Tiiu ban: cdc chdt co hogt tinh sinh hqc ISBN: 978-604-913-012-0<br />
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KET HOP MO PHONG PHAN IVLANH PEPTIT VA PHO FT-ICR-MS<br />
KHANG DINH CAU TRUC OXYTOCIN<br />
Ngo Van Ouang, Ngo Quoc Anh, Nguyen Van Tuyen, Nguyen Tien Tai, Nguyin Le Anh<br />
Vien Hda hoc<br />
18 - Hoang Quoc Viet, Cau Giay, Ha ndi<br />
Email : ngo_nhatquang@yaboo.com<br />
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Tom tat:<br />
Ky thugt khdi phd hai ldn MS/MS hay cdn ggi la Tandem MS su dung phdn<br />
mdnh bdng bdt giir dien lu (Electron Capture Dessociation - ECD) do trin thiit<br />
bi phd khdi cgng hu&ng ion cyclotron cd biin ddi Fourier (Fourier<br />
Transformation Ion Cyclotron Resonance Mass Spectroscopy - FT-ICR-MS) dugc<br />
ung dung nhu mdt gidi phdp hiru hiiu trong nghiin ciru cdc phdn^ tu sinh hgc cd<br />
kich thudc ldn vd cdu true phuc Igp nhu peptU vd protein. Ldn ddu din, ky^ thudt<br />
phdn lich hien dgi ndi tren dugc thirc^ hiin tgi Viet Nam di khdng dinh cdu true<br />
Oxytocin - Mdl peptit cd vai trd ndi tiit Id v&i chuc ndng truyin ddn cdc thdng tin<br />
tgi ndo<br />
Abstract:<br />
MS/MS or Tandem mass spectroscopy (MS), used Electron Capture<br />
Dessociation (ECD) on the Fourier Transformation Ion Cyclotron Resonance<br />
Mass Spectroscopy (FT-ICR-MS), can be a high-throughput tool for protein and<br />
peptide characterization in proteomics. For the first time in Vietnam, this<br />
advanced technique M'as successfully applied lo confirmation Ihe structure of<br />
Oxytocin - A mammalian hormonal peptide that functions as a neurotransmitter<br />
in the braia<br />
<br />
<br />
I. Md dau<br />
Mac dii kha nang iing dung nguyen ly cdng hudng ion cyclotron do Lawrence phat<br />
minh tir nhung nam dau the ky trudc de xac dinh khdi lugng phan tir vdi do chinh xac cao da<br />
dugc tien doan sdm, nhung ciing phai tdi nam 1974, nhd nhiing tien bd mang tinh each mang<br />
ve ky thuat do, phuang phap phd khdi cdng budng ion cyclotron cd bien ddi Fourier (Fourier<br />
Transformation Ion Cyclotron Resonance Mass Spectroscopy - FT-ICR-MS) mdi thuc sir ra<br />
ddi [I]. FT-ICR-MS khdng nhirng cd the dat dugc do chinh xac khdi va do phan giai khdi cao<br />
ban nhieu cap so vdi cac ky thuat MS tmyen thdng ma nd cdn cho phep thuc hien nhieu ky<br />
thuat chuyen biet de nhan dugc cac thdng tin cau tnic phong phu, nhat la khi nghien ciiu cac<br />
phan tir cd kich thudc ldn va cau tnic phirc tap, nhu peptit, protein, ADN [2].<br />
Sir ket hgp cac ky thuat ion boa mem ciing vdi cae ky thuat phan manh ion cho phep<br />
thuc hien ky thuat MS/MS (MS^), hay cdn goi la Tandem MS, cho phep xac dinh trat tu<br />
amino axit (aa) ciia cac peptit hay protein mot each thuan tien, de dang va nhanh chdng ban<br />
nhieu so vdi phuang phap Edman truyen thdng. Trong cac giai phap phan manh ion de thuc<br />
hien MS/MS, ky thuat phan manh bang bat giu: dien tir (Electron Capture Disscociation -<br />
ECD) do Zubareb va cac cdng sir phat minh ra gan day (nam 1998) [3]. Dac diem chinh ciia<br />
ECD la qua trinh phan manh xay ra do do tuong tac ciia cac electron nang lugng thap vdi cac<br />
ion da hoa tri va hau nhu chi thuc hien dugcc tren thiet bi MS dang FT-ICR. Thuc tl img<br />
