TAP CHI SINH HQC, 2012, 34(2): 253-258 PHAN T i C H HE PROTEIN HUYET THANH BENH DAI THAO Dl/OiNG TYPE 2
Nguyen Thi Minh Phuong', Pham Dire Dan, Nguyin Bich Nhi, Phan Van Chi Vien Cdng nghe sinh hgc, '*'[email protected]
TOM T A T : Trong nghicn ciiu nay, chimg toi sir dung phuong phap biSn tinh nhiet, s4c ky ai luc va kit loai albumin va Ig de phan doan protein huyet thanh benh nhan dai thao dudng type 2 (DTDT2). H5n hop protein sau khi phan doan duoc thiiy phan bang enzyme trypsin, phan tich va nhan dang bjng s k ky long 2 chieu ket noi khoi pho (2DnanoLC ESl-MS/MS) Cac protein nhan dang trong kk qua tim kiim, dugc dinh danh, phan loai dudi sir h6 tro cua cac cong CLI sinh tin hoc (ID mapping, co sd dQ lieu iren Swissprot..-) de lien loi xay dung co sd dQ heu he protein huy^t thanh, KSi qua da xac djnh dugc 468 glycoprotein, 1110 protein ciia phan doan nhiet va 4044 protein t6ng s6 trong 30 mlu huy^t thanh benh nhan DTDT2 da nghien ciru, Dong thdi, da phal hien dugc 26 protein lien quan d^n su phat sinh va phat tnen ciia benh DTDT2.
Tir khoa: Ca sd dir lieu, dai thao dudng type 2, he protein, huyet thanh ngudi, khoi pho.
MO BAU
Huyet thanh la mgt thanh phan quan trgng trong mau, chua cac protein cd ngudn gdc va chirc nang kliac nhau, tac ddng len nhieu ca quan va qua trinh sinh hgc trong ca the. Bat ky mgt thay ddi nao lien quan den berth ly ciing dugc phan anh da dang va day dCi trong he protein huyet thanh [2], Vi vay, viec sir dung huyet thanh cho muc dich nghicn ciru la each tiep can hgp ly va hua hen se mang l^i nhieu thong tin bd ich de phat trien cdng tac nghien ciiu, chan doan, chu'a tri cac benh khac nhau nhu' chiing viem nhiem, ung thu va benh trao d6i chSt trong dd cd dai thao dudng type 2 (DTDT2)[I].
Tai Viet Nam, nhung nghien cuu tren proteome huyet thanh benh DTDT2 da va dang dugc trign khai theo nhieu hudng tiep can khac nhau. Ben canh viec phan tich mire do bieu hien ciia he protein huyet thanh benh DTDT2 d cae giai doan khac nhau, chung tdi cdn tien hanh xac dmh nhan dang loan bg he protein de cd cai nhin tong the nhat ve proteome huyet thanh cua benh DTDT2 ndi chung cung nhu cac benh ly khac. Bdi vi mdi su thay ddi bat thudng ve sd lugng, thanh phan va ndng dg ciia protein huyet thanh se la ca sd cho viec phat hien cac chi thj phan tu sinli hgc (bioraarker) ddi vdi moi trang thai benh ly [9J. Trong nghicn cina nay, phuong phap bien tinh nhiet, sic ky ai luc va kit loai albumin va Ig da dugc sir dung de phan doan protein huyet thanh. Hdn hgp protein sau khi phan doan duge thuy phan b k g enzyme
trypsin, phan tieh va nhan dang bang sac ky Idng 2 chieu ket noi khdi phd (2DnanoLC ESl- MS/MS). Ket qua da xac djnh dugc 468 glycoprotein, 1110 protein cua phan doan nhiet va 4044 protein long sd trong huyet thanh benh nhan DTDT2. Trong dd, da xac dinh dugc 26 protein lien quan den su phat sinh va phat trien ciia benh DTDT2
PHUOfNG PHAP NGHIfiN CUV Vat lieu
Huyet thanh dugc thu tir mau nguyen eua benh nhan DTDT2 (30 mau dugc lua chgn tu 100 mau) d cac giai doan benh, lua tudi khac nhau dugc tuyen chgn va cung cap bdi Betih vien Ndi tiet Trung uong, bao quan d - 80°C va gia ddng d nhiet do phdng trude khi su dung.
