Application of MALDI-TOF

Mass Spectrometry

in Post-Genomic Era

99% sequence of human genome published

Genomics:

--Identification & characterization of genes & their arrangement in chromosomes

Proteomics:

--Functional analysis of gene products

Bioinformatics:

--Storage,analysis & manipulation of the information from genomics & proteomics

DNA

mRNA

t-RNA

t-RNA

t-RNA t-RNA

Ribosome

(....)

Protein

CHOPO4

(....)

Post TranslationalModifications

X

X

Active Protein

mRNAlevel expressed protein level nor does it indicate the nature of the functional protein product

圖一：DNA序列是藍圖決定細胞的表現，蛋白質卻是實際上有功能的工作者；分子階層的蛋白質及DNA分析是瞭解其功能的關鍵

Genomics genes characterization and identification

Proteomics functional analysis of gene products

Bioimformatics

cDNA microarray:

--measures changes in mRNA

--interface between genomics and proteomics

Two-dimensional gels & Mass spectrometry:

--powerful tools for identifying proteins

--post-translational modifications

--protein-protein interaction

Transgenic & knockout mouse:

--functions of novel proteins in vivo

99% sequence of human genome published

Age of functional genomics

cDNA microarray core lab

Proteomics core lab

Biological samples (C/E)

ProteinscDNA

Microarray 2-D gels/MS

Protein expression pattern

mRNA expression pattern

Bioinformatics core lab

Function study in Tg/KO mouse core lab

Strategy for proteomic study in cancer biology

Normal cells

Tumor cells

SD

S-P

AG

E

isoelectrofocusing

MALDI-TOF MS analysis

Laser-captured microdissector

digested by trypsin

Database search/mapping

(?)

Protein identified

Clinical specimens

Mass Spectrometry for Biotechnology

Gary SiuzdakThe Department of ChemistryThe Scripps Research Institute

La Jolla, California

Academic Press 1996

Reference Book

What is a mass spectrometer and what does it do?

Analogy between mass analysis and the dispersion of light

Components of a mass spectrometer

First DetectorLaser

Reflector

Second Detector

圖十一：在 MALDI-TOF MS中的反射器設計

MALDI-TOF MS(Matrix-assisted laser desorption/ionization-Time of flight) (基質輔助雷射脫附游離 -飛行時間質譜儀 )

0

40000

40 80 120 160

RPKPQQFFGLMamide

m/z

0

40000

40 80 120 160

RPKPQQFFGLMamide

m/z

M/Z

Time of Flight

Solid probe

Ion Sources and Sample Introduction

Sample probes and matrix for MALDI

MALDI matrix

# A nonvolatile solid material that absorbs the laser radiation resulting in the vaporization of the matrix and sample embedded in the matrix.

#The matrix also serves to minimize sample damage from the laser radiation by absorbing most of the incident energy and the matrix is believed to facilitate the ionization process.

Matrix-assisted laser desorption/ionization source

Calibration for MALDI

Vacuum system in MS

Mass Analyzer-Time of Flight (TOF)

Kinetic Energy = ½ mv2

v = (2KE/m)1/2

m/z

Ion Detector

Electron multiplier

Photomultiplier conversion dynodescintillation counting

1 106

Sensitivity of MALDI-TOF MS

~10 fg

1347.7 g/mole x 5 x 10 -18 mole = 6.74 x 10 –15 g

Strategy for proteomic study in cancer biology

Normal cells

Tumor cells

SD

S-P

AG

E

isoelectrofocusing

MALDI-TOF MS analysis

Laser-captured microdissector

digested by trypsin

Database search/mapping

(?)

Protein identified

Clinical specimens

The 2-D gel pattern of human kidney epithelial 293 cells expressed without (A) and with (B) the

Epstein-Barr virus oncoprotein LMP1

(A) (B)

Pick up spots of interest

Digested by trypsin

MALDI-TOF MS analysis

IEF IEF

SD

S-P

AG

E

170

116.3

66.3

55.4

29

21.5

170

116.3

66.3

55.4

29

21.5

pH 310(A)

pH 310

a

bc

a'

b'c'd

(B)

(A) (B)

1

1

2

1 3

The substrate of GSK-3 purified from pig brain

(b1)

(d2)

(a'1)

(b'1)

(b'3)

472-480

472-480526-532533-552558-565

472-480526-532533-552558-565

472-480526-532533-552558-565

533-552558-565

MALDI-TOF analysis of tryptic fingerprint from the substrate for kinase FA/GSK-3 in pig brain

