SUPPORTING APPENDIX

TABLE OF CONTENTS

Figure S1: Compartmentalized redox proteome under steady state conditions in P. tricornutum cells.

Figure S2: Redox regulation of metabolic pathways.

Figure S3: Evolutionary conservation of GOGAT reactive .

Figure S4: Expression levels of P. tricornutum redox sensitive nitrogen genes in EST libraries.

Figure S5: Redox regulation of primary nitrate assimilation in diatoms.

Figure S6: Targeting the in vivo redox sensor roGFP to different subcellular compartments in P. tricornutum cells.

Figure S7: Growth of P. tricornutum cultures under low nitrogen conditions.

Figure S8: GSH depletion under nitrogen starvation and oxidative stress conditions.

Figure S9: In vivo quantification of the oxidation state of redox sensitive in roGFP.

Table S1: Diatom redox proteome under oxidative stress. A list of P. tricornutum redox-sensitive proteins which were identified in this study. Predicted protein localization, sensitive cysteine position and degree of oxidation under steady state and H2O2 treatment are included. When the identified peptide contains more than one cysteine, the cysteine position refers to the first cysteine. P-value ranges are marked by asterisks: *** P < 0.01, ** 0.01 < P < 0.05, * P < 0.1.

Table S2: Biological function enriched in P. tricornutum redox-sensitive proteome. Significantly enriched terms (p<0.05) are included. Enrichment of Gene Ontology (GO) terms was analyzed using the Ontologizer software (http://compbio.charite.de/contao/index.php/ontologizer2.html).

Table S3. List of abbreviations.

Supplementary methods

Figure S1: Compartmentalized redox proteome under steady state conditions in P. tricornutum cells. The thiol proteome distribution under steady state condition is presented based on prediction of their subcellular localization and their level of oxidation. Chl, chloroplast; Mit, mitochondria; Nuc, nucleus; SP, signal peptide; SA, signal anchor; NT, not targeted.

TCA cycle Cofactors, Prosthetic Groups, Nucleosides and Nucleotide Calvin cycle and Nitrogen Compound Electron Carrier Biosynthesis photorespiration Metabolism

Amino Acids Biosynthesis

Secondary Metabolite Biosynthesis Pentose Aromatic Amines and Glycolysis Compound Polyamine Fatty Acid and Lipid Pathway Biosynthesis Biosynthesis Biosynthesis

Figure S2: Redox regulation of metabolic pathways. Overview of P. tricornutum peroxide-sensitive proteins that are involved in metabolic network reactions. Redox proteomics data was mapped to metabolic-map diagrams using the DiatomCyc databases at http://www.diatomcyc.org/. Each node in the diagram represents a single metabolite and each line represents a single bioreaction. Reactions catalyzed by proteins that were found as redox-sensitive (ΔOX >10%) are marked in red.

Figure S3: Evolutionary conservation of GOGAT reactive cysteines. Sequence comparison of GOGAT protein from 38 organism is shown. Highly conserved cysteines, Cys120 and Cys704, which were found as redox sensetive are highlithed. Multiple sequence alignment was done and visulized using MATLAB software. 20 nitrite reductase ornithine aminotransferase glutamate synthase 1 carbamoyl-phosphate synthase 16 serine hydroxymethyltransferase

isocitrate nadp-dependent

serine hydroxymethyltransferase glycine dehydrogenase 12 arginase glutamate synthase

8 Frequencies of ESTs of Frequencies

4

0 OS UA AA NS NR

Figure S4: Expression levels of genes encoding redox-sensitive proteins participating in nitrogen metabolism in P. tricornutum EST libraries. Expression levels in five EST libraries derived from different nitrogen conditions is presented. Level of expression is presented as frequencies of ESTs in each library. UA, Urea Adapted, AA, Ammonium Adapted, NR, Nitrate Replete, OS, Original Standard, NS, Nitrate Starved.

A B

50 50

N N

15 40 15 40

30 30

20 20

Atom % excess % Atom excess % Atom 10 10

0 0

0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 Time (min) Time (min)

Figure S5: Redox regulation of primary nitrate assimilation in diatoms. P. 15 tricornutum cells were supplied with N-nitrate under oxidative stress (150µM H2O2) 15 and control conditions (0µM H2O2) and the enrichment of N in glutamine labeled in amide and amino groups was detected using multiple reaction monitoring (MRM) applying the following parameters: 148.1>130.1 (A) and 148.1>131.1 (B).

a

Chl-roGFP H4 promoter OEE tp roGFP

Mit-roGFP H4 promoter GSIII tp roGFP

Nuc-roGFP H4 promoter H4 sp roGFP

b BF roGFP Marker Overlay

roGFP mitoTracker DAPI Chl 

 autoflorescence

Mit Nuc

10µm

Figure S6: Targeting the in vivo redox sensor roGFP to different subcellular compartments in P. tricornutum cells (a) Schematic representation of roGFP constructs. Histone H4 (H4) promoter was used for all constructs for constitutive expression of the roGFP protein. Mit-roGFP has mitochondrial transient peptide (GSIII), nuc-roGFP has a nuclear signal peptide (Histone H4), and chl-roGFP has chloroplastic transit peptide (OEE1). (b) Verification of roGFP subcellular localization. P. tricornutum cells expressing roGFP were viewed under a fluorescence microscope. Images of expression in the chloroplast, mitochondria and nucleus are shown. For each P. tricornutum strain images of bright field (BF), roGFP fluorescence (ex: 488nm, em: 525nm) and various sub-cellular markers are presented. Chlorophyll autofluorescence (ex: 500nm, em: > 650nm), mitoTracker stain (ex: 540nm, em: 630nm), and DAPI stain (ex: 350nm, em: 460nm) were used as markers for plastid, mitochondria, and nucleus localization, respectively. Scale bar: 10µm. Mit, mitochondria; Nuc, nucleus; Chl, chloroplast. 4 + N

3 - N

)

1 -

2

Cell·ml

6

10 ( Cell abandence Cell 1

0 0 10 20 30 40 50 60 70 Time (hr) Figure S7: Growth of P. tricornutum cultures under nitrogen-starved conditions. Cells from an exponential culture (~2•105 cells/ml) were washed with nitrogen-free ¯ media and resuspended in media containing 882µM NO3 (+Nitrate) or nitrogen-free media (-Nitrate). Subsequently, cell abundance was monitored over growth phase of 72hr.

1.2

1

0.8 ++ Nitrate Nitrate 0.6 -- Nitrate Nitrate

GSH (AU) GSH ++ Nitarte, Nitrate, H H22OO2

% of control % 0.4 -- Nitrate, Nitrate, H H2022O2 0.2

0 1 2 3 4

Day

Figure S8: GSH depletion under nitrogen starvation and oxidative stress conditions. Cells were exposed to nitrogen replete or depleted media and GSH level were determined with or without addition of hydrogen peroxide (150µM, 2hr) by applying the specific GSH dye monochlorobimane. Fluorescence data was analyzed by flow cytometery and presented as percentage of monochlorobimane fluorescence of control (day 1, nitrogen replete).

Figure S9: In vivo quantification of the oxidation state of redox sensitive cysteine in roGFP. Exponential P. tricornutum cells were subjected to OxICAT labeling under steady state and oxidized conditions (150μM H2O2, 20 min). Mass spectra are shown for the peptide containing the Cys204 of roGFP which is responsible for the redox properties of the protein. Peptide mass signal with lower m/z value corresponds to the thiol reduced state, labeled with light 12C-ICAT reagent (blue) and higher m/z value corresponds to thiol oxidized state, which is labeled with heavy 13C-ICAT reagents (red). The difference in m/z between the ICAT labeled pairs is equal to 9.0 Da (difference in mass between light and heavy ICAT reagent) divided by the charge state of the peptide. As seen under steady state conditions, a majority of the protein is in its reduced state, while after H2O2 treatment most of the protein is in its oxidized state and the difference in degree of oxidation between these conditions is 41%. These results were comparable to the fluorescence measurement of the roGFP.

Table S1

Protein Code Cystein FC intensity Protein annotation JGI code Peptide Name % oxidation rest % oxidation H2O2 deltaOxi p-value Localization NCBI position (H2O2 /rest)

