin-C4 At5g20500 841.0394 841.4657 -0.4263 1 27 (ac)KTISSHKshoot Other cellular procendomemb K37 Glutaredoxin-C4 At5g20500 841.0518 841.4657 -0.4139 1 29 (ac)KTISSHKshoot Unknown biologic n.a. K180 Zinc finger (C2H2 type) fa At5g14140 870.5798 870.5035 0.0763 2 41 RE(ac)KLQRroot Unknown biologic n.a. K80 Calmodulin-binding proteinAt5g10660 1596.6354 1596.8583 -0.2229 2 32 KNNAVEENQ(ac)KLLRshoot Other cellular procn.a. K7 SWAP (Suppressor-of-WhAt5g06520 1338.1996 1338.6060 -0.4064 1 25 MYTSMH(ac)KDLRshoot Unknown biologic n.a. K294 Transducin family protein /At4g38480,F999.7104 999.5713 0.1392 2 33 (ac)KKDGELLR seed Developmental pr plasma memK133 Embryo SAC developmen At4g37890 1403.5402 1403.8071 -0.2668 2 21 LCFT(ac)KVRILNR seed Developmental pr plasma memK134 Embryo SAC developmen At4g37890 1403.5354 1403.8071 -0.2717 2 17 LCFT(ac)KVRILNR seed Developmental pr plasma memK135 Embryo SAC developmen At4g37890 1403.5388 1403.8071 -0.2682 2 16 LCFT(ac)KVRILNR shoot Unknown biologic n.a. K519 Putative uncharacterized pAt4g18120 1734.2986 1733.9498 0.3488 2 20 TTLMI(ac)KNIPNKYTRroot Transcription, othen.a. K217 NAC domain containing prAt3g56530 1201.5414 1201.6918 -0.1503 1 35 AEEAENE(ac)KLKshoot Protein metabolismn.a. K131 Putative uncharacterized pAt3g49540 912.0224 912.4916 -0.4692 1 21 (ac)KAEAEPVKshoot n.a. n.a. K191 ABC transporter A family mAt3g47770, 1199.8334 1199.6332 0.2002 0 17 MVNVS(ac)KPEVRshoot n.a. n.a. K191 ABC transporter A family mAt3g47770, 1199.5068 1199.6332 -0.1264 1 16 MVNVS(ac)KPEVRshoot n.a. n.a. K191 ABC transporter A family mAt3g47770, 1199.7654 1199.6332 0.1322 0 16 MVNVS(ac)KPEVRseed Other biological p endomemb K452 AAA-type ATPase family pAt3g28580 1377.4162 1377.6558 -0.2395 2 17 SEKEGGETCL(ac)KR flower Cell organization anucleus K27 Histone H3 At3g27360 956.3624 956.5291 -0.1666 1 55 (ac)KSAPATGGVKseed Unknown biologic n.a. K7 Unnamed protein At3g21320, 1107.4484 1107.5602 -0.1118 2 16 (N-terminal-acetyl) (ox)MMMKLA(ac)KRseed Unknown biologic n.a. K8 Unnamed protein At3g21320, 1107.4484 1107.5602 -0.1118 2 15 (N-terminal-acetyl) (ox)MMMKLA(ac)KRshoot Transcription, othenucleus K66 GATA transcription factor At3g16870 1087.2172 1087.5920 -0.3748 2 39 (ac)KRQAALG(ox)MRshoot Unknown biologic n.a. K462 Transposable element genAt3g14800 1808.8456 1808.8727 -0.0270 1 26 NQDNFLSSLAANM(ac)KAKflower Other metabolic p chloraplast K10 Terpene synthase-like proAt3g14490 1162.3414 1162.6597 -0.3183 1 22 (N-terminal-acetyl)VRD(ox)ML(ac)KSSKshoot Unknown biologic n.a. K492 Pentatricopeptide (PPR) reAt3g13770 898.5332 898.4872 0.0460 1 19 AVT(ac)KEPGRseed Protein metabolismextracellula K136 Pectate lyase family prote At3g09540 1325.4122 1325.8143 -0.4021 3 22 GQ(ac)KVKITGKGLR seed Protein metabolismextracellula K136 Pectate lyase family prote At3g09540 1325.4122 1325.8143 -0.4021 3 20 GQ(ac)KVKITGKGLR shoot Cell organization aribosome K41 60S ribosomal protein L12At2g37190, 914.4862 914.5073 -0.0210 1 24 (ac)KIGEDIAKshoot Cell organization aribosome K41 60S ribosomal protein L12At2g37190, 