dung cho thay ECD la giai phap phan manh dac biet thich hgp eho cac peptit hay protein cd<br />
cdu S-S hay S-H [4].<br />
204<br />
Hoi nghi Khoa hqc ky niem 35 ndm Vien Khoa hqc vd Cong nghe Viet Nam - Hd Noil 0/2010<br />
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Tai Viet Nam, do su phat triln khoa hoc cdng nghe va san xuat ndi chung, phuang<br />
phap MS ngay cang trd nen phd biin, sd lugng thilt bi MS ciing nhu sd lugng va trinli do<br />
chuyen gia MS tang tuong doi nhanh. Dii vay, rdt gdn day Viet Nam mdi cd he thiet bi FT-<br />
ICR-MS dau tien va bien la duy nhdt ciia ca nude, ldp dat nam 2009, tai Vien Hoa Hoc, Vien<br />
Khoa hoc va Cdng nghe Viet Nam, dinh hudng vao cac bai toan phan tich phiic tap, cac bai<br />
toan phan tich cd tinh thdi sir va y nghia cao ve khoa hoc cdng nghe ciing nhu kinh te xa hdi.<br />
Mot trong cac thir nghiem ciia chiing tdi nham timg bude tim hieu kha nang iing dung<br />
phong phii va hieu qua ciia FT-ICR-MS, la iing dung ky thuat Tandem MS de khang dinh cau<br />
true mot sd peptit vira va nhd. Trong cdng trinh trudc [5], khi Viet Nam van chua cd thiet bi<br />
FT-ICR-MS, cau tnic rieng phan ciia Interleukin 11 da dugc khang dinh dua tren dir lieu<br />
Tandem MS, thuc hien tren thiet bi FT-ICR-MS cua Hoc vien Khoa hoc va cdng nghe tien<br />
tien Nhat Ban. Tiep theo, trong cdng trinh nay, thuc nghiem dugc tien hanh tren thiet bi FT-<br />
ICR-MS ciia Vien Hoa Hoc nay nham khang dinh cau tnic ciia Oxytocin (OT), mot peptit<br />
gdm 9 amino axit vdi cdu S-S (Hinh 1). Trong boa sinh, OT dugc bilt din nhu mot ndi tilt td<br />
cua ddng vat cd vu vdi chiic nang tmyen dan cac thdng tin tir nao bd tdi cac bd phan ciia co<br />
the ddng thdi cdn ddng vai trd quan trong trong qua trinh sinh san cua phu nii [6, 7]. Ve mat<br />
cau tnic, OT dugc xem la dien hinh eiia peptit chira dj td va cau S-S, la ddi tugng thich hgp<br />
cho cac ling dung cua Tandem MS bang ECD [8].<br />
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Hinh 1; Cdu true hod hgc vd trgt tu aa cua OT<br />
H. Thuc nghiem<br />
Mau: OT chuan, SKS 0106201, do sach 98,29%, do Vien kiem nghiem Dugc cung cap.<br />
Thuc nghiem MS: Mau OT dugc pha trong hdn hgp dung mdi AcetonitriIe:Nudc:Axit<br />
focmic = 49,5 : 49,5 : 1,0. Thuc nghiem MS dugc tiln hanh tren thilt bi 910-MS TQFT 7T<br />
(Hang Varian), tai Vien Hda hoc, Vien Khoa hoc va Cdng nghe Viet Nam, sir dung ngudn ion<br />
hoa mem ESI.<br />
Tinh toan: Cdng thiie phan tir dugc tinh bang phan mem Molecular Fragment Calculator<br />
MFCalc^^ (http://home.pacbell.net/ideline/^: Phdn mim PeakHunter ^^ (Varian Inc.) dugc<br />
diing de ddng nhat cac manh ion nhan dugc tir ECD Tandem MS; Md phdng phan manh ion<br />
dugc thuc hien bang chirc nang phan manh ciia phdn mim ChemBioDraw Ultra 11.0<br />
(CambridgeSoft).<br />
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205<br />
Tiiu ban: cdc chdl co hogi tinh sinh hqc ISBN: 978-604-913-012-0<br />
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III. Ket qua va thao luan<br />
1. Xac djnh cong thuc phan tu<br />
Phd thuc nghiem FT-ICR-MS the hien rd 2 tin hieu diln hinh ciia cac peptide cd kich<br />
thudc vira va nhd, bao gdm pic ion phan tir M"^ =1007,4433 va pic da dien tich M^" = 504,227<br />
