Transcript Mass Spectrometry

Mass Spectrometry
•
•
•
•
•
Widely used analytical technique
Within an accuracy of 0.01% of total weight of sample and within 5 ppm
for small organic molecules
Unequaled sensitivity
–Nanomolar range routinely (1 x 10-9M)
–Femtomolar range possible (1 x 10-15M)
–Attomolar range claimed (1 x 10-18M)
Diversity of applications
–Proteins
–Oligonucleotides
–Oligosaccharides
–Lipids
–Others
Proteomics
–Identification of proteins expressed under specific conditions
-3
fundamental parts: Ionization source, the analyzer,
the detector
-Ionization source
시료분자를 이온화시키고 더 작은 이온으로 쪼갠다.
-Mass analyzer
이온들을 mass-to-charge(m/z) ratio에 따라 선택적으로
분리
-Ion detector
이온 흐름을 그 양에 비례하게 전기적인 흐름으로 전환,
증폭시켜 signal을 생성
-Vacuum system
Basic components to MS
•Ion source
–Electrospray(ESI)
–Atmospheric Pressure Ionization (APCI)
–Chemical Ionization (CI)
–Electronic Ionization (EI)
–Matrix Assisted Laser DesorptionIonization (MALDI)
•Mass Selection
–Quadrupole
–Time of Flight (TOF)
–Magnetic Sector
–Ion Trap
–Ion Cyclotron
•Detector
–Phosphor / Photo Diode
–Multi-channel Plate (MCP)
Ion Source: ESI
Electrospray ionization(ESI)
-용액 상태의 시료를 이온화(LC-MS)
-기존의 방법으로는 얻기 힘들었던 intact
상태의 peptide나 단백질을 이온화
-한 개 이상의 전하를 띤 이온을 생성
Ion Source: ESI
Ion Source: ESI
Ion Source: MALDI
Matrix Assisted Laser Desorption
Ionization(MALDI)
Laser
m
m
+
a
a
+
m a
m
+
a m
a
a
+
m
m
m +
m
m
matrix + analyte
Sample support
Matrix
CH
CH O
3
COOH
C(CN)COOH
CH
CHCOOH
OH
HO
HO
HO
-cyano-4-hydroxycinnamic acid
2,5-dihydroxybenzoic acid
(2,5-DHB)
CH O
3
Sinapinic acid
(3,5-Dimethoxy-4-hydroxy cinnamic acid)
Laser
Sample plate
hn
1. Sample (A) is mixed
with excess matrix (M)
and dried
on a MALDI plate.
2. Laser flash ionizes
matrix molecules.
AH+
3. Sample molecules are
ionized by proton
transfer from matrix:
MH+ + A  M + AH+.
+20 kV
Variable Ground
Grid
Grid
Why MALDI?
-Less sensitive to salts
-Lower PRACTICAL detection limits
-Easier to interpret spectra(less multiple
charges)
-Quick and easy
-Higher mass detection
-Higher Throughput(1000>samples per
hour)
MALDI/TOF Mass spectrum
(M+H)+
Relative Abundance
40000
30000
(M+2H)2+
20000
10000
(M+3H)3+
0
50000
100000
m/z
150000
200000
The Mass Analyzer: TOF
Time Of Flight(TOF)
Ion Source
Flight Tube
20-25 kV
+ +
Principle: If ions are accelerated with the same potential at a
fixed point and a fixed initial time and are allowed to drift, the
ions will separate according to their mass to charge ratios.
Calibration of the mass scale
The mass-to-charge ratio of an ion is proportional to the
square of its time of flight in the analyzer (“drift time”).
2
m 2t K
 2
z
L
t
L
m
K
z
=
=
=
=
=
Drift time
Drift length
Mass
Kinetic energy of ion
Number of charges on ion
The Mass Analyzer: TOF
Ion Source
Flight Tube
+
Detector
+
+
The Mass Analyzer: TOF
Ion Source
Flight Tube
+
+
+
Detector
The Mass Analyzer: Quadrupole
Quadrupole(Mass filter)
-4개의 molybdenum 막대로 이루어져 있으
며, 한쌍(1,2)은 DC voltage, 다른 한쌍(3,4)
은 Radio frequency voltage가 가해진다.
-가해지는 전압의 진폭은 선택된 m/z에 해
당되는 ion만 ion source에서 detector까
지 통과하게 한다.
- quadropole의 전압을 바꾸면서 주어진
mass범위의 이온을 scanning 한다.