957.5068 957.5131 -0.0062 1 40 (N-term carbamylation) (ac)KIGEDIAKroot Cell organization aribosome, mK4 60S ribosomal protein L12At2g37190, 1794.7936 1794.9880 -0.1944 1 57 (ethyl)PP(ac)KLDPSQIVDVYVRshoot Developmental pr nucleus K383 ADP-ribosylation factor GTAt2g35210 1007.4118 1007.4593 -0.0475 1 29 N(ox)MAEET(ac)KKshoot Response to stresnucleus K6K9K13 Histone H4; DNA binding At2g28740 1437.7700 1437.8052 -0.0351 4 80 G(ac)KGG(ac)KGLG(ac)KGGA(ac)KRshoot Response to stresnucleus K6K9K13 Histone H4; DNA binding At2g28740 1438.0842 1437.8052 0.2791 4 89 G(ac)KGG(ac)KGLG(ac)KGGA(ac)KRshoot Developmental pr ribosome K90 60S ribosomal protein, L10At2g27530, 1476.5756 1476.7316 -0.1560 1 56 MGLSNMDVEAL(ac)KKshoot Transcription, otheIntracellularK363 FZF; transcription factor, eAt2g24500 1379.6173 1379.6835 -0.0663 3 41 (ox)M(ac)KVMREMNKRshoot Transcription, otheIntracellularK363 FZF; transcription factor, eAt2g24500 1379.9089 1379.6835 0.2253 3 36 (ox)M(ac)KVMREMNKRshoot Protein metabolismn.a. K133 Ubiquitin-conjugating enzyAt2g16740 909.5660 909.4378 0.1283 1 47 (ac)KYEAMARshoot Protein metabolismn.a. K133 Ubiquitin-conjugating enzyAt2g16740 909.4276 909.4378 -0.0101 1 47 (ac)KYEAMARshoot Protein metabolismn.a. K133 Ubiquitin-conjugating enzyAt2g16740 909.4348 909.4378 -0.0029 1 46 (ac)KYEAMARseed DNA or RNA metan.a. K764 Putative ATP-dependent DAt2g01440 1633.5230 1633.8821 -0.3591 2 22 SKCLLVGSSTNSL(ac)KRshoot Other biological p extracellula K94 GDSL esterase/lipase EXLAt1g75910 1217.8040 1217.6404 0.1636 1 13 (ac)KLFNSPSDLRshoot Developmental pr nucleus, cytK837 AGO1 (ARGONAUTE 1),eAt1g48410 1092.5326 1092.6152 -0.0826 1 17 RSTGH(ac)KPLRshoot Unknown biologic n.a. K5 Unknown protein, F2J6.14At1g43760 643.3798 643.4017 -0.0218 1 26 LSV(ac)KRflower Other biological p n.a. K7K10 Auxin-responsive family prAt1g29430 914.3324 914.5623 -0.2298 2 20 (ac)KLM(ac)KLAKroot Other biological p n.a. K7K10 Auxin-responsive family prAt1g29430 914.4764 914.5623 -0.0858 2 20 (ac)KLM(ac)KLAK flower Other cellular procmitochondriK81 CYTC-1 (Cytochrom C-1);At1g22840 1378.4898 1378.7496 -0.2598 1 61 ALYDYLLNP(ac)KKshoot Transport endomemb K283 SEC14 cytosolic factor, puAt1g19650 1162.5508 1162.6234 -0.0725 1 19 SFLDP(ac)KTVSKshoot Unknown biologic n.a. K607 Aminoacyl-tRNA synthetasAt1g18950 943.5014 943.5385 -0.0371 3 54 A(ac)KK(ox)MPRRshoot Unknown biologic n.a. K607 Aminoacyl-tRNA synthetasAt1g18950 943.4772 943.5385 -0.0613 3 42 A(ac)KK(ox)MPRRshoot Protein metabolismn.a. K204 Ubiquitin-conjugating enzyAt1g17280 1088.9732 1088.5098 0.4635 1 20 GDSEGGL(ac)KERshoot Protein metabolismn.a. K204 Ubiquitin-conjugating enzyAt1g17280 1088.9828 1088.5098 0.4731 1 17 GDSEGGL(ac)KERshoot Protein metabolismn.a. K204 Ubiquitin-conjugating enzyAt1g17280 1088.4758 1088.5098 -0.0339 1 24 GDSEGGL(ac)KERroot Response to stresn.a. K92 Putative disease resistancAt1g10920 1006.4566 1006.6148 -0.1581 3 17 GI(ac)KKHARRroot Unknown biologic n.a. K131 Putative uncharacterized pAt1g02380 913.4450 913.6072 -0.1622 3 23 KSL(ac)KRLK

