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基于质谱的蛋白质组学定量方法解

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长0102030405质谱定量蛋白质组学简介标记定量法非标记定量法DIA技术靶向定量法CONTENTS课

录质谱定量蛋白质组学简介01解

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长蛋白质组学(英语:proteomics),是以蛋白质组为研究对象,研究细胞、组织或生物体蛋白质组成及其变化规律的科学。Pictures

were

all

from

Bing.传统Western

Blot法基于质谱的蛋白质组学方法蛋白有限抗体要求高高通量需求成千上万种蛋白能筛选,能验证高分辨,更准确解

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长1.2

质谱仪工作原理Orbitrap

质谱仪工作原理图解

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长1.3

质谱仪工作示例解

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长1.4

质谱数据构成解

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长知

牌Zoom

in解

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长知

牌1.3

质谱定量法主要有哪些?利用化学分子标签的信号相应进行定量。利用峰面积进行定量。不依赖于母离子的信号相应,对一级和二级离子进行全扫描。对目标肽段进行精确定量。标记定量非标记定量靶向定量DIA新技术标记定量02解

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长知

牌1.3

标记定量法的原理两大主流标记定量方法:优点:定量准确,稳定性高,重现性好。缺点:大样本量成本高基于报告离子的定量方法。知

牌1.4

标记定量法的实验流程1234567解

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长统计分析生物信息学分析数据挖掘……Li,

C.,

et

al.,

Proteomic

Analysis

of

Liver

Proteins

in

a

Rat

Model

of

Chronic

Restraint

Stress-Induced

Depression.

BioMed

ResearchInternational,

2017.

2017:

p.1-14.Lin,

L.,

et

al.,

Application

of

iTRAQ-Based

Quantitative

Proteomics

Approach

to

Identify

Deregulated

Proteins

Associated

with

Liver

Toxicity

Induced

by

Polygonum

Multiflorum

in

Rats.

Cellular

Physiology

and

Biochemistry,

2018.

43(5):

p.

2102-2116.Liu,

C.,

et

al.,

iTRAQ-based

proteomic

analysis

to

identify

the

molecular

mechanism

of

Zhibai

Dihuang

Granule

in

the

Yin-deficiency-

heat

syndrome

rats.

Chinese

Medicine,

2018.

13(1).Chen,

Y.,

et

al.,

Transcriptomic

and

proteomic

analysis

of

potential

therapeutic

target

genes

in

the

liver

of

metformin-treatedSprague-Dawley

rats

with

type

2

diabetes

mellitus.

International

Journal

of

Molecular

Medicine,

2018.

41(6):

p.

3327-3341.Li,

J.,

et

al.,

CXCL4

Contributes

to

the

Pathogenesis

of

Chronic

Liver

Allograft

Dysfunction.

Journal

of

Immunology

Research,

2016.

2016:

p.

1-11.Garvin,

A.M.,

et

al.,

Evidence

of

Altered

Mitochondrial

Protein

Expression

After

Chronic

Ethanol

Consumption

in

the

Aged

Estrogen-

Deficient

Female

Rat

Heart.

Alcoholism:

Clinical

and

Experimental

Research,

2017.

41(7):

p.

1288-1297.Thapa,

K.,

et

al.,

Dysregulation

of

the

calcium

handlingprotein,

CCDC47,

is

associated

with

diabetic

cardiomyopathy.

Cell

&Bioscience,

2018.

8(1).Wenting

Li,

Z.Z.C.G.,

Original

Article

Proteomic

analysis

of

mitral

valve

in

Lewis

rat

with

acute

rheumatic

heart

disease.

Int

J

Clin

Exp

Patho,2015.

11(8):

p.

14151-14160.Littlejohns,

B.,

et

al.,

The

effect

of

disease

on

human

cardiac

protein

expression

profiles

in

paired

samples

from

right

and

left

ventricles.

Clinical

Proteomics,

2014.

11(1):

p.

1-14.Liu,

S.,

et

al.,

In-depth

proteomic

profiling

of

left

ventricular

tissues

in

human

end-stage

dilated

cardiomyopathy.

Oncotarget,

2017.

8(29):p.

