48 NCUBTON OF DENTURTED SMPLES NCRESED REPRODUCBLTY ND QULTY OF PROTEOMC PROFLE OF SELD-TOF MS Muharva Rakaah 1*), T. Mrawa Sudro 2 1. u of Huma Vrolog ad Cacr Bolog (HVCB), Uvr of doa, Jakara 10430, doa 2. Dparm of Mcrobolog, Facul of Mdc, Uvr of doa, Jakara 10320, doa *) E-mal: m_rakaah@ahoo.com brac Pro proflg wh hgh-hroughpu proomc cholog, SELD-TOF, a w poal ool for dago of huma da. Th advacd chqu ha cragl b ud for h dco of da bomarkr. Howvr, aalcal rproducbl a gfca challg SELD-TOF proflg ordr o hav cofdc h rul. Hr, w howd a mpl p o mprov aalcal prformac. cubao of daurad ampl ovrgh a 4 o C crad gfcal rproducbl ad qual of proomc profl of SELD-TOF MS for MC30-Cu ProChp. Th rag could b cocrd ad appl o addr rproducbl u h m. Kword: Proomc, rproducbl, SELD-TOF 1. roduco Pro proflg wh urfac-hacd lar dorpo-oao m-of-flgh ma pcromr (SELD-TOF MS) a promg approach for bomarkr dcovr. Th roluo of h advacd cholog ha b mprovd b corporag fracoao ad a var of aff capur chqu 1. Rcl, SELD-TOF pro proflg ha b appld ma proomc bomarkr rarch uch a cacr ad fcou da ha rval hgh dagoc v ad pcfc. lhough a robu plaform for pro proflg, om ourc of chcal ad o-chcal varao mak rproducg ad valdag poal bomarkr challgg. 2-3. wh a proomc aal, ll rqurd for opmzao of xprmal procdur o ur co daa oupu. Cofdc ha dffrc SELD-TOF profl par pcfcall rflc chag pro cocrao dpd par o pcra rproducbl ad qual, ad h paramr ar affcd b mulpl varabl 4. Sampl prparao procdur o of mpora facor o gv a br qual, rproducbl ad robu oupu of proomc profl of SELD-TOF MS 5. Hr, w prform a mpl p o crad robu of h chqu. W howd ha b ug MC30-Cu ProChp, cubao of daurad ampl crad gfcal h umbr of pak dco ad dcrad pak coffc of varao (CV). Th rag could b ud a a alrav chqu o addr rproducbl u h m. 2. Mhod Srum Sampl. Blood from 5 halh volur wr collcd Vacuar ub whou addv, l clo 30 mu a room mpraur ad crfug for 30 mu a 3000 rpm. Srum wr rcovrd, poold ad froz a mu 80 o C ul ud. SELD Pro Proflg. For SELD aal, w u wo ProChp rra (Cphrg Bom, c), mmoblzd aff capur (MC30) ad wak cao xchag (CM10). Srum ampl ar procd a prvoul dcrbd wh mor modfcao 6. Sampl wr prpard c b mxg 20 ul rum wh 30 ul U9 buffr (9 M ura, 2% CHPS, 100 mm Tr ph 9) a 1.5-ml mcrofug ub a 4 o C for 10 mu ad wr hakd for 30-45 mu a room mpraur. Th, 2 ul rum/u9 buffr wa dlud wh 98 ul corrpodg bdg buffr (PBS 1X + 0.5 M NaCl for MC30 ad 100 mm Na aca ph 4 +/- Tro X for CM10). Daurad ampl wr h ord (cubad) 4 o C ovrgh. For corol (ram whou cubao), daurad ampl wr pod drcl o h arra. Th ProChp rra wr ambld o a dp-wll p boprocor (Cphrg Bom, c). Pror o ampl loadg, MC30 wr coad wh CuSO 4 b 48
