1 Se a ern 20521586 arig ORL mS IN EPHESUS Sihane & The $5 hl deg acon of duy caged enc Co nd eipeptie (ACA nk by adc ule ed eure apa Fae | Opts umdrs and ene Guys brand uber! ate aacnetga (Cys and dicalet Ca appeus to alte excise feemation of Cys leading i tam to gh seeve ‘SC band vga, The ME! pce of reaticts wi AAs all identify peptides wietamo- leur dul laos (gue fh Schemes 3 and 9. (Otherwise, MS spc provide sequence ifraen ofthe product peptide, evening he cation cfthe Cy res that forms the dsalide bond in the peptide (Gehemes 2-4) Fr the doubly urged meal com Petes with both ntemoledar ad inamclcular ulde inkages, the SC boed cleavage occurs plow at the inermcleclar dul linkage under low-energy CID ditons the press being asia by hare repube been manameri prod vs pepe, Conclusion The tie dadfide aks i nin were dura land wang mass getty. The pple digest ag rents cotanng, inernceclr didi were ay dented rom Mf deubly caged Na and Co campleues by a hig abundanee of pazed produc spate by 65 mas unis. Moe dead struct! aloratin forthe peptide, iad he Testor of he dsulide ages ae the presence of additonal ineandlear dulfde rhage, was ‘ached by further activation of he proc peptides to Hey the poston of debydraodenine reskdacs, The mechanism propel for thee proces up porte by thao saas of model ens, soles feemato ofa attained soo at Cp, followed by cage of the SC hand, We Bale hi he ‘approach desbed in the present ad peeves [12] sd, which werefer oa th Rute method in prong ts determine he presine ard Testion of bth ictre and itarbleadar dsalice Tnkages in peptides and proe, Acknowledgments Tema deb ee at ail at te kan Ita and he Nes Lato Chel Pi a be Gi tec Teng. Re ape e per pu ys Bonar see Maw Spee Kune neal he Pasay Se in Pop aoe mos oan ACA, ey Cals nate: Pat apt abe pie yt Natiral Se Foto ser Gn CHE bcs, References 1 Regpst on tpttene int 2 ETT naea ea ee aie moe a baes Rea ye a Galati eines ive penn ee pre STE tp sae pee ree as REET ARERR Cepia Coheicesy meey eid cian in eh a Gee as cma 0. al. An Sari pa CA Ratan php ‘iSkin acs ate btn PP setts scram, 1 ey aca ro 1 ie rants cet an te Caatay eon a As Tl Coe is eS TRE Ria A ste Dart ng ca aera = Repent ee16m axD saver Schone Pop Chin nge means dehy ‘tne Chou pepe (ACA) Ee! by te ‘ler dae nd Optinond aad ep ie camapedng tinbavé ton of CP" bound (thy 2 howe ga 210 A)/nol compared wit intermediates inated in the cearage cf the 5-5 bord. The SC band rage ‘siniiate by erolt at Cys and sabia by intra tian fe C2* ion with the ule group The proc tranet from the Neemital amine ty the dsl ‘up allows the Ca" ion to interact mail withthe Side /earospli: aid caygen abn. Onve the poten is taser to the disulfide grup nse sper talon ofthe two Kagmens cers atthe rel of churge repibioe ad bath ae observed i the MS? spectum igure 18). The S-5 bond dearage process ‘equresunfavoable prion wafer rom he fasten tC) tthe Netersel amine (theme 6) Ir aden, the Ca" ion interacts withthe slur ato and the aide oxygen ats after the SS bund dewvage We sgt thatthe itracon between hard acid C™ ‘eh the wlaively sft base slur ao causes the process of SS hoe deavage to be ess favorable than AheS€ bond deavage [27] Asa rel highly sles he SC bond clave i elev ala a alain earth meta comple. of pepe with iter- ‘ole dul beds, ‘The Route 66 Method for Loating Disulfide Linkages in Peptides ‘Simple and fast identicaton of peptides with dsulide iskages i he papi dies of rin i eons 1 An SM tn OE ner low-energy CID conditions using Nat and G+ eomplses Diu Hg ols ae abl to bo located via further ativaton of the CID pod Te