江蘇省臨檢中心 臨床化學(xué)繼教班 6.載脂蛋白標(biāo)準(zhǔn)化-李卿

載脂蛋白標(biāo)準(zhǔn)化參考測(cè)量實(shí)驗(yàn)室李卿MoraS.Advancedlipoproteintestingandsubfractionationarenot(yet)readyforroutineclinicaluse.Circulation.2009;119:2396–2404.?

計(jì)量溯源性?

載脂蛋白標(biāo)準(zhǔn)化歷史和進(jìn)展?

我們的探索?

《醫(yī)療機(jī)構(gòu)臨床檢驗(yàn)項(xiàng)目目錄（2013年版）》，共包括檢驗(yàn)項(xiàng)目1462項(xiàng)，蛋白類約200項(xiàng)，JCTLM（BIPM+IFCC+ILAC）網(wǎng)站上列出的蛋白類的參考方法共計(jì)19項(xiàng)。?

19個(gè)項(xiàng)目中，15個(gè)是在2001年發(fā)布報(bào)告，由IFCC

Committee

onPlasma

Proteins

(C-PP)委員會(huì)發(fā)布。CNAS-GL013量值溯源要求在醫(yī)學(xué)測(cè)量領(lǐng)域的實(shí)施指南2018新修訂版溯源模型

一級(jí)參考測(cè)量程序一級(jí)校準(zhǔn)品溯源至SI單位1333參考測(cè)量程序（定義被測(cè)量）一級(jí)參考物質(zhì)溯源至SI單位23332333國(guó)際約定校準(zhǔn)品（定義被測(cè)量）且符合15194參考測(cè)量程序溯源至SI單位4232國(guó)際約定參考測(cè)量程序國(guó)際約定校準(zhǔn)品溯源至SI單位

國(guó)際一致性計(jì)劃5622223被測(cè)量溯源到制造商內(nèi)部定義的校準(zhǔn)品王軍,劉毅.ISO17511臨床檢驗(yàn)量值溯源國(guó)際標(biāo)準(zhǔn)修訂解讀[J].中華臨床實(shí)驗(yàn)室管理電子雜志,2015,(3):168-170.擾計(jì)量學(xué)溯源性建立的問題被測(cè)量定義不充分；在實(shí)現(xiàn)物質(zhì)的量的單位摩爾時(shí)，難以獲取指定化合化合物的超純物質(zhì)；校準(zhǔn)品中分析物的非均一性（異構(gòu)體、衍生物）；測(cè)量方法對(duì)校準(zhǔn)品和人樣品中的分析物有不同的特異性和選擇性；分析物在人樣品中和校準(zhǔn)品之間有微小不均一性，如免疫法測(cè)血清鐵蛋白濃度時(shí)，因分析物的微小不均一性，不同的多克隆抗體對(duì)各個(gè)異構(gòu)體的識(shí)別程度不同。人樣品基質(zhì)與校準(zhǔn)品基質(zhì)不同；被測(cè)量發(fā)生物理或化學(xué)修飾。IFCC于1991-1993年成立的載脂蛋白標(biāo)準(zhǔn)化工作組，其值由該工作組的組織方，即“美國(guó)華盛頓大學(xué)西北脂類代謝和糖尿病研究實(shí)驗(yàn)室”，使用免疫法（作為參考方法）確定。996年IFCC在美國(guó)國(guó)家疾控中心建立的載脂蛋白參考方法研究工作組發(fā)表研究成果，即使用同位素稀釋質(zhì)譜法定量apoA-Ⅰ。主要原因是免疫法可能會(huì)受交差反應(yīng)、抗體親和力減弱、抗原表位變性等因素影響。BCR：歐洲共同體標(biāo)準(zhǔn)物質(zhì)局華盛頓大學(xué)西北脂類代謝與糖尿病研究室apoA1和apoB的標(biāo)準(zhǔn)化方案Apolipoproteins

byMassSpectrometry

(WG-APO

MS)Terms

ofReferenceToachieve

standardization

ofapanel

ofclinically

relevant

serum

apolipoproteins

(apo)

A-I,

B,C-I,C-II

,

C-III,

E

and

apo

(a)

(including

qualitative

phenotyping

where

needed).

Standardizationisdone

insuch

away

that

measurement

results

are

traceable

to

SIasoutlined

inISO17511.

Othertraceability

chains

will

beused

incases

where

traceability

to

SIcannot

beachieved.Toevaluate

clinical

performance

and

clinical

utility

ofserum

apolipoprotein

panel(s)

forCVD

riskstratification

and

treatment,

incomparison

toortogether

with

contemporary

blood

lipids.Current

projectsDefine

theanalytes

/measurands

intended

tobemeasured.Development

ofprimary

and

secondary

reference

materials,

including

evaluation

ofcommutability.Development

ofan

LC-MS/MS-based

reference

method

for

the

above

mentioned

analytes

that

areunaffected

bygenetic

variants,

post-translational

modifications

and

other

factors.

The

referencemethod

will

meet

relevant

ISO

standards

(i.e.,

ISO

15195).Evaluation

ofthe

analytical

performance

ofthe

LC-MS/MS

reference

method.Assessment

ofthe

performance

of

commercially

available

apolipoprotein

assays

compared

tothereference

method

using

commutable

reference

materials

aswell

as

single

donation

samples.Any

reference

materials

and

reference

measurement

procedures

developed

will

besubmitted

toJCTLMfor

review

and

listing

onthe

JCTLM

database.Future

ProjectsEvaluation

ofclinical

performance

and

clinical

utility

ofthe

multiplexed

apolipoprotein

testaccording

totheTest

Evaluation

framework

developed

bythe

EFLM

working

group

onTest

Evaluation(Horvath

ARetal.,

CCA,

2014).質(zhì)譜法絕對(duì)定量蛋白質(zhì)白?