ROS metabolism and redox signalling alkyl hydroperoxide reductase thiol specific antioxidant mal allergen 217408144 12713 GCTLEAR 100 12.0 47.2 35.2 ** 6.64 SP peroxiredoxin 5, atypical 2-Cys peroxiredoxin 217405639 14898 VAIFGVPGAFTPGCSK 94 16.9 30.8 13.9 * 2.25 Chl thioredoxin h 217408133 56471 LAEENPDIEFVKVDVDEADDVAAHCGVR 69 35.3 45.5 10.2 ** 1.54 Cyt redoxin domain protein 217405724 22388 VVVFAIPGAFTPTCSSTHLPGYEAAYDK 98 17.8 29.9 12.1 ** 1.94 SP thioredoxin 219110721 8167 DELLDTIVGCVAK 170 22.0 37.9 15.9 ** 2.16 Chl peroxiredoxin q 217406452 21736 KPLVVYFYPADSTPGCTK 74 33.0 57.4 24.3 *** 2.77 Chl ascorbate peroxidase 219122837 47395 YGRVDASGPENCSAEGNLPDAEPGPDGK 158 12.5 34.6 22.0 * 3.77 Chl ascorbate peroxidase 219122837 47395 VDASGPENCSAEGNLPDAEPGPDGK 158 9.6 29.4 19.8 *** 3.91 Chl ascorbate peroxidase 219122832 54731 IDSNGPENCSK 125 12.3 23.0 10.7 * 2.10 Cyt dehydroascorbate reductase 219120379 36641 CSLESVLLR 167 20.5 38.5 18.1 * 2.48 Cyt cytochrome c peroxidase 219120259 13174 CPANGRLPDATQGAEHLR 127 16.6 27.3 10.7 ** 1.89 Cyt glutaredoxin 2 219123711 38124 DVLPTVYVYDHCPFCVR 73 46.2 69.5 23.3 2.64 SP and Chlorophyll biosynthesis magnesium atpase subunit d 219112735 33017 DLKVAPSQMQYLCEEAIR 372 46.4 66.5 20.1 ** 2.31 Chl ferredoxin 219127706 49401 VKIPVNCQK 106 55.8 77.6 21.8 ** 2.73 Chl fucoxanthin Chlorophyll a c binding protein 219110471 54065 LAMLAAAGCMAQELANGK 186 3.2 13.4 10.2 ** 4.60 Chl pgr5-like protein 1a 217411614 42543 CYIDTGICK 192 49.8 60.4 10.6 * 1.53 Chl ferredoxin 219127706 49401 ACSTPLPTGK 126 20.9 43.9 22.9 * 2.84 Chl ferredoxin 118411008 SDCTISVHQEDELY 88 49.2 59.5 10.3 *** 1.52 Cyt ferredoxin 118411008 VTAGEIDQSEQTFLDDDQVEAGFVLTCIAYPK 80 54.3 66.9 12.6 ** 1.69 Cyt magnesium chelatase subunit h 209582682 13265 QVGMSGTCYPDR 640 6.7 19.5 12.8 ** 3.39 Cyt pgr5-like protein 1a 217411614 42543 FLAYGPCPSCQAENR 275 29.0 52.8 23.8 ** 2.72 Chl protoChlorophyllide reductase 219117802 12155 LAQVIDDPQCTK 356 9.1 19.6 10.4 * 2.43 Chl RNA processing and modification nhp2p 209582520 36743 CEAVNVISHPLASK 17 22.3 48.0 25.7 *** 3.21 Cyt protein 219112587 25394 ILLDAPCSGLGVISR 399 5.8 23.6 17.9 *** 4.88 Cyt dna helicase 219111707 43406 DPNCLLQDER 413 9.0 24.4 15.4 *** 3.23 Cyt rnp-1 like rna-binding protein 217409158 8269 GFCFVTMPAK 43 18.3 31.9 13.6 ** 2.08 Cyt tex-like protein 219121604 47014 CQDDFVESAEAVHRPGDEVSAR 305 18.5 31.5 13.0 ** 2.04 Cyt rnp-1 like rna-binding protein 217409158 8269 QAFEQYGTVSDCFLPTDR 28 10.8 23.2 12.5 ** 2.51 Cyt atp-dependent rna helicase 219122468 47384 ICLAIANTFIDR 202 64.9 76.7 11.7 ** 1.77 Nuc protein 219112577 18258 SLATPICEVR 671 8.1 19.6 11.5 * 2.80 Cyt nucleolin 219127948 49387 SKGETLAECTK 96 21.5 32.8 11.3 * 1.76 Cyt nucleolin 219114504 QSFADCGEISQVR 43 5.6 16.7 11.0 * 3.39 Cyt protein 219112587 25394 TIADVNQCAHLQK 423 8.3 19.2 10.9 * 2.64 Cyt transcription elongation factor spt6 219124143 47879 AFEPVQLVENFCTERDDDIR 375 6.4 17.2 10.8 ** 3.02 Cyt u2 snrnp auxiliary small subunit 219126548 29958 QGYCNFLHSKPVPR 170 22.9 33.7 10.7 * 1.73 Nuc rnp-1 like rna-binding protein 217409158 8269 QAFEQYGTVSDCFLPTDRDSGR 28 20.3 30.3 10.0 * 1.68 Cyt Energy production and conversion succinate-semialdehyde dehydrogenase (NADP+) 219110691 31599 VPCTPLDELDVMIR 28 31.1 61.5 30.4 * 3.55 Cyt nadh dehydrogenase subunit 9 324309726 _ CFILK 207 30.2 52.5 22.3 *** 2.56 Cyt vacuolar proton atpase 100-kda subunit 219122580 28794 SEPMEYISLIVNEDAAHDCLADLGK 25 3.9 24.7 20.8 ** 8.17 Cyt mitochondrial tricarboxylate transport protein 209582879 18489 AAQYNGTLDCFK 235 26.8 47.3 20.5 * 2.39 Cyt signal transduction protein with cbs domains 219121815 47178 FAAFNIGCLVTTDK 66 13.6 31.7 18.1 ** 2.96 Mit aryl-alcohol dehydrogenase (NADP+) 217405284 42255 DFEAGLGEACFHHNVGLLAYSPLAAGTLSGK 224 4.6 22.4 17.9 ** 6.04 Cyt pyruvate dehydrogenase component x 219110923 31840 EGDVVMQEDVLCDIETPDFVFGLK 118 29.9 43.2 13.3 ** 1.78 Mit pyruvate dehydrogenase E1 component subunit alpha 217404765 55035 MEITCDNEYK 84 12.4 24.9 12.4 * 2.25 Mit succinate dehydrogenase flavoprotein 209583348 41812 YRPIHYYTLDEDECK 628 60.1 72.1 12.0 * 1.66 Cyt atp synthase cf1 alpha chain 118411031 SVCEPLQTGITSIDAMIPIGR 144 6.0 18.0 11.9 ** 3.42 Cyt h+ lysosomal v1 subunit g2 209583268 44050 CCEVSLEVPTAR 103 14.5 26.1 11.6 *** 2.08 Cyt malate synthase a 219118921 54478 LAGNLVENMLNKDELDDFLTSVCYPHIVTTAYDDGR 536 26.5 37.9 11.4 ** 1.68 Cyt iron-sulfur cluster assembly mitochondrial 219121569 28374 NVGSLDKNDESVGTGLVGAPACGDVMK 64 28.5 39.8 11.3 ** 1.64 Mit cytochrome b6-f complex iron-sulfur subunit 209582673 13358 GPAPLPLALAHCDVGEDGK 151 25.1 35.4 10.3 ** 1.64 SigAnc cytochrome c heme- 219124959 6437 VLCANSATLPK 109 10.9 22.2 11.3 ** 2.32 Cyt xenobiotic reductase b 219118365 50914 SGAADLACFGR 329 19.3 42.3 23.0 ** 3.03 Cyt monooxygenase fad-binding protein 219116769 45140 SCESDPFLAFDPYTLR 480 17.6 37.1 19.4 ** 2.74 Mit Cell cycle control, cell division, chromosome partitioning septum formation protein 219111777 5720 LLAADAPILSCAGGLMVEHPFVK 164 0.0 16.3 16.3 ** Cyt nucleotide-binding protein 1 