Algepeides, CIVEQCCASVCSL/FYNQHLOGSHL and GIVEQCCASVCSL/FYNQHLCGSHLVE, which por ‘eat coe ntermoleclardalfide kage and one io- tramoleculr disulfide linkage each, ied producsev- ieneng the presence of dale lakages via CID of doubly charged Na” comple (Figue 2b ard The CID ofthe deubly charged dsodated the prods resting fom two HS, eliminations Each ingly charged Na* cation may intact with etch Alalsde linkage fo ee lavage yield two HS, ‘liminators when the peptide comple are colison- ally ativatd ‘Assen a Figures 35 higher selectivity frclenage 9f5Choedsis observe foe the Ca” hound comes ‘compared with the sedited comple vi CID. The ‘Heong iteration etcen the aide cxypen soc a peeeeRS Rae Enid tes Std tee SS (tof deal aac tun tne (ACA lly interes neon te [BAP/LICW be tlaling aoa orton atin! ‘bene tale ee rer hops ae tt. Oped = sli a cited le me pore hee eed ‘hoon Scam Sa 6Se Mun Sern 2,186 eubiy Charged Dicaium Complex of GIVEQECASVCSL|EVNQHLCGSHLVE ‘re Sashows he CD spectrum of he doubly cared deaéiam band GVEQCCASVCSL/FVNOEE.COSLVE can. aalogy to ether doubly charged tet core ‘plex dipypides with intermoleular daalide bonds, ‘our daa pode ate beerved vs CID Thea dn procs at wi 1868 and wk 1826 eigte ‘orm the B cain acd ae separate by 66 mas units. ‘The Achaia peplile abo dbserved at 151. The two prods af yt 1373 and ae 13135 from the Achat peptide were dkcused the previous scsi, ‘Tae speceu ofthe peda ati 86 ls :rujer produc renga dey cet (gare) “The pat from the elnnaton of Carmina Gi dered at mt 13876 The producscompesig and 1 fagments at the dehydroinine residue ar abo ‘dsr Coabbad wih thee agar thes sy, Iragents ae the postion of the dle Linkage ‘in he Bechain peptide. The CID pathways of the dleubly charged Gesls bund GIVEQCCASVCSL) FVNGHLCGSFLVE cation summarize n Steed Seltoe SAC Bond Cleanse Prcesses Ineoloong Enolate at Cs ‘Ths uy demure lative SC deneage of doubly durged sollte and ear bound dial fide Inked dipepde eatirs under low-enemy ID condita. As een a gu cn amine produ is ede by the 5S bond clemage proces AS we ‘ropes te formato ofan eran athe Cys esd nite the seve elastin of HS, Gor singly cing etl complex popes ude kw ‘uray CID, These Cad eleavage doubly charged cllsinally actsatal mel comple, pepe s considered ob date fm an enlate Structure a he Cy resue Gum 5). At sen it Figure te and d the BS products compete Baye ‘eogments (ads) formed by ceavage between the enya (4A esd for MPD This suggets ‘hat he Hydogn ars atcha tothe salar abs = a spin ose te ssl aie {= en ae oe Scheme 3 Disc pany of duly ued eau tear pp yop a ia CIVEQCCASNCRL AVN (ESI eed fem Cn pc eed MAPTNG DELARUE BONES IN EPHES au, BET 'e nondesrie tho tm tant Or . i ee Higoe § (4 CD scram of daly chanel akin ‘teri tr emma FRAQILGSI NE py teh apodata i em Amo inc np nga rly achat, utd rm he wun of Cs a al te ‘woken prc Fite 6 shaws he DET cated change le tec nay (5) scaled wid sages lig $5 and SC bends be Ge bound dime Femi (ACA, lay an emda die EDA The optioned geomet of th coreapeing inert ae town lo te dpa. The SS ‘era lavage prc of lds bound (ACAI, echt byapposinday 132m Hower, {be pres 6 elem ovrll 6 he SC ond lage The bem spe sed wi he SC Ne cerage ae energy red by shot Selene Diccaton pote day cand dain No pep ppc fas CVECCEASICHL PQ, {CCIE ede I dpa pred by B®‘nf 1504 eonems thal the MS* product at n/t 11162 Contains a daulde group via the SC bond cleavage Products relting from terminal Asn eliinason ae nerve al ns SDS (HLS-—Aan + HO) and mis TOOLS (Asn + H,0) The products comprise bivpe leagmnonte (by and by $53) formed by clasvage 0 te ‘ini sie A signin feet