Cac hda chSt sir dung cho sac ky Idng nano deu dugc eung cap bdi cac hang cd uy tin, cd do tinh sach can thi6t cho sinh hgc phan tir. He thdng may khoi pho QSTAR®XL MS/MS {AppliedBiosystem/MDS Sciex, Toronto, Canada) ciing vdi cac trang thiet bi khac ciia Phdng thi nghiem trgng diem Cdng nghe gen, Vien Cdng nghe sinh hgc, Vien Khoa hgc va Cdng nghe Viet Nam.
Phirong phap nghien cuu Phdn doan protein trong huyet thanh
Thu nhan glycoprotein bang sac ky ai luc:
Hdn hgp glycoprotein trong huyet thanh dugc thu nhan qua cot sac ki ai lire vdi chat gia Sepharose-4B gan lectin ConA theo quy trinh
Nguyen Thi Minh Phuong. Pham Due Dan. Nguyen Bich Nhi, Phan \'^m_Chi_
da duge md ta ciia VQ Minli Thiet va nnk.
(2006) [10],
Phan doan nhi?t protein bang phuong phap bien tinh nhiet: Huyet thanli dugc Iron vdi dera can biing (EDTA 20 raM, Tris HCl pH 8,9 6,2 M, polyethylene glycol (PEG) 6000 7%) theo ti le 1:1. Hdn hgp huyet thanh va dem dugc ii va lac 10 phiit d 98''C sau dd de d nhiet do phdng trong 10 phut Mau dugc ly tam 10 phut vdi loc do 12000 vdng/phiit de thu giu: phan djch ndi.
Ndng do protein trong mau duge xac dinh bang phuong phap Bradford trude khi tien hanh cac thi nghiem tiep theo.
Kit loai Albumin va Ig. Dich huyet thanli dugc loai albumin (HSA) va y-Irnrauglobulin (IgG) bang Aurum scmra protein mini Kit (BioRad, My). Kit gdra cd col MicroSpin la hdn hgp ciia Affi-Gcl Blue va Affi-Gcl protein A.
Huyet thanh dugc hoa vao dem Aurum va dua len cot MicroSpin, phan khong bam cot duac thu lai bang ly tara 10000 vdng/phul trong 5 phut.
Thuy phan bon hup protein bdng enzyme trypsin Hdn hgp protein trong huyet thanh sau khi phan doan dugc lam klid va khu eau ndi disulfide bang dung dich khu Dhhiothreitol 5 mM d 56''C trong 1 gid, alkyl hoa gdc cysteine bang lodoacetaraidc 20 mM d nhiet do phdng, khdng anh sang trong I gid, sau dd thuy phan bang enzyme trypsin (Sigraa, My) vdi ty le enzyme/protein bSng 1/50 d 37*'C trong 16 gid.
Hdn hgp peptide duge lam khd d nhiet do phdng trong may Specdvac va hda vdi dung dich acid formic (FA) 0,1% de ngirng phan irng.
Nhgn dgng protein trong huyet thanh bang sdc ky long hai chieu ket noi khoi pho
Hdn hgp peptide sau khi thuy phan duge phan tieh qua he thdng sac ky long hai chieu trcn cot sac ky trao doi cation (SCX) d chieu thit nhat va cot ngugc pha (RP--C18) d chieu thir hai. Cot ngugc pha CI8 dugc gan vao dau dua mau Silica Tip ket ndi vdi may khdi phd QSTAR^XL bang nguon Nano-ESI (Nano- Electrospray ionization), Phd khdi MS/MS dugc phan tich bang phan mem Mascot vl.8. Ca sd dir lieu dugc dimg de tim kiem trong Mascot la NCBlm, mdt ca sd dir lieu protein loan dien vdi tren 7 trieu trinh tu khac nhau.