217409202 19821 TEASSENAGDAPANANTGCVGPTSETAGK 20 35.7 45.8 10.2 * 1.52 Cyt anaphase-promoting complex subunit 7 219112065 43701 DVVCLEGLVDAYLAAHK 374 11.8 26.0 14.2 *** 2.63 Cyt protein phosphatase 6 catalytic subunit 219125122 29561 CGNVAAILEIGGKPGNDTEGFEGSIPQR 265 29.8 51.4 21.6 ** 2.48 Nuc transport and metabolism 219127036 49056 TGQLTIQGDLLRQAQADFDSCR 369 15.7 69.7 53.9 * 18.37 Cyt arginase 219124730 38509 YGIGQVMEMALDHLCAK 212 10.0 50.4 40.4 * 8.21 Cyt cobalamin-dependent methionine synthase 219128523 23399 QMDLALCER 1232 68.2 96.9 28.7 * 13.99 Cyt ll-diaminopimelate aminotransferase 219126955 22909 VMSTAFNGASNIVQAGGLACLDDDGLK 337 30.4 55.5 25.1 *** 2.92 Chl glutamate synthase 219110034 24739 SSLDALCDKAVEEVR 709 10.4 35.0 24.6 ** 4.28 Chl 3-isopropylmalate 219111101 24772 EPGCSMCLAMNPDKLKPQER 451 26.4 46.1 19.7 ** 2.38 Chl trans-aconitate 2-methyltransferase 209582531 46736 DIGESVTLQAQPGAAENQPCTKPDYLR 146 10.1 27.2 17.1 ** 3.31 Cyt d-isomer specific 2-hydroxyacid dehydrogenase 219118215 27166 ITSCIGEMDVNIVQQINQSR 396 50.3 67.1 16.8 * 1.98 Mit glutamate synthase 219110034 24739 SENCPQPGVGMTFLPQDETRR 156 10.9 26.3 15.5 *** 2.91 Chl glutamate synthase 219110034 24739 VLQQGISALTCMEHR 120 33.9 49.0 15.1 *** 1.88 Chl diaminopimelate epimerase 219116604 34582 CTAVGMGNPHAIFFVNDLEK 206 12.5 26.5 14.0 ** 2.52 Chl diaminopimelate epimerase 219116604 34582 CMAQFLK 128 14.4 57.2 42.8 *** 7.95 Chl diaminopimelate epimerase 219116604 34582 IYNSDGSEPEMCGNGIR 122 22.8 62.9 40.1 ** 5.70 Chl diaminopimelate epimerase 219116604 34582 DKSEPSLTPEESAMLCHR 78 11.9 29.7 17.8 *** 3.11 Chl phosphoribosylanthranilate 219117203 34747 HQDHTFKPPNDESPLPHLVSCSR 113 7.0 21.0 14.0 * 3.57 Cyt ornithine aminotransferase 219119439 27726 ELCEKYNVLLIADEVQTGLGR 253 10.9 24.9 14.0 * 2.71 Mit glutamate synthase 1 219128141 51214 TGRDVAIAALLGAEEFGFATAPLVVMGCIMMR 1210 32.4 45.7 13.3 ** 1.77 Mit phosphoadenosine phosphosulfate reductase 209583097 25956 TLKTDCMINGR 191 23.5 36.6 13.1 *** 1.87 Chl serine hydroxymethyltransferase 217411875 54015 HDVSEFACLFPTVGFNEDEMTFEGEYNVDFVA 477 8.2 21.0 12.8 ** 2.95 Mit glycine decarboxylase p- protein 219124701 22187 DLALNHSMISLGSCTMK 539 2.7 18.0 15.3 ** 7.69 Mit histidinol dehydrogenase 219110091 9654 CESLEQCIAISDDIAPEHLEIQTR 217 3.4 15.6 12.2 ** 5.05 Cyt arginine biosynthesis bifunctional protein 219119917 12762 AICTTDR 104 8.2 20.3 12.0 * 2.78 Cyt dihydroxy-acid dehydratase 219119793 20547 EKADECYR 278 10.9 22.6 11.7 * 2.36 Chl procollagen-proline dioxygenase 219109501 42426 AAALDAMDDLVNCVQGGVSSR 163 54.2 65.8 11.6 * 1.60 SP 3-isopropylmalate dehydratase 219111101 24772 VFIGSCTNGR 391 12.3 23.6 11.3 *** 2.20 Chl glutamine synthetase 219123807 51092 FCYGVANR 353 14.7 25.7 11.0 ** 2.02 Chl protein 209582929 43957 NLTDNTVNSLQSHASSCSR 955 26.4 37.1 10.7 ** 1.65 Cyt serine hydroxymethyltransferase 209582997 18665 NCVPGDVSAVTPGGVR 371 7.6 17.8 10.2 ** 2.63 Cyt Nucleotide transport and metabolism phosphoribosylformylglycinamidine synthase 219111275 17772 LGRDPTDVECFDMGQSNSEHSR 223 20.6 47.3 26.6 ** 3.43 Cyt amidophosphoribosyltransferase 219130909 KPQQSNNGCESVNNDK 564 28.3 43.1 14.8 ** 1.91 Cyt amidophosphoribosyltransferase 219130909 KPQQSNNGCESVNNDKR 564 30.1 44.5 14.4 *** 1.86 Cyt adenylosuccinate synthetase 219115101 26256 YLSSMPASLDVLSNVGVEYEVLPGWSEDISNCKK 473 54.4 65.4 11.0 * 1.56 Mit phosphoribosylaminoimidazole-succinocarboxamide synthase 219111681 18080 NQKLECNILTPTTK 388 9.8 19.9 10.1 ** 2.29 Cyt Carbohydrate transport and metabolism transketolase 219124318 29260 VVSMPCQEVFLAQPASYQR 584 20.7 41.6 20.9 * 2.66 Cyt YPNLPESIHPGVLSGQAQMDLLNHAKEHGYAIPAVNC fructose-bisphosphate aldolase 28566281 22993 93 37.1 56.9 19.9 ** 2.42 SP VSNSGINACLEAAR 6-phosphogluconolactonase 219124540 38631 QVVVAACGVSDKYPQGK 223 15.2 34.3 19.0 ** 2.87 SP NADH dehydrogenase 219128931 30690 CNPIAEAELAQYLMDSVTDPTR 231 23.3 41.6 18.4 ** 2.33 SP phosphoglycerate kinase 219124885 14792 CNAVLVSHMGRPK 70 26.5 44.0 17.5 ** 2.20 Cyt fructose- -bisphosphate aldolase 219109531 42447 EIIAAELIPIIEPEVDIHSEEKEECETILK 192 20.3 35.5 15.1 ** 2.16 Cyt plastidic 219130730 AKFGGDATLIGDEGGFAPPCDNR 263 17.2 30.1 12.9 ** 2.07 Chl fructose-bisphosphate aldolase 28565371 _ CNGVLN 397 12.9 23.6 10.8 ** 2.07 Chl pyruvate kinase 219128273 55079 GIHPIVDSTLTDGNDCKRPEQAVQEAK 516 8.6 18.7 10.0 ** 2.44 Mit domain protein 209583099 33631 DCHSDNEVILSVAGTSLLPGNEHSDPPVGK 262 30.9 43.0 12.1 * 1.68 Cyt protein 219124122 42169 CGNVAAILELDENLER 274 24.0 39.2 15.2 ** 2.03 Cyt Coenzyme transport and metabolism arsenite methyltransferase 217404823 39627 MPELEPACEDFGQAVIYK 310 8.0 50.1 42.1 * 12.58 SP arsenite methyltransferase 217404823 39627 HYGIFEGCGSSLPYDLQPASTGK 379 14.1 46.9 32.7 ** 5.52 SP geranylgeranyl pyrophosphate synthase 219115519 19000 ACEQFALNIGLAFQVADDILDVTQSTEELGKTAGK 249 19.2 37.6 18.4 ** 2.55 Chl lipoic acid synthetase 219111613 18029 ELGLATVCEEAR 117 13.3 31.6 18.3 * 3.07 Mit S-adenosylmethionine-diacylglycerol 3-amino-3-carboxypropyltransferase 219110040 42872 FANLIDTICNR 156 10.0 25.6 15.6 ** 3.09 Cyt