49 addg 50 μl of 100 mm CuSO 4 o ach arra. fr 10 m agao o plaform hakr a a pd of 250 rpm for 10 mu, h arra wr quckl rd wh D war o rmovd uboud mal. To rmov uboud coppr, 50 μl odum aca oluo ph 4 wa addd o ach arra ad hak for 5 mu ad rd aga wh D war. Th arra wr h qulbrad wh 150 μl of bdg buffr (PBS). Pror o ampl loadg, CM10 arra wr qulbrad wh 150 μl of corrpodg bdg buffr (50 mm odum aca ph 4.0). rra wr pod wh 50 μl of dlud ampl for 30 m o a hakr a 250 rpm ad wahd hr m wh 150 μl corrpodg bdg buffr. fr rg wh D war, h arra wr rmovd from h boprocor ambl ad ar drd. CHC (Cphrg Bom, c, Ca.No. C300-0002) a rg aborbg marx wa prpard accordg o h maufacurr ruco 50% v/v acorl/5% v/v rfluoroacc acd ad 0.5 μl of h aurad oluo appld wc o ach po o h chp. W ud CHC bcau h marx gav b proomc profl molcular ma 3-15 kda whch bcom our ad ma ohr ud cocr dfcao of bomarkr. ProChp arra wr aalzd h ProChp Bolog Sm radr (Modl PBS, Cphrg Bom) ad h daa wr aalzd b ProChp Sofwar vro 3.0. 3. Rul ad Dcuo bomarkr ud wh SELD-TOF pro proflg h am o df pak (or pak ara) ha ar dffr bw ca ad corol ampl ad hu rproducbl of pak of hgh mporac. Dp promg, SELD-TOF aal o bologcal pcm uch a rum rma challgg du o complx. Much ha b dcud SELD-TOF cholog cocrg log-rm robu, h corbuo of o-bologcal varao o h rul ad h d for br qual corol 7. O of h mo crcal u of h chqu rproducbl of h daa ha crcal bomarkr dfcao ad valdao. Svral ud addrd h u hav b lookg a h mpac of om praalcal varabl uch a blood ampl procg 2, ad adardzao aalcal codo 8-9. To ur co daa oupu, procdur or rg proocol ha hav b opmzd ad rproducbl a urg d. Th ma hru of h work wa o dvlop hacd proocol o mmz varabl of daa caud b praalcal varabl. Hr w dcrb a mpl p o mprovd proomc profl ad rproducbl of pak dco a drmd b umbr of pak ad gal coffc of varac (CV) o a QC ampl (poold ormal rum). To calcula CV of pak, all pak dcd ach xprm wr ud. wa how ha b cubag daurad ampl ovrgh 4 o C, gral, robu of h chqu wa crad for MC30-Cu a dmorad b rproducbl ad umbr of pak dcd (Tabl 1 ad Fgur1). Th avrag of CV wa dcrad from 18,5 (whou cubao) o 13,9 (o/ cubao) (p < 0,05) ad h avrag of umbr of pak wa crad from 30 (whou cubao) o 50 (o/ cubao) (p < 0,01). For CM10, alhough cubao ram md o hav pov ffc o proomc profl qual, h mprovm wr o acall gfca. W alo foud ha om pak wr dcd hghr b crag of h cubao m (30 mu ad 4 hour h V Vru O/N), howvr corar ffc wa how om ohr pak (Fgur 2 ad 3). MC30- Cu ad CM10 ProChp wr wdl ud bomarkr ud for fcou da uch a frcatrpaooma 10, SRS 11 ad cacr lk ovara 12, ad lug cacr 6. Th wo arra gv mor pak compard o ohr Chp avalabl. Th mpl p dcrbd h work could b ak oghr wh ohr rag o addr rlabl proomc daa dvlopd b om ohr ud. Sc valdao of da bomarkr rl o opmzd ad rproducbl laboraor mhod, uch work of mporac. Furhr ud, ha focu o vgag h ffc o ohr Pro Chp arra dd. Tabl 1. rra Summar. Rproducbl ad Qual of Proomc Profl MC30-Cu ad CM10 ProChp rra wr Show crad wh Ovrgh cubao Tram MC30 Exp %CV p<0,05) a Numbr of (p<0.01) a pak w/o cubao o/ cubao w/o cubao o/ cubao 21.4 11.6 40 49 13.7 11.8 39 50 16.8 14.0 39 51 V 17.9 12.7 35 49 V 20.8 18.1 37 47 V 20.6 15.5 42 50 18.5 13.9 39 50 rra CM10 18.0 12.8 37 40 20.8 18.1 35 45 19.4 10.4 b 36 43 b a Calculad b h o-paramrc O-Wa ova b Sacall o gfca