cy alo Oecrved 1 9 5892 The MS" specu ofthe pode st mi itso. is shown in Figite 3c. Sur to he MS spo ‘ram ofthe peptide atm 1162. the spectrum eis products salting from Cerin Aan elisinatcn {nd the agent. No padi reuling fm he limiration of Hs obseFved,incheting te absence ‘64 daulfde group in the peptide. Two b-ype frag. ‘Pert yaa by at deruarine rsd nate ta poalton Of the ntermobelar diuiide lage fn the gue Al and ¢ show MS* spectia of the MS protean 1072 and ms LRT from the chat, Depte. The MS" af the proc tf: 10072 yields the fxcsive put at 10313 resulting pe ine ‘fon! HS, The MS" spectrum of the MS" peoict st nf BLA ies = and type fagments, Formation of Ju and yy hogments tre dehydroslanine read Ineate the positon of the disulfide lakage in the ‘Sian pepide. In addition, products compraing ‘ype fragments (and 2) are observed in the 2B pales of fe dey charged dale boure YQLENYCN/LVCGERGFF cation are Summarized Doubly Chargat Diculcium Complex of GIVEQCCASVCSL/FVNQHLCCSHL The MS? of the doubly charged dee Sound GIVEQCCASVCSL/FYNQHLCCSHL peptide shows two ditine products at ws 1334 and mz 12864 Figure Aah A produ separated by 6 mass ts om the prdct st 137.4 observed a me 1513.3 The MS" products at w/e 13794 and mis 13133 te aig charge fnamocachs toured pepel fons the Achainpepise with Cyx converted fos dale snd dakydrclanlng, epectvly A produc parted evi aaa ras pore cate aes por iain a vara agg cma lier chem 2. Dencion patina deny caged desion sot pete pie sin VOLENTCNPLARERGPY {Soe fam Cited ont probed by A 1 Aa SoMa Speen 281576 Siieobeiats a Fyne @) (0) slo soa ° ©) fos pe eee Sar Regaraaratircpsoraediera se ees ‘by 65 mass units fom the product at 1258 is na ‘bse inthe spectrum The prodct atm 1258.4 5 the slgly charged morwcacim tound pete with {Achydoalnine fom the Bein peptide gaze ab shows the MSt sperum of the MS prod at w/e 1510.3 Other Hat a dehydration prod et, an fon resulting fro the elimination of HS. ‘tterved asa major puts the ME specu OF tote, the MS prodct st m/s 13133 contra 9 deby- ‘Petinne wokdus cooneted fram Gye ra Be SC fond cleavage. The sipeptise CIVEQOCASVCSL/ FYAIQHE CCE contains to inrmolelar haope nd one intramolecular alfieinage rom fl ts inferred thatthe observed selective eiinton of HS from the dntramolelar daule Unkage In. the ‘chain peptide The observation of by and eae ment af Cysy, and fragient st "Cy late Be Ponitn ofthe intameleular dislide tage Figute {Cnons the MP apectum e the MSF prods a me 12584 The major prod at mf 128. real rom he limination of CHO (-30 mane uns) fam "ery The products campriing bype (by and ba and caype (Cu) fegmonts lente the eatin of he ine ‘Roleclar diulfide linkage. Otter products kom = type Fagments ate ano served. All observed CID pathways of the doubly chirged dica:ium bound GIVEQCCASVCSL/FVNOHLCGSHL cation ate su marae in Scheme 3.Ae So Maspten 200 2155 = » (e) Scho” en” oo 10 10100 "V0 70 ae igure 2.) MS! ecru of doubly dared mona pi diet pt ier oan WOLINYES/LVCGERGH (SP pect dow chmed said ‘Semen in. inalin CVEDCE ASC FINGHLC CT Sy a pls dy i ag Sip ier emate eamigeciaveat rem Beane, ‘icing mujer rodice ieg Saas bond image ‘Swe oi pa ly atid sn oly served at nfs 170.6 and mi 15365 fom the Achaia epi Dowdbiy Charged Calcium Compleres of Peptides rom Pepsin Digest of insulin. ‘The observ alum Bound pepsin digs of isl ‘Rom the ES muse spectusn summarized in Tale Abundant doubly charged calcium complexes are ob- served for three peptic digest products YQLENYCN/ TVCGERGH, GIVEQCCASVCSL/FVNQHLCGSHL, ae CGIVEQCCASVCSL/FVNQHICCSHLVE. Figues 25 show low-energy CID species