48321.解

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长相关文献 知

牌非标记定量03解

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长知

牌3.1

非标记定量法的原理

顾名思义,非标记定量法是指不使用同位素标记物,而是直接根据质谱峰的强度来对肽段或蛋白进行相对定量的方法,

文献中一般称为“label

free”。其定量原理是计算一级质谱中每个肽段的信号强度在LC-MS

色谱上的积分面积,利用MaxQuant等软件解析这些肽段的定量信息,从而获得对应蛋白质的相对定量值。此外,也可以通过计算谱图数目进行相对定量。传统定量方法:优点:成本低,无样本数限制,操作简便缺点:重现性差解

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长知

牌3.2

非标记定量法的流程解

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长相关文献 知

牌Lundberg,

K.C.,

et

al.,

Proteomics

of

skin

proteins

in

psoriasis:

from

discovery

and

verification

in

a

mouse

model

toconfirmation

in

humans.

Molecular

&

cellular

proteomics

:

MCP,

2015.

14(1):

p.

109-119.Chang,

Y.,

et

al.,

Quantitative

proteomics

reveals

differential

regulation

of

protein

expression

in

recipient

myocardium

after

trilineage

cardiovascular

cell

transplantation.

PROTEOMICS,

2015.

15(15):

p.

2560-2567.Chang,

Y.,

et

al.,

New

Mass-Spectrometry-Compatible

Degradable

Surfactant

for

Tissue

Proteomics.

Journal

of

ProteomeResearch,

2015.14(3):

p.1587-1599.Schiza,

C.,

et

al.,

Identification

of

TEX101-associated

proteins

through

proteomic

measurement

of

human

spermatozoa

homozygous

forthe

missense

variant

rs35033974.

Molecular

&

Cellular

Proteomics,

2018:

p.

mcp.RA118.001170Latosinska,

A.,

et

al.,

Comparative

Analysis

of

Label-Free

and

8-Plex

iTRAQ

Approach

for

Quantitative

Tissue

ProteomicAnalysis.

PLOS

ONE,

2015.10(9):p.

e0137048.Sebastian

Funke,

S.B.K.L.,

Original

Article

Analysis

of

the

effects

of

preservative-free

tafluprost

on

the

tear

proteome.

Am

J

Transl

Res,

2016.

10(8):

p.

4025-4039.DIA技术04解

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长4.1

DIA技术原理 知

牌DIA(Data

Independent

Acquisition)是随着定量蛋白质组学而建立的质谱扫描技术。DIA技术能够获得扫描范围内所有母离子和二级离子的信息,不会造成低丰度离子信息的丢失。Data

Dependent

Acquisition Data

Independent

Acquisition解

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长知

牌DDADIA4.1

DIA技术原理Picturewas

modified

from

matearials

of

AB

SCIEXtraining.解

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长知

牌4.2

DIA实验流程Picturewas

modified

from

Tiannan

Guo,Nature

Communication,

2015.解

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长相关文献 知

牌Egertson,

J.D.,

et

al.,

Multiplexed

peptide

analysisusing

data-independent

acquisition

andSkyline.NatureProtocols,

2015.

10(6):

p.

887-903.Hu,

A.,

W.S.

Noble

and

A.

Wolf-Yadlin,

Technical

advances

in

proteomics:

newdevelopments

in

data-independent

acquisition.

2016.

5.Nigjeh,E.N.,etal.,QuantitativeProteomicsBasedonOptimizedData-IndependentAcquisitioninPlasmaAnalysis.JournalofProteomeResearch,2017.

16(2):

p.

665-676.Smith,

P.K.,

et

al.,Measurement

of

protein

using

bicinchoninic

acid.

AnalyticalBiochemistry,1985:

p.

76-85.Xiang,L.,etal.,ComprehensiveAnalysisofAcylcarnitineSpeciesindb/dbMouseUsingaNovelMethodofHigh-ResolutionParallelReactionMonitoring

Reveals

WidespreadMetabolic

Dysfunction

Induced

by

Diabetes.

AnalyticalChemistry,

2017.

89(19):

p.

10368-10375.Wiśniewski,

J.R.,

et

al.,Universal

sample

preparation

method

for

proteome

analysis.

