Supplement S1 Supplemental Methods
Mass spectrometry conditions. Excised bands were washed with 100mM NH4HCO3, reduced with DTT, treated with fresh iodoacetamide, and digested overnight with trypsin at 37C as described by Shevchenko et al (3). Peptides were then extracted with 1 volume of acetonitrile, sonicated, and the supernatant was dried under vacuum and reconstituted in 20 l of 3% acetonitrile, 0.1% acetic acid. Peptides were loaded onto a C-18 precolumn and washed extensively with 3% acetonitrile, 0.1% acetic acid. The precolumn was connected to an integrated nano-HPLC analytical column/ESI emitter, fabricated as described by Martin et al. (2). The peptides were eluted using a linear 3-80% acetonitrile gradient at a flow rate of 50 nL/min. The LCQ Deca Max mass spectrometer (ThermoFinnigan) acquired a full scan (400-2000 m/z) followed by MS/MS spectra on the three most abundant ions, using dynamic exclusion. Using the SEQUEST algorithm in Bioworks 3.1, SR1, the MS/MS spectra were compared to a database of 50,719 entries including non-redundant proteins of S. aureus and all bacteriophages. Peptides were matched using the following rigorous criteria: Cross correlation vs Charge, +1>1.9, +2>2.7, +3>3.5; Δcorrelation>0.1.
Digestion of whole virions. Whole virion samples were denatured as described in (1) with the following modifications. The particles were disassociated in 6M urea followed by 6 freeze-thaw cycles (-80oC to 37oC). The disassociated particles were centrifuged in a Nanosep (Pall Corp, East Hill, NY.) 100K MWCO centrifugal device and the eluate was diluted to 0.75M urea and digested overnight with 36 ng of trypsin. The remainder in the cup was washed and reconstituted in 100 mM NH4CO3 , digested overnight with 36 ng of trypsin. Peptides and DNA were separated with a 10K MWCO centrifugal device. Approximately 25 μl of each eluate was loaded onto the pre-column.
References
1. da Poian, A. T., A. C. Oliveira, and J. L. Silva. 1995. Cold denaturation of an icosahedral virus. The role of entropy in virus assembly. Biochemistry 34:2672-2677.