of daubly charged Ca?" Sound peptides YQLENYCN/LVCGERGEF, GIVEGC- cas and GIVEQCCASVCSL/ FYNQHLCKSHLYE Dowliy Charged Dicalcium Complex of YQLENYCNAVCGERGFF ‘As sen in Figure 34, CID of doubly charge dsl ‘Sound dipeptide YQLENYCN/LVCGERGEF yids four Alstinet products. The products af a/s 1162 and mlz Tso.2and the products a/21097 and nz 081A ae separated by 66 mass tals. The to seb of prods Inbicae the proonce of an intermoleclar Gufie linkage in the peptide. The proc af my 1162 and i Tsb are ingly charged mosaic bound [Neha peptides with Cys converted toa disulde ac “ehjdrosaninetespctivly. via SC bond ce ages. The producs at m/s 10572 and m/ 10314 ae 9 singly charged monoclcum bourd Bhain peptie With s distiide and a debdralaine, respectively Further MS" andlais was performed 59 loate te Prllon ofthe dutfide lnkage in the dipeptice Figure 3b shows the MS" spectrum ofthe radu at wa Tile. the major produc observed at M2 10304 resus fom elimination of H,S, The MS* praduct at | alba Fe [Se we ae fh alle1 A Se i pn 2005 21768 “abe 1 Mae pepe oni the mae pcan of te Singly Charged Saliated Peptides from Pepsin Digest of Insulin As summarziat ip Tabi 1, dbundant singly charge ‘Salita peptde lone ae ‘observed for thice peptic ‘igeat produce om maul, hich coerespondt te ‘YIPKA, FYIPKA, and NYCN/LVCGERGEF. By scare ting, ME of ingiy ected pepe lget Connpenent for lw of Sess nits FS tis posable to tdenufy peptide agments with disulde linkages {GD of sedited peptises YIPKA at ms 6012 and FYIPKA at m/s 7805 yield major products resulting commer fn the elcination of terminal Als (=89 ‘nase unis).No products found fom Ue elimination GEHL, (~ts mass un). Results for CID oF monoaee “late dipeptide NYCN/LVCGERGFF at m/s 15598, lke via an intemolcular ule bowed, were pre ‘ented in our carer sty [12]. The CID of sdised NYCN/LVEGERGET yiede a major product rom HS, ‘lication. The obervedelninaten of HS isis the preemce of dildo Hnkage in the pope Disbly Charged Sodiaied Peptides fim Pepin Digest of Insin Abundaot doubly charged sisted pepide ans are ‘served forthe peptic dig NOLENYCN? LVCGERGEE, GIVEQCCASVESL/FVNQHLCOSHL, and GIVEQCCASVCSL/FVNQHLCGSHLVE (Table 1 Figure 2 sktws CID ‘pects of the cor doubly hanged sodited peptic digest frogments (Table 1) cf Inaull The GID’ cf doubly chatged moncendiated Aipeptide YOLENYCN/LVCGERGEF yieis abundart eokane poclling ers dobpdenien and comical ‘ehydration and arnmonia elimination (Figure 2), bx dui, minor product fom she elminason of ‘Aehan C-erminal Aen ls olzerved in the spect, ea sonee eae h fms tenn “the podct tm 10593 ithe singly changed, poten ste Bei pepe, with “SSH replacing SH a 6 "Figae 2 shows the CID spect of he duty starged dhodiated.dipephde’ CIVEQCCASVCSL! FVNQHLCCSHL Te dpi by one tr ‘oleclr dilfide bond between “Cys ad "Cys Ia tie nage btw cjg sl Cy, Varins pr sete sing fs debyetn (13805 a 12765} the SS bond ceavage (nf 13293), he terminal vai tna a 12465), and caer Aap res (ne HDL and nf 1053) we oar inthis Inada, CD af oly charged ‘aod ipepte ye ow it singly charged ‘monomeric at mi 12025. mh TTA. SO aa nA! The pod a ne 13S ad tds 138 3a separated by Goma i. The prods Sul tom he conversion of Cys to (202.9 tec luli group (13083) foe Acain peptide SSC bord lemape The place at ns EAE Sand Ife 133 melt fom convenion of Cys nk alter + 4A ora diol group on be Bed pope, The product foe the eiinatons of two HS, aka two die nag fe pepe “The dijpile GVEQCCASVCS./FVNQHLCGSHLVE erent se rma rings beeen Cyd * ye, long with neal Une tween Cy od Seyeh The CID oper cf day Gage ‘etd CIVEQCCASVCSL/FVNQHLCGSHILVE