PNNL Protein Complex Characterization Efforts Richard D. Smith, Joshua N. Adkins, Gordon A. Anderson, Deanna L. Auberry, Yuri A. Gorby, Eric A. Hill, Kim K. Hixson, Chiann-Tso Lin, Mary S. Lipton, Lye Meng Markillie, Uljana Mayer, Matthew E. Monroe, Ronald J. Moore, Sewite Negash, Ljiljana Paša-Tolić, Liang Shi, David L. Springer, Thomas C. Squier, Eric F. Strittmatter, Keqi Tang, and Nikola Tolić Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352 Himadri B. Pakrasi, Department of Biology, Washington University, St. Louis, MO 63130 Identifying protein complexes using Higher characterization throughput at lower cost AM T tags A component of the PNNL program is to develop an approach that provides both increased confidence, higher P rotein synthesis Nucleoba se synthesis 96% 97% Am ino acid biosynthesis throughput, and a quanti tati ve tool for characterizing protei n complexes. We have ini ti all y expl ored the utili ty of The database of Shew anell a accurate mass and time (AMT) 96% Othe r categories Transcription 94% 40% characterizing protein complexes at the peptide level using AMT tags with Q-TOF instrumentation as an alternative tags provi des the basi s for hi gh throughput characteri zati on of Cen tral intermediar y metabolism 94% to much less sensitive and lower throughput approaches based upon tandem MS (e.g., using ion trap mass protein complexes at either the peptide level or intact protein Ener gy metabolism 94% Prot ein fate spectrometers) or more expensive FTICR instrumentation that is needed for much more demanding “whole level. The intact protein level analysis is enabled by the Fa tty acid and phospholipid peptide level approach (by providing their initial me tabolism 94% proteome” analyses. T ransport and binding pr oteins 93% identifications) and also complements the information Cov erage This initial evaluation examined the highly active oxygen-evolving photosystem II (PSII) complex purified from the Hypothetical pro teins DNA metab olism 9 3% obtainable at the peptide-level with additional information on65% HT-3 strain of the cyanobacterium Synechocystis sp. PCC 6803. This initial study used high pressure capillary LC- protein modifications (e.g., chemical modifications, protein Regulator y functions 93% Q-TOF instrumentation and used AMT tags that were generated from capillary LC-MS/MS analyses. The table truncation). Regardless, the use of AMT tags can greatly Cell envelope 93% below shows a partial listing of >120 proteins that were identified, along with a measure of their relative abundances speed the analysis and potentially allows characterization S ignal transdu ction 92% based upon the integrated peak intensities for the corresponding peptides. The proteins highlighted in yellow were Cellular pro cesses times of <5 minutes per complex. Proteome coverage by 91% previously identified by Pakrasi and coworkers (Kashino et al., Biochemistry 2002, 41, 8004 – 8012). All proteins B iosynthesis of cofactors Unknown function 90 % AMT tags is indicated by the pie chart (right). 