S-adenosylmethionine-diacylglycerol 3-amino-3-carboxypropyltransferase 219110040 42872 KFANLIDTICNR 156 8.5 22.3 13.9 ** 3.12 Cyt coproporphyrinogen iii oxidase Chl precursor 219125200 15068 VPFFACGLSSVMHPK 97 22.9 35.7 12.8 ** 1.87 Cyt arsenite methyltransferase 217404823 39627 ACYDLLKPGGELYFSDVYANR 207 36.3 48.4 12.1 ** 1.65 SP gtp cyclohydrolase ii 219109725 42619 GPVICTVR 89 7.0 18.8 11.8 * 3.12 Cyt carnitine acetyl 219124371 48078 AGLIGEHSMMDGMPMVGLADHCTK 398 42.0 59.3 17.3 * 2.91 Mit Lipid transport and metabolism acetyl- carboxylase 219130617 55209 ILVDTLNAFDLCSVK 1389 6.8 24.1 17.2 ** 4.36 Cyt amp-dependent synthetase and 209583125 54151 SCQVFAEKR 94 6.2 19.0 12.8 * 3.55 Mit upf0551 protein mitochondrial-like 219114805 33756 DHQYCVDLVR 410 16.6 29.3 12.7 ** 2.08 Cyt 3-oxoacyl-(acyl carrier protein) synthase i 219129305 52648 SALRFCGINAQYAYIAMDR 79 23.0 69.3 46.2 ** 9.99 Cyt 3-oxoacyl-(acyl-carrier-protein) synthase 2 219122229 37367 QAVQDAGLGDTPETLQNPDRVGCMVGTAFGGVETFER 116 12.8 23.3 10.5 * 1.97 Cyt Translation, ribosomal structure and biogenesis alanyl-trna synthetase 219125523 15186 ATLESCGGR 918 11.1 62.2 51.2 ** 13.16 Cyt large subunit ribosomal protein L15 219120327 5769 HEAIMCPFNIGTLQTYIDMGR 76 28.5 62.2 33.8 * 4.41 Cyt ribosomal protein l18a 219119939 36226 KTTGEILDCVELVEK 64 9.4 37.1 27.6 ** 5.68 Cyt glycyl-trna synthetase 219116973 19761 EFCMGEIEHFVNPK 295 14.7 41.5 26.7 * 4.21 Cyt arginyl-trna 219114449 HGDYQCNAAMPVFAALKK 9 43.8 67.7 23.9 * 2.66 Cyt methylthioribose-1-phosphate isomerase 219129798 23811 SNGIIAMEYLAPPCIILR 524 5.9 26.3 20.4 ** 5.44 Cyt 50s ribosomal protein l15 219114835 10774 GIAAGQGATCGFGMR 33 27.6 47.4 19.8 * 2.49 Cyt predicted protein 217405743 48404 CLDYKPYLK 66 5.3 23.8 18.6 ** 5.64 Cyt translation initiation factor sui1-like protein 219119292 12868 NFSCNGAVQRDEEAGEVIQLSGDQR 77 30.8 49.0 18.2 ** 2.12 SP protein 219110179 9143 CLDSQPTEAR 175 11.8 29.6 17.8 ** 3.08 Cyt translation initiation factor sui1-like protein 219119292 12868 CITTVAGLADDLDVK 51 14.5 31.7 17.2 *** 2.73 SP translation initiation factor sui1-like protein 219119292 12868 KCITTVAGLADDLDVK 51 23.4 38.6 15.3 * 2.03 SP translation initiation factor eIF-3 subunit 5 219115896 11458 SNEPEAICLNQMIR 168 19.4 34.3 14.9 ** 2.13 Cyt rna-binding protein of the pumilio family 219122008 47058 YCHSDPASVK 158 31.7 46.0 14.3 ** 1.85 Cyt elongation factor tu 219127281 23083 VNVGDELDVIGLDQNHKTICTGVEMFK 288 17.2 31.4 14.2 *** 2.19 Mit aspartyl-trna synthetase 219129606 41016 CHDPDLVEK 390 19.2 33.1 14.0 *** 2.08 Cyt valyl-tRNA synthetase 219111249 43016 KCDLEEAIANFER 1048 6.1 20.0 13.9 * 3.91 Cyt translation initiation factor sui1-like protein 219119292 12868 CITTVAGLADDLDVKR 51 20.3 34.0 13.7 ** 2.01 SP ribonuclease ii 219112029 43677 FNAVVLGCIDPEKR 758 26.9 40.5 13.6 *** 1.85 Chl predicted protein 217405743 48404 GLLTSQLLCR 83 6.2 19.3 13.1 * 3.65 Cyt 40s ribosomal protein s27 219114298 _ LTEGCSYR 75 14.6 26.7 12.2 ** 2.11 Cyt protein 219124772 _ AETDCGHITR 172 18.0 29.9 11.9 * 1.93 Cyt translation initiation factor eIF-3 subunit 5 219115896 11458 QNGNVVEVTNCFSVPHAERGEEVAIGK 43 9.8 21.5 11.7 ** 2.50 Cyt exosome complex exonuclease DIS3/RRP44 219116793 26775 SCILENEIQALLLEHDVDHGDFDR 31 7.1 18.7 11.6 * 3.03 Cyt arginyl-trna synthetase 219119306 36013 LQAGNEEELAAWESLCAASRK 216 36.3 47.9 11.6 * 1.57 Cyt translation initiation factor if-2 219115513 10835 DAECIVVEANVEK 257 3.2 14.7 11.5 * 5.05 Cyt leucyl-tRNA synthetase 219115549 11088 NNVFSLVDGQVCADHDR 258 12.7 24.1 11.4 ** 2.19 Cyt protein 219124772 ILADAPCSGDGTSR 274 9.8 21.0 11.2 ** 2.46 Cyt ribosomal protein s12 219110859 8686 ALCDEGEVHVVMVEDGK 48 17.1 28.2 11.1 * 1.92 Cyt cysteinyl-trna synthetase 219114520 TALENFEVAICDDLSMPR 441 19.1 30.2 11.1 ** 1.84 SP 116 kda u5 small nuclear ribonucleoprotein component 219117015 45319 IYSACLGER 412 8.2 19.3 11.1 ** 2.67 Cyt translation initiation factor sui1-like protein 219119292 12868 SFLVDQEICHGESVVLHGF 111 16.6 27.0 10.4 ** 1.83 SP arginyl-trna synthetase 219119306 36013 FNQFYESCSVNKAESEEIK 533 9.7 19.9 10.2 ** 2.33 Cyt 30s ribosomal protein s5 118411075 ASVCALQNLTTK 158 5.8 16.1 10.2 * 3.09 Cyt ribosomal protein l10e 219126015 48699 RCGELSADGNIVK 187 11.6 21.6 10.1 *** 2.11 Cyt 40s ribosomal protein 219114717 33660 GLCAQAQAESLK 93 10.1 20.1 10.0 ** 2.25 Cyt nucleolar complex protein 3 homolog 219111751 43447 TVSETCDYIAELSEAILEQPDK 85 25.0 36.2 11.2 *** 1.70 Nuc Transcription snf2 super family 209583262 51604 CTLLTSLK 154 10.9 37.3 26.4 ** 4.89 Cyt p300 cbp acetyltransferase-related protein 219117946 45703 LPTCLPYFEGDYIPGEIENILETIDEK 1731 10.0 30.8 20.7 *** 3.95 Cyt DNA-directed RNA polymerase I subunit A12 209583355 10310 CSHPEVGYYTVQLR 49 17.0 34.7 17.7 ** 2.57 Cyt rna polymerase ii core subunit 219121575 13566 QELVDTCPDR 224 47.9 64.6 16.7 ** 1.98 Cyt snf2 super family 209583262 51604 NLADGCILK 9 20.1 35.8 15.7 * 2.31 Cyt p300 cbp acetyltransferase-related protein 219117946 45703 LLSLCTIHSR 2369 28.2 43.8 15.6 * 2.01 Cyt nascent polypeptide associated complex alpha chain 219126064 48728 ENDCDLVNAIMSLTK 181 6.2 21.1 14.9 * 4.15 Cyt dna-directed rna polymerase beta chain 118411018 _ CGVELTESR 88 26.8 40.7 13.9 ** 1.88 Cyt dna-directed rna polymerase i subunit rpa1 219114393 GHLLGSSGACVSER 1139 7.0 19.4 12.4 * 3.21 Cyt e1a creb-binding protein 219118433 45764 VLDDDEEELDCEFLNNR 1385 12.9 23.4 10.4 ** 2.05 Cyt saga-associated factor 29 homolog 219124229 47951 LSTGAEGCFK 189 14.5 26.6 12.1 ** 2.14 Nuc Cell wall/membrane/envelope biogenesis protein 219117311 TTCISSVETVYQR 690 22.4 35.8 13.3 ** 1.95 Cyt Posttranslational modification, protein turnover, chaperones atp-dependent chaperone 209583039 25856 LRCELDEYLAK 158 4.9 30.0 25.1 ** 8.33 Chl atp-dependent chaperone 209583039 25856 CELDEYLAK 158 5.2 29.3 24.1 ** 7.61 Chl oligopeptidase a 219110915 8670 TFNAGMMSCR 586 34.3 58.0 23.7 ** 2.72 Cyt protein geranylgeranyltransferase type-1 subunit 219118556 45841 PLTTIDLPTVFADLAPIPQQDGPHAVCR 28 49.3 70.8 21.5 ** 2.87 Cyt alpha ubiquitin conjugating enzyme 219125799 22525 IYHCNINSNGGICLDILK 76 77.6 99.0 21.4 *** Cyt t-complex protein 1 subunit alpha 219117083 26980 ALHDALCVVK 409 5.2 24.4 19.1 ** 5.84 Cyt ubiquitin carboxyl-terminal l3 219117057 41928 IGNACGTIGLLHTLLNVPEPLR 91 5.4 23.3 17.9 * 5.39 Cyt deoxyhypusine synthase 219121587 46999 SHCTGQVILGGGLVK 315 10.0 24.4 14.4 ** 2.88 Cyt ring finger protein 219130740 1905 CNKYDSDQPSSSGPVDQSDAAK 289 14.1 27.6 13.5 * 2.30 Cyt tripeptidyl peptidase ii 219117041 26948 GSGCSVFFGEPASSK 971 31.7 44.8 13.1 * 1.70 Cyt methionine aminopeptidase 1 219119660 12595 KPDYADHVAGVSECEQR 81 13.5 25.5 12.0 ** 2.17 Cyt ubiquitin-conjugating enzyme E2 M 219126435 48810 VCLNILR 149 11.2 22.9 11.7 *** 2.35 Cyt proteasome ( macropain) alpha 1 219118758 27508 SDSICVLGALK 44 45.6 57.3 11.7 * 1.62 Nuc peptidylprolyl isomerase 219118969 12254 QTDSGLVFCPMTEGEGK 13 9.5 21.2 11.6 ** 2.55 Cyt hsp70-type chaperone 118411057 17633 NQAEALCFEAEKEVSSFNDTVPENK 538 10.7 22.0 11.3 ** 2.32 Cyt ubiquitin-conjugating enzyme 219118660 20308 IHCPDIYPASPPNIR 74 15.9 26.0 10.1 ** 1.85 Cyt cathepsin A (carboxypeptidase C) 209583212 18551 QFGHGINPYALDYPVCK 297 11.1 100.0 88.9 ** SP Inorganic ion transport and metabolism rhodanese domain-containing protein 217405090 48802 SAPVLVYCASGK 121 67.3 100.0 32.7 ** Chl heat shock protein 67b2-like 219115195 44679 SVYACQAAAR 164 33.4 64.8 31.4 ** 3.66 Cyt atp-sulfurylase 219127317 42282 FTPDKPLECLK 125 17.4 44.3 26.9 * 