Shwe in Figaro prt wouing fous she ‘Sinton two HS obverved st ms 1225. Two Siely charged mancnochted product soporte by 6 fete se nerd a me 2974 and fs 3634 from he Bhain pple The other wo singly charg ‘eneodied peoduts seperated by ae un1 Shan pn 205 8 66 4A, epee. There scp refer to the ‘rater of rt the pret bo when more un one ‘peated monomer ntexsin aeons For ene, pure eM) wihtwo popes wich ae Cae V, Tike vl to lle nc refered diay ‘The dehydlaine ride 6 referred to a6 JA in daplived peptide sequences The element mba a peri forthe agent on refers othe metal cain in the dogly carga metal compen ofthe fags Forexampb, ay ramet ion complen wih Ca* is efor ty The mmol subsp fr to the ‘numero equientaino ald unis in he Fagen ens of etl conplse ofthe pepe pep Resulis and Discussion Lote-Eergy CID of Met Completes of MP2 We have rely denon the lw-enegy CID of slay cued conc Nat and C* camplesof MP, hid b te dime motel pepe (AARAAACAA): Tine by a itermolecale ule bow 12. The (ID of the morosodiated PB yield th dominant produc sung from te elininaon of HS, (66 ‘mas un) with mone reduc from theca ‘peso eS Sind SC. Adnau rete the relative abundance othe pdt fm thee tion of bere rm singly charged aalne earth etl (Ca and Mg) comple of MPL Ine pleat wade we ened thes eather sid Ws lake oubly targedcatoicomplees of MP2 antipating ‘hal thesis canbe wen erpasng CID suis of te dundrt doubly charged Na” and C* cane ‘pln bnerved th BT mas petra ofthe pple gest cid, gue 1 shows MS specte of doubly charged ‘toe complees of MD With 4 Na” fn ada Ca" Jon Thelu-enegy CID spectrin of the monoxditad peptides dh ia Figure i The CID ofthe doubly ‘hared sdateé peptide yields four dine singly charged products The pecs att HLA aed [M72 are poked rot pps aaa wf 18.5 and ns $2.3ace monosdatd prduct peptides eth pate protratd and soit procs ran MP re Sprte by 66 mass uns, corespeeding I he - Iecular weight of, This eas rom the Clad avage af the ineolenr did Inkage. The low-energy CID of doubly dargedCa* bend peptide lls Hight selective clavages of SC bends Two ‘lng charged calcu boul products eign ‘the SC bond cleavages ame observed at 794 and nt SED as wel as bao singly chug potnalad procs at wf TLS and w/e S02 Figure bl eis ‘otbl that only a noe produ from he SS bond dewage i aber a 774 in he CID pete (Pe 1). The NS? pei fhe prac a 7A and mfp 7413 are shown fn Fg 1c and, rsp tively. The MS? spectrum ofthe Ca® bound prodctat ‘Wh 7™0A eats the donna product sll ees ‘ohoptantan vt to sag > me foal ll soe 7m mem toy 20 aa ears Sy comeneresmrremiee Sheri eer eee meine oped oe eer daydeaton (gute 19. Oe hun deyeaion, he prot capris ype amen and by eed by chavages on both sides of the GA residue, The calisinaly seated prfaatd product at mf 74.5 deselies to yield brood sange of b- and y-ype fragments, Sear to the MS of the pdt at mit ‘74, tye eapents (and) formed by dene ‘on bthsdes ofthe dre ae observe in the MS? Speco of te prt ats 75 (gue 1d) Low-Enargy CID of Meal Complees of the Pepsin Digest of Insulin “The ES rms spect of pepsin yet of lin eats ight mur Son peaks ht show goad agree sent ith ere publ arlyes (abe 1) [17,1 “The mass and spent of bere metalic digs nso sin with ded seam and clue sa inl in Table 1‘sequence information [23 The ID cf sng to eely ‘aged iain ie fngrents ruling fom inte ‘oleae dsulide bond deavages [19 Although ‘hee iop-dows meade yield sequence donation of fnsulin ivduding he positon of the Cys residues, ‘ecting the poi othe orgie dale Lnkages in ‘he protin romain 2 challange. Anliis of «pepe digest ls prolered forthe invention ofthis prot tenet