90 % that were assi gned based upon more than one peptide are done so wi th very hi gh confidence. Value-added protein complex characterization at the intact protein Mass Tag Average Approx. Reference Description Descr iption fr om fromFASTA FASTAGene GeneCount Ab un Count da nc e otherwise) AbundaSLR0906 ncephotosystem MW II core light harvesting protein psbB 26 23386 45 00 0 level – initial demonstration SL L1 13 0 unknown protein 7 2 31 13 10 00 0 SL L1 63 8 hypothetical protein psbQ 12 14362 11 00 0 T he se re su l ts sh o w: SLR0144 hypothetical protein 4 1 36 78 24 00 8 To evaluate our approach, we have initially studied the well characterized yeast large ribosomal SL L0 42 7 photosystem II manganese-stabiliz ing polype ptide ps bO 19 12461 31 00 0 • Q-TOF instrumentation augmented SL L0 25 8 c yt o ch r o me c5 50 p s bV 11 11983 16 00 0 SL L1 41 8 s imilar to p ho to syst em I I o xyg en - evo lvin g comp le x 2 3K pr ote in Psb P p sb P 2 78 78 16 82 8 subunit. The 43 proteins in this complex were previously identified at the peptide level using tandem SL L1 39 0 hypothetical protein 7721321 00 0 by the use of LC elution time SLR1181 photosystem II D1 protein psbA1 4 7098 32161 MS, providing an expected set of tentative molecular weights. The constrained level of complexity SL L1 09 9 e lo ng atio n fa cto r T u tu fA 22 67 91 35 47 2 information provides sufficient SL L1 19 4 photosystem II 12 kDa extrinsic protein psbU 12 67 41 10 00 0 associated with most protein complexes (ignoring obviously low-level contaminants) allows the SLR0172 hypothetical protein guaB 3667515 00 0 specificity for application of AMT tag SSR0692 hypothetical protein 3 6445 4746 detected masses to be assigned to the various proteins, as well as (in most cases) assignment of SL L0 85 1 photosystem II CP43 protein psbC 22 62 67 34 00 0 approaches. SLR1604 cell division protein FtsH ftsH 21 61 82 57 00 0 their modification states. SL L1 41 4 hypothetical protein 7617724 00 0 SL R0 01 2 r ib ulo se bis ph os ph at e c ar bo xyla se sm a ll s ub un it r bc S 4 61 58 10 73 8 • The AMT tag approach with SLR1051 enoyl acyl-carrier-protein 7 6001 22407 Partial 2-D display SSL0426 putative transposase ISY100_t 1 5806 8867 LC-Q-TOF analysis provides SLR0228 cell division protein FtsH ftsH 19 56 46 59 00 0 Capillary LC-FTICR of SML0007 photosystem II protein Y psbY 1549236 00 suffi cient speci fi city for protei n L6B SL L1 86 7 photosystem II D1 protein psbA3 6548327 00 0 yeast ribo somal comp lex L6A SLR1311 photosystem II D1 protein psbA2 6548327 00 0 L17AB Acetylated SL L1 39 8 photosystem II reaction center W protein (Psb28 b?) psbW, psb13, ycf79 5527710 00 0 complex characterization, along with L14B SLR1128 hypothetical protein 14 50 96 28 97 8 Acetylated SL L1 02 9 c ar bo n dio xid e con ce nt ra tin g m e ch an ism p r ote in Ccm K cc m K 5 49 95 98 16 high throughput, and preliminary Acetylated L14A SL L0 84 9 photosystem II reaction center D2 protein psbD 9475329 00 0 L11A/B L25 L28 SLR0927 photosystem II reaction center D2 protein psbD2 9 47 53 29 00 0 quantitation. L35AB L32 SLR1622 soluble inorganic pyrophosphatase ppa 6 4707 15482 SLR0151 unknown protein 13 46 93 28 32 0 SLL1694 pilin polypeptide PilA1 pilA1 2 4478 14254 • The use of quantitative information Methylated SL R0 00 9 r ib ulo se bis ph os ph at e c ar bo xyla se la rg e sub u nit r bc L 11 43 37 42 57 6 Mass L23 L31A/B SL L1 46 3 c ell divis ion p ro te in F t sH ft sH 21 42 20 55 31 7 and multiple analyses (e.g., using L38 SL L1 54 5 g lu tat hio ne S- tr an sfe ra se 8 4113 24143 L36A/B SSL2245 unknown protein 1 4087 7467 SLL1106 hypothetical protein 5 3985 