shoot Protein metabolismmembrane, K227 Eukaryotic elongation fact At5g60390 4208.9291 4209.2546 -0.3255 1 42 ALDQINEP(ac)KRPSDKPLRLPLQDVYKIGGIGTVPVGRVEshoot Developmental pr n.a. K180 WD40 repeat-like At5g50230 1373.5246 1373.7224 -0.1978 1 21 EMLA(ac)KLKANGLEshoot Unknown biologic chloroplast K3 Putative uncharacterized pAt5g41110 1233.4934 1233.6023 -0.1089 0 19 MD(ac)KVNLSRTEshoot n.a. n.a. K25 Putative uncharacterized pAt4g37600 1018.3906 1018.4753 -0.0847 0 19 AQ(ac)KNMLQEshoot Transcription, otheneucleus K4 Basic helix-loop-helix (bHLAt4g16430 982.3576 982.4218 0.0642 1 21 (ox)MGQ(ac)KFWEshoot Electron transport chloroplast K41 Chlorophyll a-b binding proAt4g10340 1856.6677 1857.0723 -0.4047 0 40 LFS(ac)KKKPAPAKSKAVSEshoot Electron transport chloroplast K41 Chlorophyll a-b binding proAt4g10340 1856.7382 1857.0723 -0.3342 0 37 LFS(ac)KKKPAPAKSKAVSEshoot Electron transport chloroplast K41 Chlorophyll a-b binding proAt4g10340 1856.7816 1857.0723 -0.2907 0 49 LFS(ac)KKKPAPAKSKAVSEshoot Transcription, othenuclei K200 Transcription factor At3g11100 1373.5370 1373.7224 -0.1854 1 24 GK(ac)KQQIMIELE shoot Protein metabolismn.a. K668 Unknown protein At2g17110 1101.4838 1101.5562 -0.0724 0 22 M(ac)KMVLVGPGEshoot Transport, respon plasma memK148 PHO1-like protein At1g68740 1517.5964 1517.7838 -0.1873 0 20 LKDAFKQ(ac)KQANGEshoot Transport, respon plasma memK148 PHO1-like protein At1g68740 1517.5864 1517.7838 -0.1973 0 21 LKDAFKQ(ac)KQANGEshoot Unknown biologic n.a. K206 Mitochondrial transcriptionAt1g62010 1304.5116 1304.7340 -0.2223 0 15 (ac)KFDASLKKVVEshoot n.a. endomemb K453 AAA-type ATPase family pAt1g43910 1374.4274 1374.7870 -0.3596 1 18 FLENKKLK(ac)KGE shoot Electron transport chloroplast K37 Chlorophyll a/b binding proAt1g29910, 2058.6688 2059.0487 -0.3799 0 44 R(ac)KTVAKPKGPSGSPWYGSDshoot Electron transport chloroplast K37 Chlorophyll a/b binding proAt1g29910, 3372.1661 3372.6656 -0.4995 0 82 R(ac)K(p)TVAKPKGPSGSPWYGSDRVKYLGPFSGE

