Transcript Proteomics

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
mRNA level  expressed protein level nor does it indicate
the nature of the functional protein product
t-RNA
t-RNA
mRNA
Ribosome
t-RNA
(....)
Protein
DNA
t-RNA
Post Translational
Modifications
X
(....)
X
CHO
PO4
Active Protein
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
Proteomics
genes characterization
and identification
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
Biological samples (C/E)
cDNA microarray
core lab
Proteomics
core lab
cDNA
Proteins
Microarray
2-D gels/MS
mRNA expression
pattern
Protein expression
pattern
Bioinformatics core lab
Function study in
Tg/KO mouse core lab
Strategy for proteomic study in cancer biology
Clinical specimens
Laser-captured microdissector
Tumor
cells
SDS-PAGE
Normal
cells
isoelectrofocusing
(?)
digested by trypsin
MALDI-TOF MS analysis
Database search/mapping
Protein identified
Reference Book
Mass Spectrometry for Biotechnology
Gary Siuzdak
The Department of Chemistry
The Scripps Research Institute
La Jolla, California
Academic Press
1996
What is a mass spectrometer
and what does it do?
Analogy between mass analysis
and the dispersion of light
Components of a mass spectrometer
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)
Solid probe
Reflector
Second Detector
Time of Flight
First Detector
Laser
圖十一：在 MALDI-TOF MS中的反射器設計
M/Z
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
1
Photomultiplier conversion dynode
scintillation counting
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
Clinical specimens
Laser-captured microdissector
Tumor
cells
SDS-PAGE
Normal
cells
isoelectrofocusing
(?)
digested by trypsin
MALDI-TOF MS analysis
Database search/mapping
Protein identified
The 2-D gel pattern of human kidney epithelial
293 cells expressed without (A) and with (B) the
Epstein-Barr virus oncoprotein LMP1
IEF
(B)
SDS-PAGE
(A)
IEF
Pick up spots of interest
Digested by trypsin
MALDI-TOF MS analysis
The substrate of GSK-3a purified from pig brain
(A) (B)
10
(A)
pH
3
170
c
b
116.3
1
a
66.3
55.4
29
21.5
(B)
10
pH
3
d
170
b'
116.3
66.3
55.4
29
21.5
2
1
3
1
a'
c'
MALDI-TOF analysis of tryptic fingerprint from
the substrate for kinase FA/GSK-3a in pig brain
(b1)
472-480
526-532
533-552
558-565
(d2)
472-480
526-532
533-552
558-565
(a'1)
472-480
(b'1)
472-480
526-532
533-552
558-565
(b'3)
533-552
558-565
Data base search for the substrate of kinase
FA/GSK-3a from pig brain
(d2)
Collapsin Response Mediator Protein-2 (CRMP-2, human)
MSYQGKKNIP
VPGGVKTIEA
TTMIIDHVVP
EMEALVKDHG
NGDIIAEEQQ
YITKVMSKSS
FVTSPPLSPD
IPEGTNGTEE
IAVGSDADLV
KIVLEDGTLH
GPVCEVSVTP
PRRTTQRIVA
RITSDRLLIK
HSRMVIPGGI
EPGTSLLAAF
VNSFLVYMAF
RILDLGITGP
AEVIAQARKK
PTTPDFLNSL
RMSVIWDKAV
IWDPDSVKTI
VTEGSGRYIP
KTVTPASSAK
PPGGRANITS
*908.4 da --- 391-397
GGKIVNDDQS
DVHTRFQMPD
DQWREWADSK
KDRFQLTDCQ
EGHVLSRPEE
GTVVYGEPIT
LSCGDLQVTG
VTGKMDENQF
SAKTHNSSLE
RKPFPDFVYK
TSPAKQQAPP
LG
FYADIYMEDG
QGMTSADDFF
SCCDYSLHVD
IYEVLSVIRD
VEAEAVNRAI
ASLGTDGSHY
SAHCTFNTAQ
VAVTSTNAAK
YNIFEGMECR
RIKARSRLAE
VRNLHQSGFS
*2169.1da --- 533-552
*pI~5.95
LIKQIGENLI
QGTKAALAGG
ISEWHKGIQE
IGAIAQVHAE
TIANQTNCPL
WSKNWAKAAA
KAVGKDNFTL
VFNLYPRKGR
GSPLVVISQG
LRGVPRGLYD
LSGAQIDDNI
Amino acid sequence analysis by PSD
technique in MALDI-TOF MS
(b1)
2169
908
Tandem Mass Spectrometry
MS/MS with a TOF reflectron
mass spectrometer
Peptide fragmentation postionization
[Post Source Decay (PSD)]
Amino acid sequence analysis by PSD
technique in MALDI-TOF MS
(b1)
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)
Normal
&
Tumor
MALDI-TOF
=
Posttranslational
modification
(?)
Trypsin digest
nano-LC (check minor difference)
MS-MS(or MSn) analysis for selected peptides
Identification of modifications on protein
ESI Ion trap LC/MSn
MALDI-TOF
Aim: Set up proteomics core lab
Sample purification/preparation
(Laser-captured microdissector)
Two-dimensional gel
electrophoresis system
1o-D Isoelectrophoresis
2o-D SDS-PAGE
Quantitation of spots in 2D-gel
2D-gel image
analysis system
Image comparison between 2D-gels
Automatic spots pick-up
Spot picker and Digester
Automatic spots
digestion/extraction
MALDI-TOF & ESI Ion trap LC/MSn
MS spectrometric
MS spectrometry database
analysis system
Bioimformatics
貴儀中心
Label
&
Quantify
Isotope–Coded Affinity Tags
ICAT™
• Breakthrough approach to quantitative
protein expression analysis - 2D gel alternative
heavy reagent: d8-ICAT (X=deuterium)
light reagent: d0-ICAT (X=hydrogen)
O
NH
HN
S
biotin
tag
O
XX
N
H X XO
O
XX
O
N
XX H
linker (heavy
or light)
I
S
H
cys
reactive
group
Emerging Technology
Nature Biotechnology 17, 994-999 (1999)
The End
Label
&
Quantify
Isotope–Coded Affinity Tags
ICAT™
• Breakthrough approach to quantitative
protein expression analysis - 2D gel alternative
heavy reagent: d8-ICAT (X=deuterium)
light reagent: d0-ICAT (X=hydrogen)
O
NH
HN
S
biotin
tag
O
XX
N
H X XO
O
XX
O
N
XX H
linker (heavy
or light)
I
reactive
group
Emerging Technology
S
H
cys
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 cysteine
containing peptides.
• Applied Biosystems ‘Systems Approach’ for ICAT Technology
including ICAT reagents, software and support.