2. Martin, S. E., J. Shabanowitz, D. F. Hunt, J. A. Marto. 2000. Subfemtomole MS and MS/MS peptide sequence analysis using nano-HPLC micro-ESI fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 72:4266-4274.
3. Shevchenko, A., M. Wilm, O. Vorm, and M. Mann. 1996. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68:850-858.
Supplement S2. Tryptic peptide sequences identified by mass spectrometry.
Protein ID; ORF# AAs
function / Peptides a
ABF71627; ORF 56 / LDASIQDLQQ
1154 amino acids / ILESNFELDKLNSK
Tape measure protein / VNIDVNADNSK
AILDVDNSVANSK
QNQSAIFTTLANGLNTVK
LVALVTK
LVTAIFQLLFGK
VVSIFTQVVSTISDK
VIQGFVNVVNK
VTEYKIKATIEASVAKFKRQIDSAVKSVQRFKRVADQTKDVELNANDKKLQKTIKVAKKSLDAFSNKNVKAKLDASIQDLQQKILESNFELDKLNSKEASPEVKLQKQKLTKDIAEAENKLSELEKKRVNIDVNADNSKFNRVLKVSKASLEALNRSKAKAILDVDNSVANSKIKRTKEELKSIPNKTRSRLDVDTRLSIPTIYAFKKSLDALPNKKTTKVDVDTNGLKKVYAYIIKANDNFQRQMGNLANMFRVFGTVGSNMVGGLLTSSFSILIPVIASVVPVVFALLNAIKVLTGGVLALGGAVAIAGAGFVAFGAMAISAIKMLNDGTLQASSATNEYKKALDGVKSAWTDIIKQNQSAIFTTLANGLNTVKTAMQSLQPFFSGISRGMEEASQSVLKWAENSSVASRFFNMMNTTGVSVFNKLLSAAGGFGDGLVNVFTQLAPLFQWSADWLDRLGQSFSNWANSAAGENSITRFIEYTKTNLPIIGNIFKNVFAGINNLMNAFSGSSTGIFQSLEQMTAKFREWSEQVGQSQGFKDFVSYIQTNGPLIMQLIGNIARGLVAFATAMAPIASAVLRVAVAITGWIANLFEAHPATAQLVGVIITLVGAFRFLIAPILAVMDFLGPLAARLVALVTKFGWAKTGTLVLSKAMTSLKGPIKLVTAIFQLLFGKIGLIRNAITGLVTVFGILGGPITIVIGVIAALIAIFVLLWNKNEGFRNFIINAWNAIKTFMVNVWNVLKAVASVVWNAILTAITTAVSNVYNFIMIVWNQIVAYLQGLWNGIIAIATTVWNLLVTIITTVFTTIMTIVMTIWTAIWTFLSTIWNTIITIATTIWNLLVTVITTVFTTIMTIAMTIWNAIWTFLQTLWNTIVTVATKVWNAITTAISTALQAAWSFISNIWNTIWSFLSGILTTIWNKVVSIFTQVVSTISDKMSQAWNFIVTKGMQWVSTITSTLINFVNRVIQGFVNVVNKVSQGMTNAVNKIKSFIGDFVSAGADMIRGLIRGIGQMAGQLVDAAKNVAKKALDAAKSALGIHSPSREFMDVGMYSMLGFVKGIDNHSSKVIRNVSNVADKVVDAFQPTLNAPDISSITGNLSNLGGNINAQVQHTHSIETSPNMKTVKVEFDVNNDALTSIVNGRNAKRNSEYYL
633 amino acids / KLEEDASDFATFIR
Minor tail protein / SALAEIYGDIHAEPFK
LGLNDLVR
QDVTLGEFNR
TVTTSDGTIVHDFIDK
MTITIKPPKGNGAPVPVETTLVKKVNADGVLTFDILENKYTYEVINAIGKRWIVSHVEGENDKKEYVITVIDRKSEGDRQLVECTAREIPIDKLMIDRIYVNVTGSFTVERYFNIVFQGTGMLFEVEGKVKSSKFENGGEGDTRLEMFKKGLEHFGLEYKITYDKKKDRYKFVLTPFANQKASYFISDEVNANAIKLEEDASDFATFIRGYGNYSGEETFEHAGLVMEARSALAEIYGDIHAEPFKDGKVTDQETMDKELQSRLKKSLKQSLSLDFLVLRESYPEADPQPGDIVQIKSTKLGLNDLVRIVQVKTIRGINNVIVKQDVTLGEFNREQRYMKKVNTAANYVSGLNDVNLSNPSKAAENLKSKVASIAKSTLDLMSRTDLIEDKQQKVSSKTVTTSDGTIVHDFIDKSNIKDVKTIGTIGDSVARGSHAKTNFTEMLGKKLKAKTTNLARGGATMATVPIGKEAVENSIYRQAEQIRGDLIILQGTDDDWLHGYWAGVPIGTDKTDTKTFYGAFCSAIEVIRKNNPASKILVMTATRQCPMSGTMIRRKDTDKNKLGLTLEDYVNAQILACSELDVPVYDAYHTDYFKPYNPAFRKSSMPDGLHPNERGHEVIMYELIKNYYQFYG