Trypsin digests

Table S2. Summary of lysine modification sites identified in proteins digested with trypsin or Glu-C.

Glu-C digests

Ac-BSA BSA

Figure S1. Acetyl-BSA competition confirmed the acetylation specificity of the generic anti-LysAc antibodies.

100150

25

37

50

75

20

15

kDa

100150

25

37

50

75

20

15

kDa

100150

25

37

50

75

20

15

kDa

Time at 95°C (min) 5 7.5 10 15

Anti-LysAc

Coomassie stain

5 7.5 10 15

- Urea + UreaA

B

Figure S2. A, Anti-LysAc antibodies cross react with carbamylated lysine residues. Extended boiling of protein extracts in SDS sample buffer (+ urea) causes chemical carbamylation of protein lysine residues. This lysine carbamylation was recognized by anti-LysAc antibodies. The artifact was dramatically minimized by removing urea from the sample buffer. B, The Coomassie stained blot showed no change in protein loading at the different time points.

Coomassie Coomassie

75

150

20

25

37

50

100

250kDa IB:Anti-LysAc

pH 10.0 3.0

IB: Anti-LysTrimethyl

pH 10.0 3.0

Figure S3. LysAc is a more abundant protein modification compared with lysine trimethylation in Arabidopsis. Replicate 2-DE blots were probed with: A, anti-LysAc antibodies or B, anti-trimethyllysine antibodies. C- D, Corresponding Coomassie stained blots. Note that the anti-trimethyllysine antibodies only detected two clusters of proteins, one of which was eEF-1A that is also lysine acetylated.

A B

C D

75

150

20

25

37

50

100

250kDa

75

150

20

25

37

50

100

250kDa

75

150

20

25

37

50

100

250kDa

Figure S4. Gene ontology categorization of cellular localization of lysine modified proteins identified by LC-MS in Arabidopsis.

Spectra of LHCb1 acK37 with the phosphorylation of pT38A

Spectra of LHCb1 acK37 without the phosphorylation of T38B

Figure S5. MS/MS spectra of peptides derived from the N-terminus of LHCb1 containing acetylated Lys-37 (acK37) with (A) or without (B) phosphorylation of Thr-38. The spectra were collected from an in gel digestion of the 25 kDa band from a 1-DE gel as shown in Figure 1. Glu-C was the protease used for digestion to avoid cleavage at Lys-37. Peptides without acK37 but with N-ac-R36 were co-identified in the same peptide mixtures. However, neither double acetylation peptide (N-Ac-R36 acK37) nor null acetylation peptide (N-R36 K37) were identified in the digests.

Spectra of LHCb5 acK5 and alternative N-termini

A B

C

Figure S6. MS/MS spectra identified acetylation of Lys-41 of LHCb5. Samples were processed as detailed in the legend of Supplemental Figure S5. Spectra shown (A-C) identified peptides with alternative N-termini of LHCb5, but only the sequence starting with Leu-38 was acetylated at Lys-41 (A).

A

50kDa

anti-EF1α-acK306 peptide competition

50kDa

50kDa K227acK

K306

K306acK

BDot blot of anti-LysAc, anti-eEF1A-acK306, anti-eEF1A-

acK227 and anti-eEF1A

IB: anti-EF1α-acK306

IB: anti-eEF1A-acK227

Amount (ng)

500 1500 5000

Amount (ng)500 1500 5000

IB: anti-eEF1AeEF1A antigen peptide

Amount (ng)50 150 500

IB: anti-LysAc

Amount (ng)

500 1500 5000

K227acK

K306

K306acK

K227

K227acK

K306

K306acK

K227K227acK

K306

K306acK

K227 E

D

C

Peptide:

Peptide:

Peptide:

Peptide:

Figure S7. A, Peptide competition confirmed the sequence specificity of the anti-eEF1A-acK306 antibodies. B, The generic anti-LysAc Abs cross reacted with the acetylated forms of the Lys-306 and Lys-227 peptides on dot blots. C, The anti-eEF1A-acK306 and D, anti-eEF1A-acK227 antibodies only detected the acetylated form of the antigen peptides on dot blots. E, Detection of the eEF1A antigen peptide by anti-eEF-1Α antibodies. In addition, immunoblot signals with all four antibodies were proportional to amount of antigen present.