50 0 Da 5000 10.000 20.000 w/o cubao o/ cubao B C Fgur 1. Effc of Ovrgh cubao o Proomc Profl MC30-Cu arra. Spcra Ragd 0-20.000 Da (); Som Pak (Ragd 3000-4000 Da) wr Dcd Hghr cubad Sampl Compard o u-cubad Sampl (B); Gl Vw (C) B 30 cubao 4 h cubao o/ cubao 2800 Da 3400 2626 Da 2725 Fgur 2. Effc of cubao Tm of Daurd Sampl Proomc Profl for MC30-Cu rra. Two Pak (2872 ad 3162) wr Dcd Hghr Sampl cubad for 4 h, ad Ovr Ngh). Howvr Pak of 2968 ad 3321 wr Dcrad b Logr cubao (4 h, ad ovr gh) (); Th am Phomo wa alo Show om ohr Pak (B)
51 30 cubao 4 h cubao o/ cubao 6500 Da 6750 7000 7250 30 cubao 4 h cubao o/ cubao Fgur 3. Effc of cubao Tm Proomc Profl, Ragd 6000-7500 Da (); Gl Vw (B) 4. Cocluo For MC30-Cu, ovrgh cubao of daurad ampl a 4 o C wr how o crad rproducbl ad qual of proomc profl of SELD-TOF MS. Th wr how b gfcal dcrag pak coffc of vara (CV) ad crag umbr of pak dco, rpcvl. Th rag could bcom a mpl p o addr rproducbl u h proomc cholog. ckowldgm Th work wa fudd b R Uggula Uvra doa (RUU) gra 2008 udr Corac No. 2405/DRPM-U/N1.4/2008. W hak Fra brahm, Ph.D ad Budma Bla, Sp.MK for hr uppor h ud. Rfrc 1. Mrcha M, Wbrgr SR. Rc advacm urfac-hacd lar dorpo/ozaom of flgh-ma pcromr. Elcrophor 2000; 21(6): 1164-77. 2. Bak RE, Sal J, Car D, Barr JH, Clark P, Thompo D, Slb PJ. fluc of blood ampl procg o low-molcular-wgh proom dfd b urfac-hacd lar dorpo/ozao ma pcromr. Cl Chm 2005; 51(9): 1637-49. 3. lbrh J, Bøgbo R, Ol J, Rakov H, Gammlof S. Praalcal ad aalcal varao of urfac-hacd lar dorpoozao m-of-flgh ma pcromr of huma rum. Cl Chm Lab Md 2006; 44(10): 1243-52. 4. Jock C, Paulauk JD, Bakr D, Oll E, Blav MR, Joho KJ, Hard PL fluc of marx applcao mg o pcral rproducbl ad qual SELD-TOF-MS. Bochqu 2004; 37(1): 30-2, 34. 5. Cordgl HC, Robr SL, Took P, rmag JR, La PW, Wu W, Wldmh SE. Mulfacoral crg dg ad aal of SELD-TOF ProChp arra opmzao xprm. Bochqu 2005; 34(2): 364-5, 368-73 6. Yag SY, Xao XY, Zhag WG, Zhag LJ, Zhag W, Zhou B, Ch G, H DC. pplcao of rum SELD proomc par dago of lug cacr. BMC Cacr 2005; 20(5): 83. 7. Gurrro N, Gomz-Maclla B, Charmo S. Opmzao ad valuao of urfac-hacd lar-dorpo/ozao m-of-flgh ma pcromr for pro proflg of crbropal flud. Proom Sc. 2006 pr 27;4:7. 8. Smm OJ, Fg Z, dam BL, Baz LL, Bgb WL, Campo D, al. Evaluao of rum pro proflg b urfac-hacd lar
52 dorpo/ozao m-of-flgh ma pcromr for h dco of proa cacr:. m of plaform rproducbl. Cl Chm 2005; 51(1): 102-12. 9. Bo J, d Bor D, va Dj-Vr MP, Wodzg WK. Sadardzao of calbrao ad qual corol ug urfac hacd lar dorpo ozao-m of flgh-ma pcromr. Cl Chm ca 2006; 366(1-2): 249-56. 10. Papadopoulo MC, bl PM, graoff D, Sch, Tarll E, Bll B, al. ovl ad accura dagoc for huma frca Trpaooma. Lac 2004; 363: 1358 63. 11. R, Y. Th u of proomc h dcovr of rum bomarkr from pa wh vr acu rpraor drom. Proomc 2004; 4: 3477 3484. 12. Prco EF, rdka M, H B, al. U of proomc par rum o df ovara cacr. Lac 2002; 359: 572 77.