ABF71632; ORF 61 / YVDENEYNFK636 amino acids / EVFAHNGEQIK
Minor tail protein / LYHDYSTLDAFTK
NSLNFVEVR
PANPNYLQGFDIK
RIDIGGVNNNFK
ALLIGVTIGPGNNR
VKPLPIQNPAYLSDITEVGHYYIYTQDTQNALDFPLPK
VIDIFNK
IVGLDFYITTEESNR
NNFPSAHQFLVR
MDNKLITDLSRVFDYRYVDENEYNFKLISDMLTDFNFSLEYHRNKEVFAHNGEQIKYEHLNVTSSVSDFLTYLNGRFSNMVLGHNGDGINEVKDARVDNTGYDHKTLQDRLYHDYSTLDAFTKKVEKAVDENYKEYRATEYRFEPKEQEPEFITDLSPYTNAVMQSFWVDPRTKIIYMTQARPGNHYMLSRLKPNGQFIDRLLVKNGGHGTHNAYRYIGNELWIYSAVLDANENNKFVRFQYRTGEITYGNEMQDVMPNIFNDRYTSAIYNPIENLMIFRREYKASERQLKNSLNFVEVRSADDIDKGIDKVLYQMDIPMEYTSDTQPMQGITYDAGILYWYTGDSKPANPNYLQGFDIKTKELLFKRRIDIGGVNNNFKGDFQEAEGLDMYYDLETGRKALLIGVTIGPGNNRHHSIYSIGQRGVNQFLKNIAPQVSMTDSGGRVKPLPIQNPAYLSDITEVGHYYIYTQDTQNALDFPLPKAFRDAGWFFDVLPGHYNGALRQVLTRNSTGRNMLKFERVIDIFNKKNNGAWNFCPQNAGYWEHIPKSITKLSDLKIVGLDFYITTEESNRFTDFPKDFKGIAGWILEVKSNTPGNTTQVLRRNNFPSAHQFLVRNFGTGGVGKWSLFEGKVVE
ABF71633; ORF62 / GTGVLNFAVTK607 amino acids / GAYISDELTIVDAINGR
Minor tail protein / LQYVIPNEFLK
VHAQAFFTQNGSDNVVVER
SIQDTIEAVGK
GIQQIEIK
QNEAIQAITATQTSATQAVTAEFDK
IVEK
MVVDNFSKDDNLIELQTTSQYNPVIDTNISFYESDRGTGVLNFAVTKNNRPLSISSEHVKTFIVLKTDDYNVDRGAYISDELTIVDAINGRLQYVIPNEFLKHSGKVHAQAFFTQNGSDNVVVERQFSFNIENDLVSGFDGITKLVYIKSIQDTIEAVGKDFNQLKQNMADTQTLIAKVNDSATKGIQQIEIKQNEAIQAITATQTSATQAVTAEFDKIVEKEQAIFERVNEVEQQINGADLVKGNSTTNWQKSKLTDDYGKAIESYEQSIDSVLSAVNTSRIIHITSATDAPSFKDIGTVDTPKEDGVDDGSDIPVAPNTLGKSGVLVVYVVDDSTARATWYPDDSNDEYTKYKISGTWYPFYKKNDGDLTKQFVEETSNNALNQAKQYVDDKFGTTSWQQHKMTEANGQSIQVNLNNAQGDLGYLTAGNYYATRVPDLPGSVESYEGYLSVFVKDDTNKLFNFTPYNSKKIYTRSITNGRLEQQWTVPNEHKSTVLFDGGANGVGTTINLTEPYTNYSILLVSGTYPGGVIEGFGLTALPNAIQLSKANVVDSDGNGGGIYECLLSKTSSTTLRIDNDVYFDLGKTSGSGANANKVTITKIMGWK
ABF71613; ORF 42 / VNEFETDTDLR511 amino acids / VLSDYYEGK
Portal protein / NLVELTR
SLGLDLSIYGK
SDAMSTFIIYDNTVE
ENSFESHSFERMPITEFSNNER
VITLIDLYDNAESDTANYMSDLNDAMLLIK
GNLNLDPVEVRK
ETEGSVDGGYIYK
QYDVQGTEAYKDR
LNSDIHMFTNTPNMK
LFGLEQR
EGLFTK
LLETILK
SLIEELK