Nature

Methods,

2009.

6(5):

p.359-362.Escher,C.,

et

al.,Using

iRT,a

normalized

retention

time

for

more

targeted

measurement

of

peptides.

PROTEOMICS,

2012.

12(8):

p.1111-1121.Piazza,

I.,et

al.,

A

Map

of

Protein-Metabolite

Interactions

Reveals

Principles

of

Chemical

Communication.

Cell,

2018.

172(1-2):p.

358-372.e23.Reitsma,

J.M.,

et

al.,

Composition

and

Regulation

of

the

Cellular

Repertoire

of

SCF

Ubiquitin

Ligases.

Cell,

2017.

171(6):

p.

1326-1339.e14.von

Ziegler,

L.M.,

et

al.,

Subregion-Specific

Proteomic

Signature

in

the

Hippocampus

for

Recognition

Processes

in

AdultMice.Cell

Reports,

2018.

22(12):

p.

3362-3374.Latonen,

L.,

et

al.,

Integrative

proteomics

inprostate

canceruncovers

robustness

against

genomic

andtranscriptomic

aberrations

during

diseaseprogression.Nature

Communications,

2018.

9(1).Rapid

mass

spectrometric

conversion

of

tissue

biopsy

samples

into

permanentquantitative

digital

proteomemaps,

2015

April

;

21(4):

407–413.doi:10.1038/nm.3807.靶向定量05解

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长5.1

靶向定量的原理 知

牌PRM:平行反应监测(Parallel

Reaction

Monitoring,

PRM)技术是一种基于目标肽段质谱信号获取,以此来进行蛋白质定量的技术。解

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长知

牌5.2

靶向定量实验流程Peterson,A.C.,etal.,Parallelreactionmonitoringforhighresolutionandhighmassaccuracyquantitative,targetedproteomics.Molecular&

cellular

proteomics

:MCP,

2012.

11(11):

p.

1475

-

1488.Ronsein,G.E.,etal.,TargetedProteomicsIdentifiesParaoxonase/Arylesterase1(PON1)andApolipoproteinCsasPotentialRiskFactorsforHypoalphalipoproteinemia

in

Diabetic

Subjects

Treated

with

Fenofibrate

and

Rosiglitazone.

Molecular

&

cellular

proteomics

:

MCP,

2016.

15(3):p.

1083

-

1093.Taumer,

C.,

et

al.,

Parallel

reaction

monitoring

on

a

Q

Exactive

mass

spectrometer

increases

reproducibility

of

phosphopeptide

detection

in

bacterial

phosphoproteomicsmeasurements.

Journal

of

Proteomics,

2018.Lawrence,

R.T.,

et

al.,

Plug-and-playanalysis

of

the

human

phosphoproteome

by

targeted

high-resolution

mass

spectrometry.

Nature

Methods,

2016.

13(5):

p.

431-434.Tang,

H.,

et

al.,

Multiplexed

Parallel

Reaction

Monitoring

Targeting

Histone

Modifications

on

the

QExactive

Mass

Spectrometer.

Analytical

Chemistry,2014.

86(11):

p.

5526-5534.Gallien,

S.,

S.Y.

Kim

and

B.

Domon,

Large-Scale

Targeted

Proteomics

Using

Internal

Standard

Triggered-Parallel

Reaction

Monitoring

(IS-PRM).Molecular

&cellular

proteomics

:MCP,2015.

14(6):

p.

1630

-

1644.Martinez,E.,etal.,Targetedproteomicsidentifiesproteomicsignaturesinliquid-biopsiesoftheendometriumtodiagnoseendometrialcancer

and

assist

in

the

prediction

of

the

optimal

surgical

treatment.

Clinical

Cancer

Research

An

Official

Journal

of

the

American

Association

forCancer

Research,

2017.

23(21):

p.

clincanres.0474.2017.Thomas,

S.N.,

etal.,Multiplexed

TargetedMass

Spectrometry-BasedAssays

forthe

Quantificationof

N-Linked

Glycosite-ContainingPeptides

in

Serum.Analytical

Chemistry,

2015.

87(21):

p.

10830-8.Xue,

T.,et

al

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