Humans MGKEKTHINIVVIGHVDSGKSTTTGHLIYKCGGIDKRTIEKFEKEAAEMGKGSFKYAWVL 60 Yeast MGKEKSHINVVVIGHVDSGKSTTTGHLIYKCGGIDKRTIEKFEKEAAELGKGSFKYAWVL 60 arath MGKEKFHINIVVIGHVDSGKSTTTGHLIYKLGGIDKRVIERFEKEAAEMNKRSFKYAWVL 60

Domain I

Alignment of LysAc sites in eEF-1A proteinFigure S8

arath MGKEKFHINIVVIGHVDSGKSTTTGHLIYKLGGIDKRVIERFEKEAAEMNKRSFKYAWVL 60 ***** ***:******************** ******.**:*******:.* ******** Humans DKLKAERERGITIDISLWKFETSKYYVTIIDAPGHRDFIKNMITGTSQADCAVLIVAAGV 120 Yeast DKLKAERERGITIDIALWKFETPKYQVTVIDAPGHRDFIKNMITGTSQADCAILIIAGGV 120 arath DKLKAERERGITIDIALWKFETTKYYCTVIDAPGHRDFIKNMITGTSQADCAVLIIDSTT 120 ***************:******.** *:***********************:**: . . Humans GEFEAGISKNGQTREHALLAYTLGVKQLIVGVNKMDSTEPPYSQKRYEEIVKEVSTYIKK 180 Yeast GEFEAGISKDGQTREHALLAFTLGVRQLIVAVNKMDSVK--WDESRFQEIVKETSNFIKK 178 arath GGFEAGISKDGQTREHALLAFTLGVKQMICCCNKMDATTPKYSKARYDEIIKEVSSYLKK 180 * *******:**********:****:*:* ****:. :.: *::**:**.*.::** Humans IGYNPDTVAFVPISGWNGDNMLEPSANMPWFKGWKVTRKDGNASGTTLLEALDCILPPTR 240

238

Identified LysAc sites:

K Humans

K Arabidopsis

Yeast VGYNPKTVPFVPISGWNGDNMIEATTNAPWYKGWEKETKAGVVKGKTLLEAIDAIEQPSR 238arath VGYNPDKIPFVPISGFEGDNMIERSTNLDWYK------------GPTLLEALDQINEPKR 228 :****..:.******::****:* ::* *:* * *****:* * *.* Humans PTDKPLRLPLQDVYKIGGIGTVPVGRVETGVLKPGMVVTFAPVNVTTEVKSVEMHHEALS 300 Yeast PTDKPLRLPLQDVYKIGGIGTVPVGRVETGVIKPGMVVTFAPAGVTTEVKSVEMHHEQLE 298 arath PSDKPLRLPLQDVYKIGGIGTVPVGRVETGMIKPGMVVTFAPTGLTTEVKSVEMHHESLL 288

Domain IIeEF1A-acK227

site

Predicted CaM binding site for eEF1A in Arath

Arabidopsis dimethylation sitesK

*:****************************::**********..:************ * Humans EALPGDNVGFNVKNVSVKDVRRGNVAGDSKNDPPMEAAGFTAQVIILNHPGQISAGYAPV 360 Yeast QGVPGDNVGFNVKNVSVKEIRRGNVCGDAKNDPPKGCASFNATVIVLNHPGQISAGYSPV 358 arath EALPGDNVGFNVKNVAVKDLKRGYVASNSKDDPAKGAANFTSQVIIMNHPGQIGNGYAPV 348 :.:************:**:::** *..::*:**. .*.*.: **::******. **:**

Domain III

eEF1A-acK306site

in Arath

Humans LDCHTAHIACKFAELKEKIDRRSGKKLEDGPKFLKSGDAAIVDMVPGKPMCVESFSDYPP 420 Yeast LDCHTAHIACRFDELLEKNDRRSGKKLEDHPKFLKSGDAALVKFVPSKPMCVEAFSEYPP 418 arath LDCHTSHIAVKFSEILTKIDRRSGKEIEKEPKFLKNGDAGMVKMTPTKPMVVETFSEYPP 408 *****:*** :* *: * ******::*. *****.***.:*.:.* *** **:**:*** Humans LGRFAVRDMRQTVAVGVIKAVDKKAAGAGKVTKSAQKAQKAK 462 Yeast LGRFAVRDMRQTVAVGVIKSVDKTEK-AAKVTKAAQKAAKK- 458

site

Q Qarath LGRFAVRDMRQTVAVGVIKSVDKKDPTGAKVTKAAVKKGAK- 449 *******************:***. ..****:* *

A(ac)KK(ox)MPRR Mr (calc)=943.5385, delta=-0.0613, charge=+2, Mascot Score:42

Figure S9. LC/MS-MS spectra with annotation (arrows) identifying indicative b- and/or y-ions for acetylated residues.