KET QUA VA THAO LU^N
Phan doan he protein trong huyet thanh ngirdi
Nhu da biet, cd rSt nhilu md, ca quan ddng vai trd quan trgng trong sir phat trien ciia benli DTDT2 va cae hgi chung ciia chung, Dg xac dinh duac dac diem sinh ly ciia qua trinh gay benh tao nen su phat trien benh va tim ra eac lieu phap de chiia trj se phai nghicn ciiu nhieu hon nira. Dac biet, phan tich dac diem cac protein dac hieu md, te bao dudi cac dieu kien gidng nhu DTD va kich thich cua radi truong khac nhau dan tdi su phat tnen cua DTD; phan tich cae mau tir cac benh nhan DTD do khang insulin hoac sai hdng trong qua trinh dieu tiet dudng va so sanh chiing vdi cac ca the klide manh. Cd nhieu md va ca quan quan trgng lien quan den DTD nhu: tuyen tuy, gan, mo ca, mo md, than, nude tieu, tira... tuy nhien, so sanli voi eac rad da trinh bay d tren, huyet thanli cd nhieu thuan lgi cho nghicn cuu dieu tri benh. Benh nlian ed the cung cap raau mau tdt hon so vdi sii' dung cac md sinh thiet. Hon nira, huyet thanh la mdt ngudn cung cap gia trj trong phan tich proteomic, day la nai xay ra cac qua trinh sinh ly va benh ly trong co the. Tuy nhien, he protein trong huyet thanh rat phirc tap gdm nhieu protein ham lugng Idn thudng gay can trd cho viec phan tich va nhan dang cac protein cd ndng do thap hon nen viec phan doan protein de nghien cuu la rat can thiet. Trong nghicn ciru nay, chiing tdi su dung ba phuong phap de phan doan protein: phuong phap bien tinh nhiet, sac ky ai luc va kit loai alburain va IgG. Nong do protein sau khi phan doan duoc xac dinh bang phuang phap Bradford va dien di tren gel SDS-PAGE I2,6%_(hinh anh diSn di klidng trinh bay d day). Ket qua cho thSy, trong thanh phan protein thu dugc, ndng do eiia albumin va mgt so protein ham lugng Idn khac da giam dang ke, tao dieu kien thuan lgi cho viec nghien cuu va nhan dien nhirng protein cd nong do thap.
Nhan dang, xay dung co- sd dir Ueu he protein huylt thanh benh nhan DTDT2
Xdc dinh vd nhgn dgng protein trong huyet thanh benh BTDT2 bdng sdc ky long hai chiiu kel noi khoi pho
TAP CHI SINH HQC, 2012. 34(2): 253-258 Hdn hop protein thu dugc sau khi phan
doan dugc ket tiia b^ng acetone l^nh vdi ty le protein:acetone/l:4 vS the tich, de qua dem d - ZO^C. Sau dd hdn hgp protein dugc thuy phan bang enzyme trypsin va phan tich tren b? thdng sac ky Idng hai ehilu nano ket ndi khdi phd dudi su hd trg ciia phin mem Mascot vl.8 tren CO dir li?u NCBInr vdi hon 7 trieu trinh tu khac nhau. Chi nhirng protein cd diem sd ciia ion peptide Idn hon 35 radi dugc xac dinh. Trong tdng sd 30 mau benh DTDT2 duge phan tich va nhan dang, chiing tdi deu thu dugc cd tir 100- 999 protein trong mdi raau huyet thanh nghien cuu, tity thugc vao timg phuong phap phan doan protein. Cae protein nhan dang dugc bao gdm cac loai immunoglobin, cac thanh phan bd the, protein lien ket, enzyme, thu the va mdt sd protein khac, cd sir phii hgp ve hau het eac thanh phan ca ban trong huyet thanh.