MLKVNEFETDTDLRGNINYLFNDEANVVYTYDGTESDLLQNVNEVSKYIEHHMDYQRPRLKVLSDYYEGKTKNLVELTRRKEEYMADNRVAHDYASYISDFINGYFLGNPIQYQDDDKDVLEAIEAFNDLNDVESHNRSLGLDLSIYGKAYELMIRNQDDETRLYKSDAMSTFIIYDNTVERNSIAGVRYLRTKPIDKTDEDEVFTVDLFTSHGVYRYLTNRTNGLKLTPRENSFESHSFERMPITEFSNNERRKGDYEKVITLIDLYDNAESDTANYMSDLNDAMLLIKGNLNLDPVEVRKQKEANVLFLEPTVYVDAEGRETEGSVDGGYIYKQYDVQGTEAYKDRLNSDIHMFTNTPNMKDDNFSGTQSGEAMKYKLFGLEQRTKTKEGLFTKGLRRRAKLLETILKNTRSIDANKDFNTVRYVYNRNLPKSLIEELKAYIDSGGKISQTTLMSLFSFFQDPELEVKKIEEDEKESIKKAQKGIYKDPRDINDDEQDDDTKDTVDKKE
ABF71639; ORF 68 / IKNDGVDLGDIGCR390 amino acids / FYTEDENTASIR
Tail, fiber? / LHLFMEDGSIFK
NEPLIIDDVVKGFITYK
LFLEKEEEK
IHVANFSFNIVDSGIESAVAK
LVDDAITR
DNATDLLSK
EKIDKDVISYIEK
NGTVVSINPDTK
DTDIK
AEPELLDK
INIANVEGLEDK
ELAEAIQNNSISESVLQQIGSK
YVLSSQAFTK
IWTGTENEYNYIYQK
DPNTLYLIK
MYKIKDVETRIKNDGVDLGDIGCRFYTEDENTASIRIGINDKQGRIDLKAHGLTPRLHLFMEDGSIFKNEPLIIDDVVKGFITYKIPKKVIKHAGYVRCKLFLEKEEEKIHVANFSFNIVDSGIESAVAKEIDVKLVDDAITRILKDNATDLLSKDFKEKIDKDVISYIEKNESRFKGAKGDKGEPGQPGAKGEAGKKGEQGAPGKNGTVVSINPDTKMWQIDGKDTDIKAEPELLDKINIANVEGLEDKLQEVEKIKDTTLNDSKTYTDTKIAELVDSAPESMNTLRELAEAIQNNSISESVLQQIGSKVSTEDFEEFKQTLNDLYAPKNHNHDERYVLSSQAFTKQQADSLYQLKSASQPTVKIWTGTENEYNYIYQKDPNTLYLIKG
ABF71618; ORF 47 / KFTFWADKPGAYWVGEGQK324 amino acids / LGVILPVTK
Major capsid protein / KFDEAGILNQGNNPFGK
SIAQSIEK
GDFTQDNIIDLEALLEDDELEANAFISK
NSDSLDGLPVVNLK
RGELITGDFDK
LIYGIPQLIEYK
IDETAQLSTVK
ATMHVALHIADDK
MEQTQKLKLNLQHFASNNVKPQVFNPDNVMMHEKKDGTLMNEFTTPILQEVMENSKIMQLGKYEPMEGTEKKFTFWADKPGAYWVGEGQKIETSKATWVNATMRAFKLGVILPVTKEFLNYTYSQFFEEMKPMIAEAFYKKFDEAGILNQGNNPFGKSIAQSIEKTNKVIKGDFTQDNIIDLEALLEDDELEANAFISKTQNRSLLRKIVDPETKERIYDRNSDSLDGLPVVNLKSSNLKRGELITGDFDKLIYGIPQLIEYKIDETAQLSTVKNEDGTPVNLFEQDMVALRATMHVALHIADDKAFAKLVPADKRTDSVPGEV
ABF71624; ORF 53 / IILFR193 amino acids / VLYEAWEVESR
Major tail protein / YFQGFHNK
GFATLPEAVTK
FHDTTK
EDALTSEDLTAIPQPK
MANMKNSNDRIILFRKAGEKVDATKMLFLTEYGLSHEADTDTEDTMDGSYNTGGSVESTMSGTAKMFYGDDFADEIEDAVVDRVLYEAWEVESRIPGKNGDSAKFKAKYFQGFHNKFELKAEANGIDEYEYEYGVNGRFQRGFATLPEAVTKKLKATGYRFHDTTKEDALTSEDLTAIPQPKVDSSTVTPGEV
ABF71617; ORF 46 / ENDEGTEITFTPEQQKK206 amino acids / VDEILER
Capsid scaffolding protein / KMLSEAEVDSSDEVVNLVVTDTAEQTK
SNVEAFSNAVKK
QSPLTGGDSFNHSTKNK
PQNLAEIAR