A(ac)KK(ox)MPR Mr (calc)=943.5385, delta=-0.0371, charge=+2, Mascot Score:54

AEEAENE(ac)KLK Mr (calc)= 1201.6918, delta=- 0.1503 , charge=+2, Mascot Score:35

 

AVT(ac)KEPGR Mr (calc)= 898.4872 , delta=0.0460, charge=+2, Mascot Score:19

 

GDSEGGL(ac)KER Mr (calc)= 1059.5560, delta= 0.4635, charge=+2, Mascot Score:34

ESAD(ac)KLSLR Mr (calc)= 1059.5560, delta= -0.1630, charge=+2, Mascot Score:22

 

GHYLNATAGTCEE(ox)MI(ac)KR Mr (calc)= 1949.9088, delta= -0.3632, charge=+3, Mascot Score:73

  

GI(ac)KKHARR Mr (calc)= 1006.6148, delta= -0.1581, charge=+2, Mascot Score:17

   

GITIDIALW(ac)KFETTK Mr (calc)= 1776.9662, delta= -0.1027, charge=+2, Mascot Score:65

 

 

G(ac)KGG(ac)KGLG(ac)KGGA(ac)KR Mr (calc)= 1437.8052, delta= 0.2791, charge=+2, Mascot Score:89

 

GPTLLEALDQINEP(ac)KRPSDKPLR Mr (calc)= 2628.4235, delta= -0.3014 , charge=+4, Mascot Score:91

GQ(ac)KVKITGKGLR Mr (calc)= 1325.8143, delta= -0.4021, charge=+2, Mascot Score:22

         

IV(ac)KSSRVQISR Mr (calc)= 1313.7779, delta= -0.2943, charge=+2, Mascot Score:28   

   

   

(ac)KTISSHK Mr (calc)= 841.4657, delta=-0.4139, charge=+2, Mascot Score:29

(ac)KTISSHK Mr (calc)= 841.4657, delta=-0.0479, charge=+2, Mascot Score:27

(ac)KAEAEPVK Mr (calc)= 912.4916, delta=-0.4692 , charge=+2, Mascot Score:21

(N-terminal carbamylation) (ac)KIGEDIAK Mr (calc)= 957.5131, delta=-0.0062, charge=+2, Mascot Score:40

(ac)KIGEDIAK Mr (calc)= 914.5073, delta=-0.0210, charge=+2, Mascot Score:24

(ac)KKDGELLR Mr (calc)= 999.5713, delta=0.1392, charge=+2, Mascot Score:33

(ac)KLFNSPSDLR Mr (calc)= 1217.6404, delta=0.1636, charge=+2, Mascot Score:13

(ac)KLM(ac)KLAK Mr (calc)= 914.5623, delta= ‐0.2298, charge=+2, Mascot Score:20

  

(ac)KRQAALG(ox)MR Mr (calc)= 1087.5920, delta=-0.3748 , charge=+2, Mascot Score:39

(ac)KSAPATGGVK Mr (calc)= 956.5291, delta=-0.1666, charge=+2, Mascot Score:55

  

(ac)KVGE(ac)KR Mr (calc)= 799.4552, delta=-0.0214, charge=+2, Mascot Score:27

 

(ac)KYEAMAR Mr (calc)= 909.4378, delta=0.1283, charge=+2, Mascot Score:47

KN(ac)KLSPR Mr (calc)= 883.5239, delta=-0.4929, charge=+2, Mascot Score:17

LCFT(ac)KVRILNR Mr (calc)= 1403.8071, delta= -0.2717, charge=+2, Mascot Score:17

    

LGGID(ac)KR Mr (calc)= 799.4552, delta= 0.0559, charge=+2, Mascot Score:23

 

 

 

 

LGGID(ac)KR Mr (calc)= 799.4552, delta= -0.0113, charge=+2, Mascot Score:26

 

LQV(ac)KGASRLK Mr (calc)= 1140.698, delta= -0.206, charge=+2, Mascot Score:29

 