3.74 Cyt atp-sulfurylase 219127317 42282 SSVTGDDFYGAYDAQECAEK 309 9.5 35.0 25.5 ** 5.10 Cyt atp-sulfurylase 219127317 42282 ALDDALVSEGGIVLVHPTCGPTQADDISGEVR 221 14.0 36.0 22.0 *** 3.43 Cyt atp-sulfurylase 219127317 42282 AGLPDDKDVVAFQCR 188 20.8 39.0 18.2 ** 2.44 Cyt atp-sulfurylase 219127317 42282 CYGTSQIEHPGTLMVATER 128 28.7 47.0 18.2 * 2.14 Cyt atp-sulfurylase 219127317 42282 DVVAFQCR 188 17.7 35.2 17.4 ** 2.50 Cyt atp-sulfurylase 219127317 42282 NFGCTHFIIGR 279 15.5 32.6 17.1 ** 2.65 Cyt sulfite ferredoxin dependent - sulfite reductase 219110645 9538 EDAMELMKCIVATQR 344 37.4 53.5 16.1 ** 1.92 SP atp-sulfurylase 219127317 42282 AIATVELTDKFTPDKPLECLK 125 31.0 46.5 15.5 * 1.89 Cyt 4-nitrophenyl phosphatase 209582772 13322 YGNVSISDNVEEADFILLHGCEVMR 126 15.4 30.4 15.0 ** 2.38 Cyt 4-nitrophenyl phosphatase 209582772 13322 ILKPCLDR 162 9.1 21.2 12.1 * 2.71 Cyt Secondary metabolites biosynthesis, transport and catabolism (+)-neomenthol dehydrogenase 219119049 45918 GAVPPCWLMMDEDVAHQPTGR 239 31.3 68.4 37.1 ** 4.71 Cyt zeta-carotene desaturase 209583239 10438 TEKIENIDGAILALSCNGMNSVVSSSPDLAR 304 13.9 32.0 18.1 ** 2.89 Cyt short-chain dehydrogenase reductase sdr 209583215 10567 TVPEGASTQVYLAANPGKGGSYYIDCK 252 33.1 48.9 15.7 ** 1.89 Cyt 9-cis-epoxycarotenoid dioxygenase 219111673 43374 EGCLNPYEIVEFPILDESQNGK 451 0.0 12.1 12.1 * Chl 2og-fe oxygenase 219117806 45605 FLHYPACELDDTSAESTDRPIR 181 47.3 74.5 27.1 * 4.77 Cyt General function prediction only methionine aminopeptidase 2 219110707 17304 FDCPHVPLR 328 5.2 46.3 41.1 ** 14.84 Cyt nad -linked oxidoreductase-like protein 217410977 43684 CDDHPPLLPLR 185 5.6 33.0 27.4 ** 8.42 SP cold shock domain-containing protein e1-like isoform 2 219119668 46078 DFVDEGACHAAPTQFLTPSDHPLNIGSATSDEYLDNESADIDPKR536 6.3 30.4 24.1 * 6.09 Cyt vacuolar protein-sorting protein bro1 219125540 48529 AALEDDGLADCHEYHATLLEFEK 86 30.3 51.6 21.3 *** 2.48 Cyt nitrite reductase 219120092 13154 GGVAEENEGVSDDPTAQICSCNDVTR 462 37.9 58.4 20.5 * 2.26 Cyt rna-binding protein rnp24 219114855 44395 CKGQALLAFATEEAAEK 281 13.1 30.9 17.9 * 3.08 Cyt phosphoglycolate phosphatase 219110307 43120 HNLHPITEQIYHEHTVYCTPER 109 1.8 19.5 17.7 * 13.47 SP nad -linked oxidoreductase-like protein 217410977 43684 DEAMLADNGIEIAKPFPLDIYYIHAPTCWK 177 14.6 31.9 17.3 ** 2.75 SP carbamoyl-phosphate synthase 219130749 24195 STMINYNPETVSTDYDECDRLYFEELSK 980 24.6 41.1 16.5 ** 2.14 Cyt short chain dehydrogenase family protein 209583016 33463 GVDQTSNELCVDLEHAIK 99 10.2 26.7 16.5 * 3.28 Cyt vacuolar protein-sorting protein bro1 219125540 48529 WLDSDQEVSFQPSNPLQFVLPKPDLSAAACR 50 58.1 74.3 16.3 * 2.01 Cyt autophagy-related protein 3 219109812 41667 CIEDEFDILDETGEIMDVPK 115 9.2 25.0 15.8 * 3.32 Cyt nitrite reductase 219120092 13154 DMGLESHIIEFADILMCR 195 16.8 32.4 15.7 * 2.32 Cyt ribosomal protein s8e 219119093 12394 CTFVGDSFTR 153 6.1 20.9 14.7 ** 4.05 Cyt sorting and assembly machinery component 50 putativ 219126220 39419 ALSFGGLCRPPTLSDR 293 20.4 34.9 14.5 ** 2.09 Cyt carbamoyl-phosphate synthase 219130749 24195 VPCEVVAYPTDTSGAR 1358 28.0 42.4 14.4 *** 1.90 Cyt methionine aminopeptidase 2 219110707 17304 GFVIEDMECSHYMK 319 19.1 33.5 14.4 ** 2.15 Cyt rnp-1 like rna-binding protein 217403538 16633 IYVGNIAFECHEDDILAEFSR 15 20.5 34.0 13.5 ** 1.99 Cyt leucyl aminopeptidase 219116426 1884 AIGALQPEGVEAHFVVAACENMINEK 263 12.3 25.3 13.0 *** 2.42 Cyt predicted protein 219130471 50470 LPCDSVLHEHTTLLR 271 17.1 29.8 12.7 * 2.04 Cyt phosphoglycolate phosphatase 219110307 43120 DLNLRPADCVYIGDSPSDGK 231 20.2 31.9 11.7 * 1.86 SP polyribonucleotide nucleotidyltransferase 219119866 20608 VIFHSLPGQEQDVPPICIETGK 91 29.2 40.1 10.9 ** 1.62 SP protein 219110715 DVVDAIMEACQLALDDFSK 443 22.5 33.3 10.8 * 1.76 SP chp-1 rar1 homologue 219118284 35396 GGPYPDCR 148 12.4 22.8 10.4 ** 2.08 Cyt U4/U6.U5 tri-snRNP-associated protein 3 219115593 44549 EICEANELR 324 7.3 17.6 10.4 * 2.77 SP kelch repeat protein 219111009 42836 LEPIPDPIHGLPCAR 20 7.5 17.7 10.2 ** 2.65 Nuc Function unknown protein rcc2 219130523 50520 DVVVTAGAHCSLIR 519 35.8 63.6 27.8 *** 3.14 Cyt 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase 219110343 25000 FTCDEER 187 14.2 30.6 16.4 ** 2.69 Nuc trna methyltransferase 112 homolog 209582793 36955 DGIPNMVLEEEDCEHVRY 127 12.0 24.1 12.1 * 2.39 Cyt condensin-2 complex subunit H2 219130131 50280 EFDPHSDFLLLDDVLPTDNTDDCR 146 8.5 20.5 12.0 * 2.80 Nuc pre-rrna processing protein utp22 219114203 EPLVVDPHCSLTEEDYSQLLAHFDGVR 1000 6.9 18.4 11.5 * 3.03 Nuc Signal transduction mechanisms 2og-fe oxygenase 209582792 46896 LHFDSGDDELPAGEEAQCR 242 7.6 25.5 17.9 ** 4.12 SP sterile alpha motif and leucine zipper containing kinase AZK 219124527 2607 RPLDDAFAGGTDGAYEECVAILQR 60 39.5 56.5 17.0 ** 1.99 Cyt phospholipase C, delta 219128795 49771 KEESLENEVTELHAQCSEASEAAK 879 6.0 23.0 17.0 ** 4.67 Cyt protein phosphatase 219111757 9890 TNSLVLCVLDGHGEHGDGVSQAFR 36 13.8 28.6 14.8 ** 2.50 Cyt guanine nucleotide binding protein (g protein) beta polypeptide 2-like 1 217407008 28694 YWLCAATDDSIK 256 28.0 41.5 13.5 * 1.88 Cyt programmed cell death-involved 217403713 50049 VQVAEALKEYFDSCDADEVIR 176 19.4 32.4 13.0 * 1.99 Cyt snf1-related protein kinase catalytic subunit alpha kin10 219110215 8773 CAYELILDHK 318 31.7 44.4 12.7 * 1.78 Cyt cAMP-dependent protein kinase regulator 219115103 10848 AIVTGKPPVCNIVAQTDGR 226 9.3 20.9 11.6 ** 2.59 Cyt phosphatidylinositol kinase 219130811 17109 LFVSGFEECR 90 26.0 37.5 11.5 * 1.71 Cyt predicted protein 217404573 39894 SLQCVGDPNGPGTALVTLPHAEAAAK 56 14.3 30.5 16.2 * 2.77 Cyt uracil phosphoribosyltransferase 219115743 26336 IVTACVDEGLNDDKFIVPGVGDYGDR 153 12.7 24.3 11.5 * 2.20 Cyt uracil phosphoribosyltransferase 219115743 26336 HPLDPTTICAVSIIR 38 11.6 21.7 10.0 * 2.12 Cyt Intracellular trafficking, secretion, and vesicular transport protein transport protein SEC31 219121600 47010 ASASCYASLGTAPEFPYR 796 30.3 49.7 19.5 * 2.41 Cyt signal recognition particle b subunit 219116829 34810 QTETTQHYEETVLLCGPPGAGK 120 21.6 39.1 17.5 ** 2.34 SigAnc vesicle-associated membrane protein 7 219130089 16798 IMQTTCEK 57 72.0 85.6 13.6 * Cyt protein 217411085 42501 YTASCVSSDTVR 78 17.3 30.0 12.7 *** 2.05 SP gtp-binding protein sar1 219118115 54420 DKGPDSSSSVRPIELYMCSVIR 172 50.3 62.8 12.5 * 1.68 Cyt beta-adaptin-like protein a 219122474 54730 HVDTLTTICPGVFDDEYR 338 17.9 29.1 11.2 ** 1.88 Cyt AP-1 complex subunit gamma-1 219119217 54511 ALASPNPDICQK 358 23.6 34.6 11.0 ** 1.71 Cyt predicted protein 217409422 45340 SEPILELECVK 930 6.0 16.6 10.6 ** 3.13 Cyt golgi reassembly-stacking 219130368 50387 LICLTPR 104 9.7 19.8 10.1 * 2.31 Cyt protein transport protein sec61 subunit alpha 219129623 41031 FLHLIRPVMCVLPEVASPDR 99 12.0 26.2 14.2 ** 2.61 Cyt Defense mechanisms protein 219127994 40135 NTEQSSSCISLDNLQNKR 42 7.5 18.0 10.4 * 2.71 SP Cytoskeleton nephrocystin 3 219129071 49839 SECLLAEVR 782 11.2 58.1 46.9 * 12.76 Cyt class vii unconventional myosin 239925803 13833 CETLVTELLEAVAEK 2204 19.2 38.4 19.2 ** 2.68 Cyt serine threonine protein kinase 219123928 47992 DVYTDSVCIVHDEQAEK 303 9.6 28.2 18.6 ** 3.72 Cyt protein 219116739 34728 AGYEVIAHSGLLPLDCK 226 4.6 20.6 16.0 * 5.40 Cyt predicted protein 217411839 42911 AVSSQEEQECGKAPPSETGVDEVAQGK 340 21.9 37.4 15.5 ** 2.13 Cyt predicted protein 217411839 42911 APECVEDDSK 375 24.0 37.4 13.4 * 1.86 Cyt superkiller protein 3 219128363 49558 DCFVPHAVLASR 334 45.6 57.8 12.2 * 1.65 Cyt tubulin alpha 219119808 54534 CGINYQAPMQVPGGDLAQVK 353 8.9 19.8 10.9 * 2.55 Cyt