(Hinh 2). Cac thanh phdn ciia ldp ddng vi (Isotopic Cluster - IC) tren phd FT-ICR-MS da tach<br />
nhau rdt rd ret, gdm pic don ddng vi A (Monoisotopic Ion) cd cudng do ldn nhat va cac thanh<br />
phdn A+n (n>l) cd gia tri m/z ldn hon nhung cudng do nhd ban (Phan gian IC tren binh 2).<br />
Vdi chi do quet phd cham hon dl dat dugc do phan giai khdi cao ban, chiing ta cd the nh^n<br />
dugc cdu tnic ddng vi tinh tl (Isotopic Fine Stmcturre - IFS), cac thanh phan A+n bi tach tiep<br />
(Phan gian IFS tren hinh 2).<br />
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IFS<br />
n5.200WFHM<br />
IC<br />
46,500WFHM<br />
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k 1<br />
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r-rf-I-TTjTifTTT<br />
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Hinh 2: Phd FT-ICR-MS ciia OT v&i phdn gidn IC vd IFS cita pic M'^=1007,443<br />
Ve CO ban. cac thanh phan A+n (n>l) cua IC la sir thay the n ddng vi C bang n ddng vi<br />
13<br />
0 va dugc goi la cac pic caebon. Ngoai sir thay the ' C ndi tren, sir thay the cac ddng vi nang<br />
ciia cac nguyen td kbac cd mat trong cau tnic ciing tao thanh cae pic IC.<br />
Vdi do phan giai chua dii cao (46.500WFHM), A+2 la mot pic don. Khi thuc hien<br />
quet cham de dat do phan giai cao ban (115.200WFHM), tren phd xuat hien IFC, thanli phan<br />
A+2 tach thanh 2 pic. ky hieu lan lugt theo chieu tang m/z la A+2L va A+2H, vdi do tach Am/z<br />
0,005. Theo tinh toan ly thuyet, thir tu xuat hien cac thanh phan IFS ke tir phia m/z nhd se la<br />
34c<br />
S - '*0 - "C vdi do tach Am/z(''S-"0) = 0,009 va Am/z(''S - "C) = 0,011. Trong pham vi<br />
gidi ban ciia dp chinh xac khdi >2ppm, chiing tdi ddng nhat pic A+2Lvdi thanh phdn ^''S, cdn<br />
pic A+2H la chdng chap ciia pic caebon va thanh phdn '^0.<br />
S\r cd mat S trong phan tir dugc khang dinh them khi phan tich ty le cudng do cac<br />
thanh phan IFC. Ty Ie Iy thuyet [9] va thuc nghiem ciia thanh phdn ^''S vdi gia thuyit S=2<br />
tuang img cho (A+2)/A la 9,01 /8,8 va (A+4)/A la 0,18/0,19. Sir phii hgp nay cho phep khdng<br />
dinh phan tir ciia mau nghien cim cd chiia 2 nguyen tir S.<br />
Bude tiep theo la xac dinh cdng thirc phan tir dua tren dii lieu thuc nghiem<br />
(M"" =1007,443) xa ket qua phan tich so bd IC va IFS dat dugc d phan tren. Tinh toan dugc<br />
thuc hien tren phan mim MFCalc^^, vdi dii lieu va cac gia thilt sau: M^' = 1007,443, do<br />
chinh xac khdi 2ppm. la peptit vdi 9aa (nonapeptide) va cd 2 nguyen tir S trong cdu tnic.<br />
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206<br />
Hoi nghi Khoa hqc ky niem 35 ndm Vien Khoa hqc vd Cong nghe Viet Nam - Hd Noi 10/2010<br />
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Bdng 1; Dir lieu xdc dinh cdng thuc phdn tu theo MFCalc<br />
Fragment Maes = 1007.443300<br />
T o l e r a n c e s +/_ o 002000<br />
Elements S e l e c t e d = C[ia-9S],H[29-&91,H15-35],0[S-27], 5 [ 2 - 2 ]<br />
Huniber Found = 11<br />
<br />
S4"59"30=llS 1007.44209100 -0.00120900 ^29'^69''l7^16^2 1007.44477700 + 0 00147700<br />
^25''65''23'^16^: 1007.44205300 -0.00120700 ^39"63''ie^l0^2 1007.44158900 -0 00171100<br />
'^26"6l"27'^12^2 1007.44343300 +0.00013300 '-4o''69''ll'^15^2 1007.44159100 -0 00170900<br />
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