14625 di fferent wash condi ti ons) wi ll be SLR0670 hypothetical protein 11 39 72 26 50 4 SLL1543 hypothetical protein 1 3921 13433 needed with this approach to better SLR1645 photosystem II 11 kD protein (Psb27 b?) psbZ 7378191 00 0 200 400 600 800 1000 1200 1400 SLR1165 sulfate adenylyltransferase 13 37 24 35 44 2 qualify which proteins are part of the SMR0007 photosystem II PsbL protein psbL 1368046 00 Spectrum Number SSL 25 98 photosystem II PsbH proteinpsbH 3360957 00 complex in contrast to being non- SLR1855 unknown protein 20 34 92 56 78 0 SSR 34 51 cytochrome b559 alpha subunit psbE 3332378 00 specifically associated. SLR0752 enolase 13 28 77 37 74 0 Large Riboso mal Su bu nit Coverage by In tact Prot ein An alysis Spectrum number SLR1218 hypothetical protein YCF39 ycf39 1 2874 14339 SL L1 02 8 c ar bo n dio xid e con ce nt ra tin g m e ch an ism p r ote in Ccm K cc m K 5 28 69 90 31 SLL1212 GDP-mannose 4,6-dehydratase 5 2866 33526 rpL1 rpL2 rpL3 rpL4 rpL5 rpL6 rp L7 rpL8 rpL9 rpL10 SLL1835 hypothetical protein 2 2852 23386 AB AB A/B A/B A/B A/B A/ B SL L0 08 0 N -a ce tyl- ga m m a- glu ta m yl-p ho sp ha te r ed uc tas e a rg C 6 28 42 30 99 2 Ac & 1 Me 1 Me Ac SLL0680 phosphate-binding periplasmic protein precursor (PBP) 10 2770 32463 Loss of Met Lo ss of Met Loss of Met Lo ss o f Me t Lo ss of M et L os s o f Me t Loss o f M et L os s of M et L os s of M et Advantages of intact protein proteomics: SSL1498 hypothetical protein 2 2728 5191 rpL11 rpL12 rpL13 rpL14 rpL15 rpL16 rp L1 7 rpL18 rpL19 rpL20 SLR0149 hypothetical protein 5 2717 14913 A/B AB A/B A/B AB A/B A/B AB AB A/B SLR1619 hypothetical protein 3 2716 22510 Ac 6 Me Ac Ac • Augments pepti de level anal yses SL L1 45 0 n itr at e/n itr ite tr an sp or t s yste m s ub str at e- bin din g pr ot ein n rt A 13 26 78 39 71 7 Loss of Met Lo ss of M et Lo ss o f M et L os s of M et Lo ss of Met Loss of Met Lo ss of Met Lo ss o f Me t Loss of Me t SLL1654 hypothetical protein 1 2430 11805 rpL21 rpL22 rpL23 rpL24 rpL25 rpL26 rp L2 7 rpL28 rpL29 rpL30 SL L1 04 3 p o lyrib on uc leo tid e nu cleo tid yltr an sf er as e 18 24 14 63 13 0 A/B A/B AB A/B A/B A/B • L e ss co m pl e xi ty SLR0476 unknown protein 1 2405 11675 Ac & 4 Me SL L1 85 2 nucleo side diphosphate kinase 3 22 97 13 53 9 Loss of Met Loss of Met Loss of Met Lo ss of M et L os s o f Me t Loss of Met Loss of Met Loss of M et L os s of M et rpL31 rpL32 rpL33 rpL34 rpL35 rpL36 rp L3 7 rpL38 rpL39 rpL40 SL R1 21 6 M g 2+ t ra ns po rt pr ot ein 5 2280 41448 A/B A/B A/B AB A/B A/B AB • Much more information on protein SL L1 55 5 t wo -co m po n en t h ybr id se ns or a nd re g ula tor 4 22 59 33 63 9 SLL1641 glutamate de ca rboxylase 4215143022 Lo ss of Met Loss o f Met L os s of Met Lo ss o f Met Loss of Me t L os s o fMe t Loss o f M et L os s of M et Lo ss of Met Fragment, 77- 1 28 modification states SLR0723 hypothetical protein 5 2128 33603 rpL41 rpL42 rpL43 SLR1618 unknown protein 3 2123 23523 AB AB AB • 42 of the 43 core l arge ribosomal subunit proteins (58 of SLL0290 polyphosphate kinase ppk 13 2115 67003 2 Me 1 Me the 64 possible core larg e subunit pro tein iso forms) are • Potenti all y more quanti tati ve, faster, and SL L0 01 8 f ru cto se -b isp ho sp ha te a ldo las e, cla ss II fb a A, f da 8 21 01 31 61 0 Lo ss of Met Lo ss of Met i den ti fied in a si ngl e an aly sis.