Not Assign to KOG protein 217407189 47103 LVCGGCLDFK 93 10.0 58.7 48.7 ** 13.56 Chl fatty acid desaturase 27883911 48423 DCFEPDTAK 117 3.1 36.6 33.5 * 17.69 SP protein 219127938 49381 CIPQLLPEQGLNR 409 6.2 39.1 32.9 ** 9.88 Cyt N-acetylmuramic acid 6-phosphate etherase 219109688 42590 YSEQPYPSESEFAAPENVCLK 517 13.3 44.4 31.1 *** 5.18 SP protein 219114883 44423 DGECVSVTGTHLGHNIPDK 178 30.2 59.4 29.2 ** 3.43 Chl dna primase 219109991 42826 FAVSPEVTLSSDHPVEGDGNPCDSTR 1031 10.2 37.3 27.1 * 5.25 SP SEC-C motif domain protein 219118109 45498 AKAETSQCAPGQELTVLEK 201 15.2 42.1 26.9 * 4.20 Chl protein 219114883 44423 DGECVSVTGTHLGHNIPDKSGNR 178 44.0 70.7 26.7 ** 3.05 Chl galactonate dehydratase 219116066 44938 CASHDDFDLDR 600 34.1 59.4 25.3 ** 2.97 Chl protein 217407189 47103 CKFEGDK 53 30.4 54.9 24.4 * 2.79 Chl protein 219126087 48742 TLHCGGANGSNQSR 242 16.6 41.0 24.4 ** 3.44 Cyt protein 219118144 35262 WGCDTDTADR 40 28.9 53.1 24.1 *** 2.84 Cyt predicted protein 217406401 48002 EDALDILTCLVER 1345 12.6 36.3 23.7 * 4.03 Cyt fatty acid desaturase 27883911 48423 GCTAVEQR 464 9.4 32.3 23.0 ** 4.60 SP plastid lipid associated protein 217403480 55153 NDILICKPDEA 224 14.3 36.9 22.6 * 3.59 Chl protein 217405994 48121 VQVGFPGLGCR 1695 20.6 43.1 22.5 * 2.90 Cyt atpase subunit 8 324309735 NSRVELDCSYTNAN 64 14.1 36.5 22.3 * 3.57 SP protein 217410683 43725 SATDCAEGECSIDDVSELLFELK 69 69.5 90.7 21.3 ** 4.30 Chl glycerol-3-phosphate acyltransferase 219129317 3262 NPELEQGNEIDFYEFGCEFFR 183 44.2 64.7 20.6 * 2.25 SP protein 217411623 42563 AEVLLDEAEGVQQCRPK 206 6.5 26.9 20.4 * 5.40 Cyt myosin heavy chain 217405979 48094 SWVAVLDANDRDEQAQDLLETCR 186 6.3 26.5 20.1 *** 5.27 Chl protein 217410123 44671 TCNIVLGQLTDSPRPDAAGAAER 107 12.8 32.5 19.7 * 3.35 Chl 3-deoxy-7-phosphoheptulonate synthase 219110605 24353 YHTFCDPR 470 12.4 30.9 18.5 ** 3.18 Chl predicted protein 219117401 45289 IGGCSILLQEGK 77 7.0 25.4 18.3 * 4.57 Chl predicted protein 217407604 46566 QPAKSLGVNCVIPEGDEDEEDLTDAESVTGLLR 1527 18.7 36.8 18.0 * 2.55 Cyt predicted protein 217408771 45641 ISIVHPNDDEEDIDFPIIVDCGPDKEDT 851 5.9 23.7 17.8 ** 4.93 Cyt protein 219116504 44936 VDGGGFCGIR 118 14.4 31.9 17.5 *** 2.76 SP protein serine phosphatase 219117077 45376 HPVAFYCTAGK 381 7.5 24.8 17.3 * 4.17 SP protein 219125984 48680 GSCTFVQTSVMR 297 23.3 40.2 16.9 ** 2.18 SP phosphoribosylglycinamide synthetase 219116056 44930 GPVLVEVNCR 442 12.5 29.2 16.7 ** 2.87 Cyt predicted protein 219119175 46011 CGFDIEELR 266 5.3 21.9 16.6 * 5.04 Cyt protein 219126087 48742 DAVLYELSCLISDPGSQR 144 7.8 24.4 16.6 ** 3.83 Cyt metal-binding cluster containing protein 219109814 31741 VTNCAAYHFVETK 59 48.1 64.7 16.6 ** 2.01 SP protein 217412033 43219 HCFGGYGSGR 1122 25.6 41.9 16.3 * 2.09 Cyt protein 219124264 47974 AGYDVLAHSGLFPQDCK 226 11.9 28.2 16.2 ** 2.90 Cyt protein 209582667 36662 FGVDVCEHLDTLPSK 203 29.7 45.7 16.0 *** 2.00 Cyt protein 217404138 49642 AGACLAEHAQHGHMLDANVPVYTPR 268 8.4 24.1 15.7 * 3.50 Cyt plastid lipid associated protein 217403480 55153 LLAACSEDKPDR 80 31.2 46.7 15.5 ** 1.96 Chl protein 219129818 41064 CPDVDALTVELDELR 127 7.0 22.4 15.4 * 3.92 Mit predicted protein 219111483 43205 TYGGFVDAVCDK 176 12.6 27.9 15.3 ** 2.68 Cyt protein 219118144 35262 CLPNGEVVSVDGTHLGHNLPDGK 136 24.6 39.5 14.9 ** 2.01 Cyt protein 209582991 44060 RSPAEIGEAECEVGAK 93 37.3 52.1 14.9 ** 1.90 Cyt protein 217409655 34590 ATPPAPFDECFSAGGDDKENAAPAGIGK 239 11.0 25.9 14.9 * 2.86 SP atp synthase alpha subunit 219110551 31465 LSESCTAVLAASEGYLDQAVDHGALAGTQQGR 609 8.4 23.1 14.7 ** 3.28 SP predicted protein 217411948 32218 ILCLAPSR 668 24.1 38.8 14.7 * 1.93 Cyt protein 217409960 44401 KQICESALQAK 235 12.1 26.8 14.7 ** 2.65 SP protein 219114883 44423 WNCDFDTADR 87 14.9 29.2 14.2 ** 2.35 Chl predicted protein 217407690 46415 TGTELVCDELR 148 15.0 28.9 13.9 ** 2.31 Chl protein 217409960 44401 VGMDSEDRFQLLTTDECEGTETK 69 9.5 23.3 13.9 ** 2.91 SP rna polymerase alpha subunit 118411080 IQSGACHGQFLINSLNPGQGITIGNQLR 19 13.9 27.6 13.7 ** 2.36 Cyt mannosyl-3-phosphoglycerate phosphatase 219117333 45240 EEDGCSDVAANLR 124 12.8 26.2 13.4 *** 2.42 Cyt protein 219114817 33767 YCINLVSVAGRPTNTV 224 32.2 45.7 13.4 ** 1.77 Cyt predicted protein 217407729 36446 DLFLQEVHGEFDCGDNGDDVDELTPHETR 167 13.9 27.0 13.2 ** 2.29 Cyt protein 217409960 44401 AALDAGGCVLR 160 9.0 22.1 13.1 * 2.90 SP protein 217406229 47994 DKPICLAVR 115 16.8 29.3 12.5 * 2.09 Cyt isocitrate nadp-dependent 219116198 45017 ILGDCLNK 704 27.4 39.6 12.1 * 1.69 Mit predicted protein 217405841 38494 CQAGDVILFDRR 71 12.2 24.1 12.0 * 2.31 Cyt predicted protein 217406316 38174 SLCATVTDHADGNQR 303 13.5 25.2 11.7 * 2.13 Cyt protein 219129818 41064 CHDQNLDEQVVQDAAALAAR 257 29.3 40.8 11.5 ** 1.67 Mit protein 217408834 45399 LCDIAAGR 1184 12.3 23.7 11.5 ** 2.22 Cyt protein 217403013 50546 AVHDDEAVLSVLTICK 308 24.8 36.1 11.3 * 1.73 SP taurine catabolism dioxygenase 219128469 49639 ACLDPLHSVSARPTVDGQK 146 8.1 19.4 11.3 * 2.77 Chl 2-dehydro-3-deoxyphosphoheptonate aldolase 219110605 24353 AFFDVHDEMGSHPGGVHLEMTGEDVTECTGGISGVSEDTLNDR450 7.0 18.3 11.3 ** 2.93 Chl rna 2 -o-ribose methyltransferase 219128640 EPTCLPEDTFAVLVHK 11 10.9 21.8 10.9 *** 2.27 Cyt ketol-acid reductoisomerase 219129854 30967 GVAFMVDNCSFTAK 463 17.7 28.5 10.9 * 1.86 Chl predicted protein 217407772 46555 IADREHDALLLEGVDAAPDEVSQDCR 202 13.3 24.0 10.7 ** 2.07 Mit predicted protein 217407772 46555 EHDALLLEGVDAAPDEVSQDCR 202 12.0 22.7 10.7 ** 2.15 Mit protein 219123743 38156 AGYEVIAHSGLLLLDCK 289 10.1 20.7 10.6 *** 2.30 Cyt predicted protein 209583288 44091 YTLCPNTVYAIGR 52 52.1 62.6 10.5 ** 1.54 SP 2-epi-5-epi-valiolone synthase 219110901 8772 CVDYGHTFSK 189 8.4 18.7 10.3 ** 2.52 Cyt 4-hydroxy-3-methylbut-2-enyl diphosphate reductase 209402471 41845 EHYMEFDTICDATQER 337 10.9 21.2 10.3 ** 2.19 Chl predicted protein 217411700 42689 AFCGRPDDVVDER 1521 9.5 19.6 10.1 * 2.34 Cyt Table S2 Name GO term p-Value iron-sulfur cluster binding GO:0051536 0.000588059 peroxidase activity GO:0004601 0.00111426 oxidoreductase activity, acting on peroxide as acceptor GO:0016684 0.00111426 antioxidant activity GO:0016209 0.00151824 peroxiredoxin activity GO:0051920 0.00309082