哪一個(gè)肽段可以代表這個(gè)蛋白？?

哪一個(gè)或一些肽段的色譜及質(zhì)譜行為可以用來(lái)定量？?

如何將這個(gè)肽段從整個(gè)蛋白中分離出來(lái)？?

分離肽段的時(shí)候如何考慮蛋白的序列和空間結(jié)構(gòu)？267個(gè)氨基酸，分子量30777.83質(zhì)譜法絕對(duì)定量蛋白質(zhì)組學(xué)介紹個(gè)肽段可以代表這個(gè)蛋白？常用工具網(wǎng)站或公共數(shù)據(jù)庫(kù)一個(gè)或一些肽段的色譜及質(zhì)譜行為可以用來(lái)定量？?

可能的候選代表性肽段；?

同時(shí)參照已發(fā)表的文獻(xiàn)中使用的多肽；?

找公司合成多肽；?

LC-MS/MS條件優(yōu)化：色譜柱選擇，流動(dòng)相，洗脫梯度，離子源參數(shù)，四級(jí)桿參數(shù)，離子對(duì)選擇等。CEIon1PeptideProteinm/zIon2Ion3(eV)(quantifier)VQPYLDDFQK*apoA-I626.8181025.5765.4228.1AKPALEDLRTEVIPPLIENR*TGISPLALIKapoA-IapoB506.8640.9506.8141513813.4838.5741.5716.4951.6854.6288.2443.2654.5apoB將這個(gè)肽段從整個(gè)蛋白中分離出來(lái)？酶R：精氨酸K：賴氨酸P：脯氨酸D：天冬氨酸E：谷氨酸sp|P02647|APOA1_HUMAN

Apolipoprotein

A-I

OS=Homoapiens

GN=APOA1

PE=1

SV=1AVLTLAVLFLTGSQARHFWQQDEPPQSPWDRVKDLATVYVDVLKDSGRDYVEGSALGKQLNLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQAAVLTLAVLFLTGSQAR

HFWQQDEPPQSPWDR

VKLATVYVDVLK

DSGR

DYVSQFEGSALGK

QLNLK

LNWDSVTSTFSK

LR

EQLGPVTQEFWDNLEK

ETEGQEMSK

DLEEVK

AK

VQPYLDDFQK

K

WQEEMELQK

VEPLR

AELQEGAR

QK

LHELQEK

LSPLGER

DR

AR

AHVDALR

THLAPYSDELR

QR

LAAR

LLK

ENGGAR

LAEYHAK

ATEHLSTLSEK

AKPALER

QGLLPVLESFK

VSFLSALEEYTK

K

LNTQ267個(gè)氨基酸，分子量30777.83apoA1

peptides

of

S1apoA1

peptides

of

S23.50E+063.00E+062.50E+062.00E+061.50E+061.00E+065.00E+050.00E+00E+06E+06E+06E+06E+06E+06E+05E+000246810121416182022242602468101214161820222426apoAAKDVYIS-VQIS-AKQGLL-A1

VQVSFL+K-A1

VSFL-A1apoA1

peptides

of

S43.80E+063.30E+062.80E+062.30E+061.80E+061.30E+068.00E+053.00E+05‐2.00E+05024681012141618202224apoAAKDVYIS-VQIS-AKQGLL-A1

VQVSFL+K-A1

VSFL-A1AKPALEDLRapoA1

peptides

of

S5+06+06+06+06+06+06+05+00024681012141618202224Evaluation

ofInterspecimen

Trypsin

DigestionEfficiency

PriortoMultiple

Reaction

Monitoring-Based

Absolute

Protein

Quantification

withNativeProtein

CalibratorsAKDVYIS-VQIS-AKQGLL-A1

VQVSFL+K-A1

VSFL-A1VQAverage

response

of

50246810

12

14

16

18

20

22

24

262016ClinChemEvaluation

ofInterspecimen

TrypsinDigestion

Efficiency

Prior

toMultipleReaction

Monitoring-Based

Absolute

ProteinQuantification

with

Native

ProteinCalibrators縱軸表示峰面積的自然對(duì)數(shù)，其中VSFLSALEEYTK(VS)為左軸；VSFLSALEEYTKK(VS-K)為右側(cè)縱軸；橫軸表示酶解時(shí)8.00E+057.00E+056.00E+055.00E+054.00E+053.00E+052.00E+051.00E+050.00E+008.00E+057.00E+056.00E+055.00E+054.00E+053.00E+052.00E+051.00E+050.00E+00apoB

peptides

of

S1apoB

peptides

of

S20246810

12

14

16

18

20

22

24

260246810

12

14

16

18

20

22

248.00E+057.00E+056.00E+055.00E+054.00E+053.00E+052.00E+051.00E+050.00E+00apoB

peptides

of

S3apoB

peptides

of

S48.00E+057.00E+056.00E+055.00E+054.00E+053.00E+052.00E+051.00E+050.00E+00apoB

peptides

of

S50246810

12

14

16

18

20

22

240246810

12

14

16

18

20

22

24

260246810

12

14

16

18

202apoBTEVISTEV

FPETGISVSL3.50E+053.00E+052.50E+052.00E+051.50E+051.00E+055.00E+040.00E+00apoC

peptides

of

S13.50E+053.00E+052.50E+052.00E+051.50E+051.00E+055.00E+040.00E+00apoC

peptides

ofS2apoC3

GWVapoC2

TYLapoC1

TPDapoC1

EFGNapoC2

ESLSapoC3

DALS02468101214161820222426024681012141618202224apoCDALSEFGNESLSGW