The Mass Analyzer: Quadrupole
Detectors
Ions are detected with a Microchannel Plate
-1000 V
-100 V
D= 6-25 u
+
Primary Ion from
Flight Tube
L
Ions are detected with a Microchannel Plate
-1000 V
-100 V
+
D= 6-25 u
L
Ions are detected with a Microchannel Plate
-1000 V
-100 V
D= 6-25 u
Multification by secondary emission
+
e-
Secondary emissive materials:
Beryllium oxide, magnesium oxide etc
L
Ions are detected with a Microchannel Plate
-1000 V
-100 V
D= 6-25 u
ee- e-
+
e-
L
Ions are detected with a Microchannel Plate
-1000 V
-100 V
D= 6-25 u
ee- e-
+
e-
L
Ions are detected with a Microchannel Plate
-1000 V
-100 V
D= 6-25 u
eee-
e-
e-
e- e
e-
+
e- e-
ee-
~103
Amplification
L
Tandem MS(MS/MS)
Tandem MS(MS/MS)
Tandem MS(MS/MS)
MALDI TOF MS
Vacuum
lock
Vacuum system
Sample Analyte Acceleration
plate molecules
grids
in matrix
Drift tube
Mass spectrum
Ion detector
High resolution TOF-MS with ion reflector
MALDI ion
source
Ion
detector
The reflector focuses ion of same
mass but different velocity on
detector; high resolution is obtained
Ion reflector
HiRes mass spectrum
TOF/TOF-MS/MS with
CID
LID
MALDI ion
source
Parent ion
selector
Ion
detector
MS/MS spectrum of daughter ions
is measured in a single acquisition;
no pasting of segments;
low sample consumption,
high speed, high sensitivity
Ion reflector
Daughter ion mass spectrum
Why interested in MALDI-TOF MS
 분자량 측정
 큰분자량 물질 분석
 혼합물 분석 : 한 종류의 성분이 아닌 몇 종류가 혼재해 있어도 분석이 가능함
 미량분석 : 매우 민감하여 미량의 시료도 분석 가능 함 : 펩타이드 경우 fmol 분석 가능
 데이터 분석이 쉬움 : 분자 구조가 깨어 지지 않고, 보통 다 전하를(multiple charging)띠지 않으므로 데이
터가 다른 질량 분석기에서 보다 단순하여 해석이 용이함
 염의 영향이 적음 : 생체단백질 분리에 이용되는 완충용액, 염 등에 LC/MS 보다 영향을 덜 받음
 분석이 신속함 : 시료와 Matrix 섞어 sample plate에 떨어뜨려 용액을 말리는 시간(약 5~10 분), MALDITOF 로 분석하는 시간 (1분 이내)
 기기 사용 및 유지하기 위한 비용이 저렴 : LC/MS, GC/MS 처럼 질소 또는 아르곤 가스를 사용하지 않고, 미
량의 Matrix와 ACN, TFA등을 이용함으로 시약 비용도 저렴함
Mass Spectrometry 분석
-base peak
-parent peak
-radical cations
-Isotopes
Peptide Sequencing
Biomolecule Analysis
*과거에는?
-Electrophoresis, chromatography,
ultracentrifugation
-Not very precise
*MS이용하면?
-Proteins, oligonucleotides,
oligosaccharides, lipids
-Detect modifications and sequences
Peptide Mass Fingerprinting
• Analytical technique for protein
identification (protein sequence)
• Unknown protein of interest cleaved into
peptide by protease
• Collection of peptides resulting from this
cleavage comprise a unique identifier of the
unknown protein
• Mass measured with MALDI-TOF
and ESI-TOF
• in silico compared to the genome
• Computer programs translate the known
genome of the organism into proteins
• Theoretically cut the proteins into peptides with
the same protease (ex.Trypsin: K or R)
• Calculate the absolute masses of the peptides
from each protein
• the masses of the peptides of the unknown
protein vs the theoretical peptide masses of
each protein encoded in the genome
• Results statistically analyzed to find the best
match
In Gel Digestion & Mass Spectrometry
Trypsin Digest
Cut out 2D-Gel Spot
Protein
Peptides
Peptide Mass Fingerprinting
N
K
K
R
K
K
K
R
Trypsin
K
R
Protein
N
K
K
K
R
K
R
R
R
C
K
K
R
C
Tryptic peptide mixture.
Masses measured by MS.
Every peptide has a basic Cterminus.
A protein can be identified in a database by matching masses of a
subset of the tryptic peptides against calculated values.
intact
protein
enzyme
peptide
fragments
MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVS
PFDHSRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMVW
EQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIK
SYYTVRQLELENLTTQETREILHFHYTTWPDFGVPESPASFLNFLFKVRE
SGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVL
LEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELSHEDLE
PPPEHIPPPPRPPKRILEPHNGKCREFFPNHQWVKEETQEDKDCPIKEEK
GSPLNAAPYGIESMSQDTEVRSRVVGGSLRGAQAASPAKGEPSLPEKDED
HALSYWKPFLVNMCVATVLTAGAYLCYRFLFNSNT
Peptide Mass Fingerprinting
2D-Gel
Database
“Spot removal”
In Silico Digestion
In Gel Digestion
MS
848.1
1272.5
492.6
883.2
2978.9
812.6
1432.3
3127.1
996.8
702.4
164.9
2748.2
Is identical to
848.3
1272.7
493.2
882.6
2978.3
364.1
948.9
3128.8
3514.2
2837.1
263.9
147.4
1429.7
199.6
142.3
640.8
Post Translational
Modifications(PTM’s)
• PTM’s are very important in signaling as well as
metabolic pathways (e.g. phosphorylation)
• Often we want to know not only which
modification a protein has undergone, but
exactly where in the sequence the modification
lies.
• Many of the search engines allow for “variable”
modifications, but very few at one time
(combinatorialy explosive)
• There is great opportunity here for robust
searches that find PTM’s reliably!
Protein sequence Analysis
For sequencing of an entire Protein…??
Divide and Conquer !!!
Deduction of Full Amino Acid Sequence of a Protein
by Overlapping the Sequences Obtained from individual Peptides
Edman Degradation Sequentially Removes One Residue at a Time
from the Amino End of a Peptide up to 50 times
Each round
can be complete
within 1 hr and
the Edman degradation
can be repeated
up to 50 cycles
in Practice.
Lymphomas and Leukemias
Regulatory Mutations
Normal expression
Overexpression
Her2 protein
Her2 protein
Messenger
RNA
Chromosome 17
Her2 gene
Her2 gene amplification