DNA integration GO:0015074 0.00437147

DNA-directed RNA polymerase activity GO:0003899 0.00709049

RNA polymerase activity GO:0034062 0.00709049 oxidoreductase activity, acting on single donors with incorporation of molecular oxygen, incorporation of two atoms of oxygen GO:0016702 0.00709049 dioxygenase activity GO:0051213 0.00709049 oxidoreductase activity, acting on single donors with incorporation of molecular oxygen GO:0016701 0.00709049 oxidoreductase activity, acting on a sulfur group of donors, disulfide as acceptor GO:0016671 0.00807816 glutamine metabolic process GO:0006541 0.00816087 fructose-bisphosphate aldolase activity GO:0004332 0.0153554 triglyceride lipase activity GO:0004806 0.0153554 sulfurtransferase activity GO:0016783 0.0153554 glycerol ether metabolic process GO:0006662 0.0153554 organic ether metabolic process GO:0018904 0.0153554 nuclear speck GO:0016607 0.0268218 arginyl-tRNA aminoacylation GO:0006420 0.0268218

3-oxoacyl-[acyl-carrier-protein] synthase activity GO:0004315 0.0268218 arginine-tRNA ligase activity GO:0004814 0.0268218 valine-tRNA ligase activity GO:0004832 0.0268218 glycine hydroxymethyltransferase activity GO:0004372 0.0268218 fat cell differentiation GO:0045444 0.0268218 response to zinc ion GO:0010043 0.0337523 aldehyde-lyase activity GO:0016832 0.0337523 cell redox homeostasis GO:0045454 0.0363294 aspartate family amino acid biosynthetic process GO:0009067 0.0461619 sulfur compound biosynthetic process GO:0044272 0.0474623 metal ion binding GO:0046872 0.0484837 Table S3. List of Abbreviation

1,3-BPG 1,3-bisphosphoglycerate

2-P-glycolate 2-phosphoglycolate

3PG 3-phosphoglycerate

3-PHP D(-)3-Phosphohydroxypyruvate

3-P-serine 3-phospho-L-serine

ACC Acetyl-CoA carboxylase

APS Adenosine 5'-phosphosulfate

APX Ascorbate peroxidase

Chl Chloroplast

CHL Mg-chelatase

CPSIII Carbamoyl-phosphate synthase 3

CTP Mitochondrial citrate transport protein

DAHP synthase 3-deoxy-7-phosphoheptulonate synthase

DAP epimerase Diaminopimelate epimerase

DHAR Dehydroascorbate reductase

DTT Dithiothreitol

EF-Tu Elongation factor thermo unstable

ETC Electron transport chain

FBA Fructose-bisphosphate aldolase FC Fold change

FCP Fucoxanthin chlorophyll a c binding protein

Fd Ferredoxin

G3P Glyceraldehyde-3-P

GDC Glycine decarboxylase

GOGAT Glutamate synthase

Grx Glutaroredoxin

GS Glutamine synthase

IDH Isocitrate dehydrogenase

KAS II 3-oxoacyl-acyl-carrier-protein synthase

LIAS Lipoic acid synthetase

METH Methionine synthase

Mit Mitochondria

MS Malate synthase

NT Not targeted

Nuc Nucleus

PDHE Pyruvate dehydrogenase

PGDH Phosphoglycerate dehydrogenase

PGK Phosphoglycerate kinase

PGLP Phosphoglycolate phosphatase

PGR5 Proton gradient regulation PKM Pyruvate kinase

POR Protochlorophyllide oxidoreductase

Prx Peroxiredoxin

R5P Ribose-5-phosphat roGFP Redox-sensitive GFP

ROS Reactive oxygen species

SA Signal anchor

SAT Sulfate adenylyltransferase

SHMT Serine hydroxymethyltransferase

SiR Sulfite reductase

SP Signal peptide

SSDH Succinate-semialdehyde dehydrogenase

SQR Succinate-coenzyme Q reductase

Tkl Transketolase

Trx Thioredoxin

TS Threonine synthase

ValA 2-epi-5-epi-valiolone synthase

SUPPLEMENTARY METHODS

Culture growth: P. tricornutum, accession Pt1 8.6 (CCMP2561 in the Provasoli-

Guillard National Center for Culture of Marine Phytoplankton) was purchased from the

National Center of Marine Algae and Microbiota (NCMA, formerly known as CCMP).

Cultures were grown in f/2 media at 18ºC with 16:8 hours light:dark cycles and light intensity of 80μM photons•m-2•sec-1 supplied by cool-white LED lights. All experiments were performed with exponentially growing cultures at ~5•105 cells/ml. For Nitrate- depletion experiments exponential growing cultures were collected by centrifugation

(4,000g , 10 minutes, 18ºC), washed and resuspended in FSW + f/2 media devoid of

NaNO3.

Plasmid preparation: The roGFP sequence was ligated in frame to either mitochondrial transit peptide (GSIII) (1), nuclear signal peptide (H4) (1) or chloroplast transit peptide (OEE) (2) and cloned into the transformation vector pPha-T1 (3) where the fcpA promoter was replaced with the P. tricornutum histone H4 promoter for constitutive expression (1).

Transformation of P. tricornutum: Cells were transformed using the Bio-Rad

Biolistic PDS-1000/He Particle Delivery System (Bio-Rad, Hercules, CA, USA) fitted with 1550 psi rupture discs as previously described (4).

Measuring roGFP oxidation: Using fluorescence microscopy, oxidized and reduced forms of roGFP were observed at excitation of 405nm and 488nm. 525nm Emission intensities were collected and ratio images were created by dividing the 405nm image by the 470nm image pixel by pixel using MATLAB, displayed in pseudocolor. Flow cytometry was also used to calculate the degree of oxidation, whereby roGFP was measured in the green channel (525nm) following excitation at 405nm (oxidized) and

488nm (reduced). Leakage of chlorophyll autoflorescence to the green channel was subtracted by measurement of WT cells in parallel to roGFP transformants, the degree of oxidation of roGFP and GSH redox potentials were calculated according to (5).

GSH detection: Cells were stained with the GSH dye Monochlorobimane (Sigma), solubilized in DMSO at final concentration of 1µM. Samples were incubated at room temperature in the dark for 40 minutes before analysis by flow cytometry (ex: 405nm, em: 455/50nm).

Fluorescence microscopy: Microscopy images were obtained on IX71S1F-3-5

Motorized inverted Olympus microscope equipped with a 60x objective, filter systems for DAPI (ex: 350/50nm, em:460/50nm), oxidized roGFP (ex:405/20nm, em:525/50nm), reduced roGFP, (ex:470/40nm, em:525/50nm), MitoTracker (ex:560/40nm, em:630/75nm), and chlorophyll autofluorescence (ex:500/20nm, em:650nm LP). Images were captured using an EXi Blue™ (Q Imaging) camera.

Targeting roGFP to various organelles: Organelle-specific markers were used to confirm roGFP localization. WT and mit-roGFP cells were stained with MitoTracker Red

CMXRos (Invitrogen, USA), solubilized in DMSO to a final concentration of 100nM according to manufacturer’s instructions. Samples were incubated in the dark for 15 minutes before mitochondria detection. Localization of MitoTracker (ex:560/40nm, em:630/75nm) was overlaid with that of roGFP (ex:470/40nm, em:525/50nm). WT and nuc-roGFP cells were fixed (30 minutes in 1% paraformaldehyde) and stained with 1µM DAPI (Sigma). Nuclei were detected by DAPI (ex:350/50nm, em:460/50nm), and compared to roGFP (ex:470/40nm, em:525/50nm) localization. WT and chl-roGFP were viewed and roGFP (ex:470/40nm, em:525/50nm) localization was overlaid with chlorophyll autofluorescence (ex:500/20nm, em:650nm LP).