MEENKLKFNLQFFADQSDDPDEPGGDGKKGNPDKKENDEGTEITFTPEQQKKVDEILERRVAHEKKKADEYAKEKAAEAAKEAAKLAKMNKDQKDEYEREQMEKELEQLRSEKQLNEMRSEARKMLSEAEVDSSDEVVNLVVTDTAEQTKSNVEAFSNAVKKAVNEAVKVNARQSPLTGGDSFNHSTKNKPQNLAEIARQKRIIKN
ABF71615; ORF 44 / AIENELKR331 amino acids / EINAFIVK
Minor head protein / YGDFAGVTLQEAQK
IIDEFDVK
VFSDQAGILGEGVQVAK
SLLYTESAR
GLNGEIFK
LPNKNTQEYWEERGRKAIENELKRDKTKAEEIERILNMMIKRIEKEINAFIVKYGDFAGVTLQEAQKIIDEFDVKAFQEEAKRLVENKDFSDRANEELKKYNTKMYVSREQMLKIQIEFLIAYATAQTELSMREYFESTAYRVFSDQAGILGEGVQVAKEVIDTIVDTQFHGVVWSERLWTNTEAMKQEVEEIIANVVIRGRHPNEYVKDMRKHLNKFEGTARQKTAAIKSLLYTESARVHAQSSIDSMKEISPEGYYMYIAKIDNRTTKVCKGLNGEIFKVKDAKIGVNFYPMHINCRSDCALLPKSMWPKKPSKKRKTKYFGGKVKSGD
110 amino acids / SNAYELNDFKEYEAIIDNYFNAR
Structural protein
MTTLADVKKRIGLKDEKQDEQLEEIIKSCESQLLSMLPIEVEQIPERFSYMIKEVAVKRYNRIGAEGMTSEAVDGRSNAYELNDFKEYEAIIDNYFNARTRTKKGRAVFF
ABF71638; ORF 67 / TWGNADAMAQK632 amino acids / YTISSQEVNYPEYIYHFIVEGNR
Virion associated hydrolase / NAQTMSSVESLYNSR
YKQDVEYPHFYVDR
HNIWAPR
YEIPLSIENLK
VDDSIWR
DKYEALEK
VMVDNK
NADIANVR
QPQIITETSPYTFK
KSNAWGWANATR
YQGVSVSALNK
NKGWTSPAK
GIYFTR
MGLPNPKNRKPTASEVVEWALYIAKNKIAIDVPGSGMGAQCWDLPNYLLDKYWGFRTWGNADAMAQKSNYRGRDFKIIRNTKDFVPQPGDWGVWTGGWAGHVNIVVGPCTKDYWYGVDQNWYTNNATGSPPYKIKHSYHDGPGGGVKYFVRPPYHPDKTTPAPKPEDDSDDNEKNNKKVPIWKDVTTIKYTISSQEVNYPEYIYHFIVEGNRRLEKPKGIMIRNAQTMSSVESLYNSRKKYKQDVEYPHFYVDRHNIWAPRRAVFEVPNEPDYIVIDVCEDYSANKNEFIFNEIHAMVVAVDMMAKYEIPLSIENLKVDDSIWRSMLEHVNWNMIDNGVPSKDKYEALEKALLNIFKNREKLLNSITKPTVTKSRIKVMVDNKNADIANVRDSSPTANNGSASKQPQIITETSPYTFKQALDKQMARGNPKKSNAWGWANATRAQTSSAMNVKRIWESNTQCYQMLNLGKYQGVSVSALNKILKGKGTLNNQGKAFAEACKKHSINEIYLIAHAFLESGYGTSNFANGRYGAYNYFGIGAFDNDPDYAMTFAKNKGWTSPAKAIMGGASFVRKDYINKGQNTLYRIRWNPKNPATHQYATAIEWCQHQASTIAKLYKQIGLKGIYFTRDKYK
131 amino acids / WTINGVEPNKSYQVTIENVR
Minor structural protein, tail?
MEGNFKNVKKLIYEGEEYTKVYAGNIQVWKKPSSFVIKPLPKNKYPDSIEESTAKWTINGVEPNKSYQVTIENVRSGIMRVSQTNLGSSDLGISGVNSGVASKNINFSNPSGMLYVTISDVYSGSPTLTIE
a For those proteins identified by both SDS-PAGE and by analysis of whole virion particles, the peptides found in bands excised from the gels are shown.
Peptides identified by mass spectrometry are indicated in the predicted amino acid sequence of each protein by underlining and italics.