LSV(ac)KR Mr (calc)= 643.4017, delta= -0.0218, charge=+2, Mascot Score:26

   

 

 

MVNVS(ac)KPEVR Mr (calc)= 1199.6332, delta= 0.2002, charge=+2, Mascot Score:17

   

 

 

 

MYTSMH(ac)KDLR Mr (calc)= 1338.6060, delta= -0.4064, charge=+2, Mascot Score:25

  

(N-terminal-acetyl) (ox)MMMKLA(ac)KR Mr (calc)= 1107.5602, delta= -0.1118, charge=+2, Mascot Score:16

   

N(ox)MAEET(ac)KK Mr (calc)= 1007.4593, delta= -0.0475, charge=+2, Mascot Score:29  

 

 

 

   

NVAV(ac)KDLK Mr (calc)= 927.5389, delta= 0.0505, charge=+2, Mascot Score:36

   

 

 

NVAV(ac)KDLK Mr (calc)= 927.5389, delta= 0.1735, charge=+2, Mascot Score:32

 

(deamination)N(ac)KWIPCVEFELEHGFVYR Mr (calc)= 2365.1201, delta= 0.4520, charge=+3, Mascot Score:87

   

 

RE(ac)KLQR Mr (calc)= 870.5035, delta= 0.0763, charge=+2, Mascot Score:41

RSTGH(ac)KPLR Mr (calc)= 1092.6152, delta= -0.0826, charge=+2, Mascot Score:17

SEKEGGETCL(ac)KR Mr (calc)= 1377.6558, delta= -0.2395, charge=+2, Mascot Score:17

      

SFLDP(ac)KTVSK Mr (calc)= 1162.6234, delta= -0.0725, charge=+2, Mascot Score:19

 

  

SKCLLVGSSTNSL(ac)KR Mr (calc)= 1633.8821, delta= -0.3591, charge=+2, Mascot Score:22

  

TTLMI(ac)KNIPNKYTR Mr (calc)= 1733.9498, delta= 0.3488, charge=+3, Mascot Score:20

   

 

 

VGYNPD(ac)KIPFVPISGFEGDNMIER Mr (calc)= 2735.3265, delta=-0.2531, charge=+3, Mascot Score:57

    

(N-terminal-acetyl)VRD(ox)ML(ac)KSSK Mr (calc)= 1162.6597, delta= -0.3183, charge=+2, Mascot Score:22

   

ALDQINEP(ac)KRPSDKPLRLPLQDVYKIGGIGTVPVGRVE

Mr (calc)= 4209.2546, delta= -0.3255, charge=+4, Mascot Score:42

 

AQ(ac)KNMLQE Mr (calc)= 1018.4753, delta= -0.0847 , charge=+2, Mascot Score:19

 

 

   

 

 

 

FLENKKLK(ac)KGE Mr (calc)= 1374.7870, delta= -0.3596, charge=+2, Mascot Score:18

   

 

GK(ac)KQQIMIELE Mr (calc)= 1373.7224, delta= -0.1854, charge=+2, Mascot Score:24  

 

 

 

     

(ac)KFDASLKKVVE Mr (calc)= 1304.7340, delta= -0.2223, charge=+2, Mascot Score:15

 

 

    

 

 

LKDAFKQ(ac)KQANGE Mr (calc)= 1517.7838, delta= -0.1973, charge=+2, Mascot Score:21

     

(ox)MGQ(ac)KFWE Mr (calc)= 982.4218, delta= 0.0642, charge=+2, Mascot Score:21  

 

 

 

    

 

 

M(ac)KMVLVGPGE Mr (calc)= 1101.5562, delta= -0.0724, charge=+2, Mascot Score:22  

 

 

 

 

    

 

MD(ac)KVNLSRTE Mr (calc)= 1233.6023, delta= -0.1089, charge=+2, Mascot Score:19

 

 

   

 

 

 

R(ac)KTVAKPKGPSGSPWYGSD Mr (calc)= 2059.0487, delta= -0.3799, charge=+3, Mascot Score:44

 

 

   

 

 

 

R(ac)K(p)TVAKPKGPSGSPWYGSDRVKYLGPFSGE Mr (calc)= 3372.6656, delta=-0.4995 , charge=+4, Mascot Score:82