acidic pH premerves the oigial doalide ‘nods [2]. Furthemene pepsin atic a wide ange of ‘walle Unages pelding pod pple lnvaling ‘ceavage between ot Cys mids [18 Our previ ‘sudy reported tha the destmspry ination (ESD ‘mas spac rom the pepsin digest ol isin shows legit aujr ion peaks, comprising ie singly charged ‘thes doubly changed i peat [2] Inthe pre fnvestgatn, we examine doubly chayed NA" and CP coaipleas of the dase ad toda pape (AARAAACAA), (MP2) Ther, we denenstate fut ‘olson avai of ctione Ni and Ca™ come plies of Lvl pple diet fegmant allows for Straightforward analy the dls iskages inthe fate roel, The mechassns and nests ofthe ‘hserved reactions are futher examined by mens of ‘compuatoal edeling Sucre of MPL ad eslin ‘examina in ths study are shown in Scheme 1 Experimental (aun dod (CC) nn ro bie pers, ‘nde (pepsin. om porcine sterack mio, and sodiue code INC wer picked fom Sa ‘Aldsidh (Lois, MO, USA). The madd pepede "AARAAACAA ws pus fon Bonen {Concord CA, USA Alsen ater, ethan, and Tervene) mere from END Chemie be. {Gidbsown, NJ USA) Forte maton oan tener lela did heed png a diner of the male ‘pide AARAAACAA, {Onl pple in 00 ater was mixed wih eM 90 pf enaene ‘Teseltion mixture wa sine vigor 3) in st oc temperate The aque soli was ex traced afer entugaton. The sample solution wat slut to an eppropte onzetation for ES with 5050 water/methunolsuvert The prodec core sent snngnmen Anteldadbttede Seer Rerrrrrveranrere sag ‘She Sac of del epi andinn amine hey 1 ean pete 2 sponding tothe diver pepde MP2 lied via « deli boa wasconfered by ES mas spectone- ric analysis. All metal complex samples nee prepared by dialing blinds aroun of tal ode ad the analyte sample nthe solvent Taal concen: tion ofthe sample slain was var rom 101 20 ‘aM. Papi digo of instlin were prepa By inc betlag 01 mg of della fo bonne pares with (1 ig of pep from porcine stomach econ in water contin 1% acetic acy volume at 37°C for 6h Thun pepe was toned using & Mesos ‘enrages (Miipre, Bilrcy, MA, USA) fed ‘than Uae Y¥-10 mentrane. The supe lator wis died to an apprpeltecaceaaton fr ESL Met complex samples were prepared dchng pM neal chloe lathe dled pep digest sletion. Experiments were pesirmed on an LCQ Deca in leap sass spectente (TMS, Therafianigan LIC, Sen Jose, CA, USA) in pate mode Electospeay volige pares of 4.5 LV ply volage of 8 V, asd ely 275°C wet at fo ESL The temperate of the MSanalyaerwasatout 5% before the expesiment and about 24°C daring he experiment ‘The pressure was estate to be approximately 10°> tort He inde the tap. Mlabcomplexed pepdes of Imterst were lite and fragmento va low-energy ‘CID Continous scaten of sete oe alowed by ‘CID (NS) Was performed uni the ac ofthe ated te was bt. The BSlras spect report in sty were obtained by avemping seamed spca “The mechs and energetis of te S—C bond and $5 bend cleavage rearins were evahsaled ting denty funcnal theory (DFT) clelabons, Canidae structures fr the Ca™ complex of the dipepide (ACA, bnked by 4 disulfide bond were [generated using the ANBER (Assisted Model Bud {ig and ergy Refinement force fl by ajectng staring canormatin to 200 pu of dynamics at 500 K, then cooig to 50 K over a varble period of tine wing HypesChem 752 (Hypercabe, Gaines, FL, USA). For intermediates, 20) ps’ of drramic skull was eset 20 K followed by cling to 50 K over variable period of tne. The charge isbn ofthe compen was assigned atthe PHO level in each annealing vesuence. Over 10 races were generated and tpl 10 candidate racures ‘were determined. The sutures were eteriained wing DFT with dhe candaate stocttes ‘The DET