Xay dimg ngan hdng du lieu hi protein huyet thanh benh DTDT2
Hdn hgp protein tdng the, protein ben nhiet va glycoprotein trong huyet thanh ciia 30 mau benh DTDT2 sau khi nhan dang se dugc loai bd nhthig protein triing nhau (ve ten protein va sd dang ky tren NCBIru), chiing tdi da xac dinh dugc 942 glycoprotein, 1906 protein trong phan dogn nhiet va 5451 protein tdng the. Sau dd, nhung protein nay tiep tuc dugc sang lgc theo nguyen tSc ehi nhiing protein cd sd manh peptide bat cap > 2 va tan sd xuat hien d cac mau > 2 mdi dugc xac dinh la protein trong hdn hgp huy6t thanh nghien ciin. Ddng thdi, dudi su
ho trg cua cdng cu ID mapping va co sd diJ lieu glycoprotein cua SwissProt [14, 15], ca sd du lieu he protein huyet thanh benh DTDT2 d Viet Nam da dugc xay dung vdi 468 glycoprotein, 1110 protein ben nhiet va 4044 protein tdng sd trong huyet thanh cua 30 mlu benh DTDT2 (bao gdm ca 3 giai doan benh). Ca sd dir lieu dugc th§ hien dudi dang bang dir lieu dugc sSp xep nhu sau doi vdi mdi protein: sd dang ki ciia protein, ten protein dugc sap xep theo thu tu ABC, sd dang ki Uniprot ciia protein (dugc link vdi trang web cd day dii cae thdng sd va ket qua nghien einj ve protein nay), chirc nang cua protein, vj tri ciia protein trong te bao, cac bien ddi eiia protein (neu cd) va lien quan den su phat sinh, phat trien ciia benh, dac biet la DTDT2 va eac benh chuyen hoa khac (neu co).
Nhu v§y, h? glycoprotein (468 protein) chiem sd lugng it so vdi he protein tdng the vdi 4044 protein. Sd lieu nay hoan loan hgp ly, bdi vi chi mgt phan protein dugc tao ra trong te bao thuc hien cae cai bien sau djch ma trong dd cd qua trinh glycosyl hda. Ket qua 4044 protein tdng the dugc nhan dang ciing phii hgp vdi nhimg cdng bd nam 2005 ciia 35 phdng thi nghiem trong du an proteome huyet tuong nguai ciia HUPO (Human Proteome Organization), vdi sd lugng protein la 3020 va mgt cdng bd khdng chinh thuc nam 2009, con sd nay la khoang 5000 protein trong huyet thanh. Day la nhimg dan lieu dSu tien ve co sd du: lieu he protein huyet thanh benh DTDT2 d Vi?t Nam.
Bdng 1. Ty le cac protein lien quan dSn benh DTDT2 va cac b?nh chuy6n hda khac h-ong he protein huyet thanh benh nhan DTDT2
Cff sa dff iieu
He protein tons the He glycoprotein He protein ben nhiet
Protein lien quan den benh DTDT2 So lirong
protein 22
8 18
Tong so protein
4044 468 1110
Tyle 0,54%
1,71%
1,67%
Protein lien quan den cac benh chuyen hoa khac So lugng
protein 66
7 25
Tong so protein 4044
468 1110
Tyle 1,63%
1,50%
2,25%
Ben canh dd, trong ca 3 ca sd dii: lieu he va cac benh chuyen hda khac thudng la bien protein huyet thanh benh DTDT2, chung tdi da chiing ciia DTDT2 nhu tim mach, beo phi..., phan tich va xac djnh dugc nhieu protein cd lien Trong do, he protein tdng the cd 22 protein lien quan den tien trinh phat trien ciia b?nh DTDT2 quan dSn benh DTDT2, chifira 0,54% va 66
Nguyen 7hi Minh Phuong. Pham Due Dan. Nguyen Bich Nhi, Phar2^yan_Clu_
protein lien quan den mdt sd benh chuyen hda khac, ehiem 1,63%. Cdn he glycoprotein lan lugt la 1,71% va 1,51%, he protein bin nhiet la 1,67% va 2,25% (bang 1),
Nhu vay, ty le protein lien quan den benh DTDT2 d he glycoprotein la cao nhSt, chiem 1,71% va thap nhat la he protein tdng the vdi 0,54%. Ty le protein lien quan den cac benh chuyen hda khac Ihi cao nhit d he protein ben