Redox Proteomic analysis:

Identification of peroxide sensitive cysteines: Proteins were extracted from P. tricornutum cells treated with 150µM H2O2 for 20 minutes or untreated cells using 10%

TCA-acetone. For protein precipitation, cells were collected by centrifugation from

250ml culture and 500µl of 10% TCA-acetone was added to the cells. Then, cells were sonicated gently and kept at -20oC for 1hr. After centrifugation (10 minutes, 10000g) the supernatant was discarded and the pellet was washed twice with 5% TCA acetone and stored at -20oC for 1hr. Precipitated proteins were collected by centrifugation (10 minutes, 10000g) and the pellet was dried under nitrogen flow. To avoid non-specific thiol oxidation, cells and proteins were kept under nitrogen during the extraction.

Subsequent to extraction, proteins were dissolved in denaturing buffer (50mM Tris, pH=8.5 and 0.1% SDS) and subjected to thiol trapping according to the OxICAT methodology established by Leichert et al. (6) using the cleavable ICAT reagent kit for protein labeling (AB Sciex, Foster City, CA, USA). In accordance with the OxICAT methodology, reduced and oxidized cysteines are differentially labeled with heavy and light forms of the ICAT reagents which consist of iodoacetamide (IAM) that specifically reacts with reduced cysteines and biotin residues that can be used for peptide purification on avidin affinity columns. Free thiol groups (-SH) are labeled with the IAM-based light

12C-ICAT reagent. Then, oxidized thiols are reduced with Tris (2-carboxyethyl) phosphine (TCEP) and subsequently reacted with isotopically heavy 13C-ICAT reagent.

Following labelling, proteins undergo tryptic digestion and purification using biotin- affinity tag.

The experiment was repeated three times and only peptides that were identified and found as redox sensitive at least in two experiments were considered as redox sensitive.

The difference in degree of oxidation between cells in steady state and oxidative stress conditions and P-values based on T-Test were calculated for each peptide. We defined cysteines as redox-sensitive if their degree of oxidation under oxidative stress conditions was at least 10% higher than under steady state conditions (ΔOX), and were at least 1.5- fold more oxidized upon H2O2 treatment. The roGFP was used as an internal standard to examine the OxiCAT method. Fig S9 presents the mass spectra of Cys204 which was engineered into GFP in order to make it sensitive to the redox state of the GSH pool (7).

Liquid Chromatography: Each sample was loaded using nano-Ultra Performance

Liquid Chromatography (nanoAcquity; Waters, Milford, MA, USA) in high-pH/low-pH reversed phase (RP) configuration. Samples were loaded onto a C18 column (XBridge,

0.3x50mm, 5μm particles, Waters). Buffers used were: A) 20mM ammonium formate, pH=10, and B) ACN. Peptides were eluted off the column in a step gradient of 9.3%B,

12.3%B, 14.3%B, 16.3%B, 18.6%B, 21.9%B and 65%B. Buffers used in the low pH RP were: A) H2O + 0.1% formic acid and B) acetonitrile + 0.1% formic acid. Desalting of samples was performed online using a reverse-phase C18 column (0.18x20mm, Waters).

Peptides were separated using a C18 T3 HSS nano-column (0.075X150mm, Waters) at

0.4 µl/minute and eluted off the column using the following gradient: 5% to 30%B in 60 minutes, 30% to 95%B in 5 minutes, maintained at 95% for 7 minutes and then equilibration.

Mass Spectrometry: The nanoLC was coupled online to a quadrupole ion mobility time-of-flight mass spectrometer (Synapt G2 HDMS, Waters). Data was acquired using

Masslynx v4.1 in data independent acquisition (HDMSE), as described in Valentine et al.

(8). A reference compound (Glu-Fibrinopeptide B; Sigma) was infused continuously for external calibration using a LockSpray and scanned every 30 seconds.

Data Processing, Searching and Analysis: Raw data processing and database searching was performed using Proteinlynx Global Server (IdentityE) version 2.5.2.

Database searching was carried out using the Ion Accounting algorithm described by Li et al. (9).

Data was searched against a combined target and reversed (decoy) database and the

CRAP list of common laboratory contaminants. Trypsin was set as the , one missed cleavage was allowed. Each raw data file was searched 4 times with the following modification settings: heavy or light ICAT as fixed modifications; then with heavy or light ICAT as variable modifications.

Raw data was then imported into Rosetta Elucidator System, version 3.3 (Rosetta

Biosoftware, Seattle, WA, USA). Elucidator was used for alignment of raw MS1 data in

RT and m/z dimensions as described (10). Aligned features were extracted and quantitative measurements obtained by integration of three-dimensional volumes (time, m/z, intensity) of each feature as detected in the MS1 scans. Light and heavy pairs were then identified using Elucidator, allowing 4 modified cysteines per peptide, with the heavy peak having a mass shift of 9.03 m/z. Criteria for the unbiased detection of light and heavy pairs included ±0.2 minutes in the time domain and ±15ppm in the mass domain. Following detection of the pairs, the search results were imported for annotation and the minimum identification score was set to achieve a maximum global false discovery rate of 1%.

All mass spectrometry data, including raw data, processed spectra and identifications have been deposited to the ProteomeXchange Consortium

(http://proteomecentral.proteomexchange.org) via the PRIDE partner (11) with the dataset identifier PXD000191.

Database creation: The protein database to be used in Mass Spectrometry analysis was built by combining three publically available protein databases: The National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/protein/?term=tricornutum), Doe Joint Genome Institute

(JGI, http://genome.jgi.doe.gov/Phatr2/Phatr2.download.html) and TrEMBL

(http://www.ebi.ac.uk/uniprot/database/download.html). Redundancy was removed at the protein sequence level using the cd-hit software with default options (http://weizhong- lab.ucsd.edu/cd-hit/) (12).

Protein functional annotation and enrichment analysis: Gene Ontology (GO) protein annotation was performed using the Blast2GO software

(http://www.blast2go.com/b2ghome) (13). In addition, GO annotations were downloaded from B2G-FAR (14) and from JGI. The three data sets of GO annotation were integrated using homemade PERL scripts. KEGG annotations were downloaded from the web server for metagenomic analysis (WebMGA, http://weizhong-lab.ucsd.edu/metagenomic- analysis/) (15). GO enrichment was assessed utilizing the Ontologizer application

(http://compbio.charite.de/contao/index.php/ontologizer2.html) (16). Mapping of P. tricornutum redoxome into metabolic pathways was done using the DiatomCyc tool and by manual annotation.

Predication of subcellular protein localization was made using the HECTAR tool (17) which is designed to predict subcellular targeting in heterokonts. The tool assigns proteins to five different categories of subcellular targeting: chloroplast transit peptides, mitochondrion transit peptides, signal peptides (periplastid space or secretory pathway) and type II signal anchors (membrane bound proteins). Proteins which do not possess any

N-terminal targets were further analyzes for a possible nuclear localization based on

Blast2GO annotation. Organelle specific enrichment probability for the proteins whose redox state changed was calculated by applying a hypergeometric test using the phyper R function.

15N Labeling of Glutamate and Glutamine:

15N-labeleling and amino acid extraction: Exponentially growing P. tricornutum cultures were collected by centrifugation and re-suspended in media containing sodium nitrate-15N (Sigma) as the sole nitrogen source. For determination nitrate assimilation under oxidative stress condition, cells were treated with 150µM H2O2five minutes before the addition sodium nitrate-15N. Amino acids were extracted from the cells according to

Scharff et al, (18). 50ml cells were collected by centrifugation and frozen in liquid nitrogen. Amino acid were extracted by addition of 1ml of MeOH:CHCl3:water (12:5:3

[v/v]), sonication for 30 seconds and centrifugation (2,000g, 5 minutes, 5°C). 1.16 ml of

CHCl3 and 0.25 ml of water were then added to the extract. Samples were vortexed and centrifuged (2,000g, 2 minutes, 5°C) to facilitate phase separation. The polar phase was lyophilized, re-suspended in 150µlL of 10mM Ammonium acetate: Acetonitrile (50:50

[v/v]), centrifuged and injected to UPLC-MS.

Liquid Chromatography: Amino Acid extracts were analyzed using Ultra Performance

Liquid Chromatography (UPLC) (Acquity; Waters, Milford, MA, USA). Glutamine and

Glutamate were separated using a Hilic column (2.5 Hilic, 2.0x1000mm, Cosmosil) at a flow rate of 0.4 ml/min and eluted off the column isocratically with 10mM ammonium acetate: Acetonitrile (40:60 [v/v]). Run time was set to 20 minutes. Glutamine and

Glutamate were eluted at 1.1 minutes and 6.4 minutes correspondingly.

Mass Spectrometry: The UPLC was coupled online to a triple quadrupole mass spectrometer (TQS, Waters, Milford, MA, USA). Data was acquired using MassLynx v4.1 (Waters, Milford, MA, USA). Optimization of ionization parameters was performed by direct infusion of authentic standards of Glutamine and Glutamate. For MS, argon was used as the collision gas with flow 0.22 ml/minute. Cone voltage was 25V, the capillary was set to 3 kV, source temperature was 150°C, desolvation temperature was 500°C, desolvation gas flow was 700 L/minute, source offset 50, cone gas flow was 250

L/minute, collision energy was 14eV). Glutamine and 15N-glutamine were detected using multiple reaction monitoring (MRM) applying the following parameters: transitions

147.1>130.1 (not labeled) 149.1>131.1 (15N double labeling), 148.1>130.1 and

148.1>131.1 (15N amino/amide labeled glutamine), according to Scharff-Poulsen, et al. (19). Glutamate and 15N-glutamate were detected using the following transitions

148.1>130.1 (not labeled), 149.1>131.1 (15N labeled). Raw data processing was performed using TargetLynx (Waters, Milford, MA, USA). Calculations of 15N enrichment was performed as described in Scharff-Poulsen, et al. (19)

References

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