caklaons were performed using Jaguat 460 Sehodinger, Ine, Prd, OR, USA) wikzing the Bech tee pacamete factional 83) 24] cam Dined with the corelation funcional of Le, Yang, aed Parr (LYP) [25]. asing the LACVP basis set ‘Thecwodynanse properties were calculated sans lng iden gas at 29815 K. "The onsets propa by Reepaof and hl sian [26] wat wef the patent and agent hs, ‘The pebus dehydodanine ad 3 of pare os (M1) refer to poptide ions containing #dsulide bond andMapping Disulfide Bonds in Insulin with the Route 66 Method: Selective Cleavage of S—C Bonds Using Alkali and Alkaline Earth Metal Enolate Complexes Hagh 1. Kian’ and J. L. Beauchamp Nags Litt hepa, Cabinet Tey, ate, Callers US ‘Spl and Gt etic of dead Unges bs lla demonstrated with a ‘it ug the Rese 6 nto. Tiss accom ied by celle eerste ng lestspaylonzaton doubly urged ctone Na* and Ca™ complees tin of gly and ‘cepleces ot CEES) Cla chain Sony dred tl pl wit init duel Unlape is tS of gly carpe ped Erection of mann ek Ss HS i gy a et ebstlied woe on Slowed by cera ‘he products ied soqurc nformatn “inka in th pop cg ht Gl peptides ASVCSL/V 565 sus ur suing rom sete SC boa la Takige ander bu-energy eollacn-edueed dash sepsctied by 6 mcs unt ening frm alactve SC band cleavages tothe cl figs it oF ie seh of peptes with disalide Tina by Temaion ef age cf SC bod Fare acta of Tails loading the poston ol he dsiide us Fv ape rye cape SNCRUCCSML jpeg ata demoed the activa the pase comping the A la eves th rem fs ond dle bly a ntarolec ee a a Experimental ced ewe mechani deta forthe selective ce tis ofthe ladle ed ene popes prove ofthe SC bend. Am Soe Nas 5 2105, 20, 157-ge) © 200 Publ by Ese Ion bea of American Sit Inte ds ar ne of he mot pant jst astona masiaie FTA) proces Recah unique rol deter the ‘hc dire stead abs of hs, USL Ahough ain PT ste a pep have eon ef ining ner mse pect QMS) cit] dade bos arent rnd cance by MS studies of rut pps P Seve! stules sched lee devas te 8 and SC had ‘de kage get aps 1126. naires WP", CP and 2) came ples oft ahi aton pty ied fo $5 and 5 bond cesages under dleton capa doco (ECD) condos [1] nf under Suited oesaane iradation olson nied scan SORECD) cnlies [5 Secive SS bend clara in ati gl) complens 0! pep {ees ender lnergy CID conditions wee ah et me Bec Ci egy Np ey ohne is etme (Spend e Cams a foe, CWI i epee “Tae sn topic Lewate, Co fta Tee Berend ts or sicrse tiered (© 2a. by Haver no blo Ameen cy or Ma cre. naan sth psms 203000 pr [316 We ave erty rept what call the Route €© metid for lean due nds in “This metadoloy i based on the highly Sate last of HS 6s ns) ey charged wilted and allie ath metal Mg and ‘Co bound pptile aon wih dale Inkages unde CID ction [2] The proces initaed stating wit metatbized enolate ann ot Cys fale by ceaage ofthe 5—C bod. Faroe NS? Spec reveal adit dels of he pepe ttre In he regen betwen the sey fred deal rie A) eas. Inthi study, we rp application ofthe Rte 6 rebar ioc the postion of suis in inulin. Ths peptide homone, widely wed as 2 rode ssh forthe entiation of dilde bonds Along with ere efrmatin, ese chlege of thee cel spaced dle bea, including oth intramolslar and ities kage 2-19, 4 nan of sthes have rere sequece ais of inulin wing mass speeromeny [9-2], Came sequence aay of ksi was densa sng therelced pt wh lowe encgy CD E2} BOD ok the onde Beha of rl yes alot compete abled ane Cee 1 208 ets fe 2 ‘eva! athe 1 sepia 3aCTD GLUE Stee te< A feat y ~ Ps = ‘ GENERAL - CHEMISTRYCOLLEGE CHEMISTRY Eighth Edition sad Mey) | JEROME L. ROSENBERG — The perfect aid lor better grades! 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