nhiet va thip nhit d h? glycoprotein (bang I) Trong sd nhiing protein da dugc nhan daiig (d ea he glycoprotein, he protein ben nhiet va he protem long the), chung tdi da phan (ich va xac dinh dugc 26 protein cd lien quan den lien trinh phal trien ciia benh DTDT2. Dang chu y, 10 protem chi thiy xuit hi?n trong ket qua phan tich cua mau huyet thanh b?nh nhan DTDT2 ma khdng thay trong mlu ngudi binh Uiudng (bang 2), Bdng 2. Sd l u g n g pro
S T T 1 2 3 4 5 6 7
8
9 10
Sd d a n g ky Iren N C B I n r gi 1556192
gi|450466l gi|l 1225607
gi|2506805 gi|189650 gi|7549809 gi|52000783
gi|28412326
gi| 1480871 gi|4323l7l
ein CO lien quan den tien trinh phal trien cua b e n h dai thao d u d n g type 2 T e n protein
51C protein Intcrlcukin 1 receptor
accessory protem iso form 1 Interleukin 1 receptor accessory protein-like 2
Laminin subunit alpha-2 precursor Plasma ceil membrane
glycoprotein PC-1 Plastin 3 Protein phosphatase 1 regulatory subunit 3A
Peroxisome proliferator-activated
receptor gamma coaclivalor lbeta-2a Sulfonylurea receptor Type 1 diabetes autoantigen ICA12
So manh peptide b i t cap
10
%
17 18 5 4 6
10
9 27
Vitri Mang te
bao Te bao chat
Mang Tieu phan
riboxom Mang te bao Ngoai bao Te bao chat
Nhan
Ty the Nhan, ludi
noi chat Chirc n a n g Thu the, enzyme Lien kel
protein Thu thS Thu th^
Ho^t tinh enzyme Lien ket ion canxi Hoat tinh enzyme Hoat tinh enzyme,
thu thg Enzyme oxi hoa khir
Hoat tinh enzyme
Bien ddi sau djch ma Photphoryl, glycosyl hoa
Photphoryl hoa, acetyl hoa
Glycosyl hoa Glycosyl hoa Glycosyl hoa Lien ket disufidc Photphoryl hoa
Photphoryl hoa
Acetyl hoa Photphoryl hoa
Ddng vai trd quan trgng trong vi?e dieu kliien do nhay cua insulin la 51C protein (Phosphatidylinositol-3,4,5-trisphosphate 5- phosphatase 2). Kaisaki et al, (2004) [4] ciing da khang djuh nhirng bien ddi ciia gen ma hda cho 51C protein that su lien quan den benh DTDT2. Interleukin I receptor accessory protein isoforra: Claus ct al. (2007) [3] chimg minh rang, su bieu hien ddi khang ctia interleukin I receptor giam trong dao tuy cda benh nhan DTDT2 va ndng dg glucose cao kich thich san xuat interleukin 1 p trong te bao P ciia
tuyen tuy dan den bai tiet insulin kem va giam sir phat trien ciia te bao. Sulfonylurea receptor (SUR): thu the SUR la ragt trong hai tieu phin thudc hg ATP-binding cassette (ABC); SURl la dudi dan vj chirc nang ciia ATP sensitive potassium channel (K^TP) ciia i^ bao beta; KATP ddng vai trd quan trgng qua trinh tiet insulin. Vi vay, nhimg bien ddi xay ra d protem nay cd th6 anh hudng den qua trinh khang msulin, mdt trong nhung nguyen nhan quan trong din din benhDTDT2[6].
Lamimn subunit alpha-2 precursc- !a mdt
TAP CHI SINH HOC, 2012, 34(2) 253-258 protein tim thiy trong chat ndn ndi bao, mdi
phan ciia protein nay dugc coi la tiin chit dk xay dimg cac ca quan ngi bao. Nghien cuu gin day ciia Tae-sun et al. (1999) chiing minh ring, s\r thay ddi^ ndng do ciia protein nay cung cd lien quan den su xuit hien ciia benh DTDTl va DTDT2 [8], Peroxisome proliferator-activated receptor gamma coactivator lbcta2 (PPARy);
cac thu the boat hda su sinh san nhanh ciia peroxisome (PPARs) la eac y6u td djch ma, dieu khien boat ddng cua gen, ddng vai trd quan irgng trong viec dieu hda ndng do glucose va lipid.
Chat kich ddng PPARy thudng dugc sir dung nhieu trong dieu trj benh DTD dl cai thien dg nhay insulin va de dieu hda dudng d muc binh thudng, thdng qua viec cai thien qua trinh tiet insulin [12], Plasma cell membrane glycoprotein PC-1: la ragt glycoprotein dugc bieu hien d nhieu rad va uc chg tin hieu insulin d muc do thu thg. PC-1 d cac rad dich insulin dong vai trd quan trgng trong su phat trien khang insulin cua benh nhan beo phi va DTDT2.