Data base search for the substrate of kinase FA/GSK-3from pig brain

MSYQGKKNIP RITSDRLLIK GGKIVNDDQS FYADIYMEDG LIKQIGENLI VPGGVKTIEA HSRMVIPGGI DVHTRFQMPD QGMTSADDFF QGTKAALAGG TTMIIDHVVP EPGTSLLAAF DQWREWADSK SCCDYSLHVD ISEWHKGIQE EMEALVKDHG VNSFLVYMAF KDRFQLTDCQ IYEVLSVIRD IGAIAQVHAE NGDIIAEEQQ RILDLGITGP EGHVLSRPEE VEAEAVNRAI TIANQTNCPL YITKVMSKSS AEVIAQARKK GTVVYGEPIT ASLGTDGSHY WSKNWAKAAA FVTSPPLSPD PTTPDFLNSL LSCGDLQVTG SAHCTFNTAQ KAVGKDNFTL IPEGTNGTEE RMSVIWDKAV VTGKMDENQF VAVTSTNAAK VFNLYPRKGR IAVGSDADLV IWDPDSVKTI SAKTHNSSLE YNIFEGMECR GSPLVVISQG KIVLEDGTLH VTEGSGRYIP RKPFPDFVYK RIKARSRLAE LRGVPRGLYD GPVCEVSVTP KTVTPASSAK TSPAKQQAPP VRNLHQSGFS LSGAQIDDNI PRRTTQRIVA PPGGRANITS LG

*908.4 da --- 391-397 *2169.1da --- 533-552 *pI~5.95

Collapsin Response Mediator Protein-2 (CRMP-2, human)

(d2)

(b1)

Amino acid sequence analysis by PSD technique in MALDI-TOF MS

2169

908

Tandem Mass Spectrometry

MS/MS with a TOF reflectron mass spectrometer

Peptide fragmentation postionization [Post Source Decay (PSD)]

(b1)

Amino acid sequence analysis by PSD technique in MALDI-TOF MS

2169

908

Amino acid sequencing

Determination of protein molecular weight

LC/MS

ESI Ion Trap LC/MSn

(電噴霧離子井液相層析多重質譜 )

Posttranslational modification (?)

Trypsin digest

nano-LC (check minor difference)

MS-MS(or MSn) analysis for selected peptides

Identification of modifications on protein

Normal Tumor

MALDI-TOF =&

ESI Ion trap LC/MSn MALDI-TOF

Two-dimensional gel electrophoresis system

Sample purification/preparation

(Laser-captured microdissector)

1o-D Isoelectrophoresis

2o-D SDS-PAGE

2D-gel image

analysis system

Quantitation of spots in 2D-gel

Image comparison between 2D-gels

Automatic spots pick-up

Spot picker and Digester

MALDI-TOF & ESI Ion trap LC/MSn

MS spectrometric

analysis system

Automatic spots digestion/extraction

MS spectrometry database

Bioimformatics

Aim: Set up proteomics core lab

貴儀中心

Isotope–Coded Affinity TagsICAT™

• Breakthrough approach to quantitative protein expression analysis - 2D gel alternative

reactive group

biotin tag

linker (heavy or light)

heavy reagent: d8-ICAT (X=deuterium)light reagent: d0-ICAT (X=hydrogen)

S

HN NH

O

NH O

ONH

IO OX

X

X

X

X

X

X

XSH

cys

Label&

Quantify

Emerging Technology

Nature Biotechnology 17, 994-999 (1999)

The End

Isotope–Coded Affinity TagsICAT™

• Breakthrough approach to quantitative protein expression analysis - 2D gel alternative

reactive group

biotin tag

linker (heavy or light)

heavy reagent: d8-ICAT (X=deuterium)light reagent: d0-ICAT (X=hydrogen)

S

HN NH

O

NH O

ONH

IO OX

X

X

X

X

X

X

XSH

cys

Label&

Quantify

Emerging Technology

ICAT Approachto Quantitative Protein Expression Analysis

Determine differences in protein expression by measuringrelative intensities of light vs. heavy

ICAT™Protein ID and Quantification Process

ProICAT provides Protein Identification and Quantitation information

ICAT™ Features & Benefits

• Overcomes some of the limitations of 2D Gels.• Ability to quantify membrane proteins.• ID and quantify low abundance proteins.• Broader range of protein MW or pI.

• Allows for greater automated/higher throughput approach in the simultaneous quantification and identification of proteins.

• Reduces complexity of analysis of protein digest -only cysteinecontaining peptides.

• Applied Biosystems ‘Systems Approach’ for ICAT Technology including ICAT reagents, software and support.