Da hinli cua PC-1 cung dugc chiing minh la CO lien quan den su kliang insulin, mdt trong nhiing nguyen nhan chinh dan den DTDT2 [7].
Bile salt-activated lipase: la mgt protein dugc ma hoa bdi gen CEL trong ca the ngudi. Khuyet t^t trong CEL la ragt nguycn nhan ciia benh tieu duong klidi phat ctia tre loan rdi loan chiic nang ngoai tiet 8 (M0DY8), eon dugc ggi la benh tieu dudng va rdi loan chiic nang tuyen tuy ngoai tiet [5]. Eukaryotic translation initiation factor 2-alpha kinase 3: la mgt enzyrae d ca the ngudi ma hda bdi gen EIF2AK3. Khuyet tat trong EIF2AK3 la nguyen nhan cua hgi chiing Wolcott-Rallison (WRS), cdn dugc ggi la loan san epiphyseal vdi hdi chiing DTD khdi phat sdm [13], Protein phosphatase 1 regulatory subunit 3A (PPPRS3): la mgt enzyme thugc nhora kinase, Xia J et al. (1998) chung minh ring da hinh ciia PPPRS3 lien quan den khang insulmvaDTDT2[ll].
KET LUAN
B:ing eac phuang phap phan doan protein kel hgp vdi sic ky Idng hai chieu ket noi khoi
phd, da phan tich va nhan dang dugc 468 glycoprotein, II10 protein trong phan doan nhiet va 4044 protein tdng sd tu 30 raau huyet thanh benh nhan DTDT2. Trong dd, da xac djnh duoc 26 protein ed lien quan den su phat sinh, phat trien ciia benh DTDT2 va 10 protein ehi thay xuat hi?n trong ket qua phan tich eiia mau huyet thanh benh nhan DTDT2 ma khdng thay trong mau ngudi binh ihudng.
Loi cam on: Cdng trinh dugc de tai KC 04- 14/06-10 va Dg tai Dgc lap cap Nha nude so 03/2011 PTNTD/HD-DTDL, phdng Thi nghiem trgng diem Qudc gia, Bd Khoa hgc va Cdng nghe tai trg.
TAI LlEV THAM KHAO
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Cellular Proteomics, 1: 845-867.
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Polymorphisms in type II SH2 doraain- containing inositol 5-phosphatase (INPPLl, SH1P2) are associated with physiological abnormalities of the metabolic syndrome.
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14-19.
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Am, Soe, Nephorol., 10: 1931-1939.
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Characterization of the Low Molecular Weight Human Serum Proteome. SAIC- Frederick Inc., Laboratory of Proteomics and Analytical Technology, Mass Spectrometry Center, National Cancer
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Phan tich cac glycoprotein trong huygt thanh ngudi. Tap chi Cdng nghe sinh hgc, 4(1): 13-22.
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Diabetes, 47:1519-1524.
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ANALYSIS OF SERUM PROTEOIME FROM TYPE 2 MELLITUS PATIENT Nguyen Thi Minh Phuong, Pham Due Dan, Nguyen Bich Nhi, Phan Van Chi
Institute of Biotechnology, VAST SUMMARY
Semm is an exceptional and special proteome in many respects. It contains a lot of essendal information for the study and disease diagnosis. However, the analysis of serum is analytically challenging due to the high dynamic concentration range of constituent protein species. Therefore, the iractionation of serum proteome is very necessary. In this study, a combination of different methods; depletion by Aumm Serum Protein MiniKit, thermostable fraction, affinity chromatography, was used to separate proteins from type 2 diabetes mellitus (T2DM) serum. The protein fractions were then digested by trypsin and analyzed by 2DnanoESI-LC- MS/MS, The proteins were named, classified and used to buih database by using bioinformatics tools including ID mapping, Swiss-Prot.... It was shown that 468 glycoproteins, 1110 proteins in thermostable fraction, and totally 4044 proteins from 30 T2DM semm samples were identified. Moreover, 26 proteins were found positively associated with T2DM.
Keywords: 2DnanoLC ESI-MS/MS, database, human serum, proteome, type 2 diabetes mellitus, Ngdy nhan bdi: 25-8-2011
