liquid chromatography mass spectrometer 液相層析質譜儀 · 2015-04-07 · (computed &...
TRANSCRIPT
LC-MS of 貴重/共同儀器中心
設備
LC-QqQ MS
液相層析-三段四極柱式質譜
LC-Q-TOF MS
液相層析-四極柱-飛行時間式質譜
游離方式
ESI電灑游離、APPI大氣壓力光游離
田家炳光電大樓 617室
2
chromatography
separation (+ enrichment)
mass spectrometry
separation + detection + identification
isotope analysis
qualitative analysis ; quantitative analysis
3
LC - MS 強調儀器硬體上的連接LC / MS 強調方法、技術上的結合
Why are they combined together ?
Mass spectrometer overview
5
sample
inlet
ion
source
mass
analyzer
ion
detector
data
system
gas phase
ions
vacuum
mass spectrum
ion sorting
by m/z
Ion & spectrum
mass to charge ratio , m/z
single charged, multiple charged
mass of atom/molecule
accurate mass & monoisotopic mass
average mass & nominal mass
mass resolution
parent ion
daughter (product, fragment) ion
6
8
rela
tive
in
ten
sity
m/z
A
B 單一同位素質譜圖
原始質譜圖
986.42 100 987.56 100
987.42 50.1 988.57 50.7
988.42 21.1 959.57 17.7
989.42 6.9 990.57 4.9
990.42 1.9 991.57 1.1
991.42 0.4 992.57 0.1
mass massrelative
abundance
relative
abundance
A = C41H66N10O14S2 B = C41H77N15O11S
0
50
100
985 986 987 988 989 990 991 992 993 994
0
50
100
985 986 987 988 989 990 991 992 993 994
保留訊號峰A = 986.42
B = 987.56
Ion sources used in LC-MS
ionization
high-energy exciting or attach other ion to the sample
hard or soft ionization
properties of sample : mass , stability…
other concerns
continuous sample introducing
large amount of solvent large amount of vapor
operate under atmospheric pressure
9
what kind of data
you want ??
11
ESI
needle tip
sampling
cone (to
analyzer)
solvent of high polarity :
water, ACN (acetonitrile, CH3CN), methanol…
with volatile acid (H+ source) :
formic acid, acetic acid…
min. flow rate :
~20 nL/min
ESI :
M + nH+ [M + nH]n+
multiple charged ions
advantage?
disadvantage?
very soft
van der Waals complex
ion is possible !!
sample
small to macro molecules
high polarity
12
sample : 17 kDa protein
14
solvent dopant
dopant ion react with sample
ionization of sample
energy of photon
enough to ionize
sample or dopant
not enough to ionize
O2 , N2
atmospheric pressure chemical
ionization , 大氣壓化學游離 , APCI
corona電暈放電
inductively coupled plasma ,
感應耦合電漿 , ICP
8,000 - 10,000 K argon plasma
inorganic elemental analysis
15
16
ESI
APCIAPPI
can be applied to
small & medium molecules
that insoluble in water
small molecules to macro molecules
(peptide, protein, DNA… )
that soluble in water
Mass analyzer used in LC-MS
principles of analyzing ions with different m/z
different trajectories in electric/magnetic field
the same kinetic energy , different velocity
motion in electric/magnetic field induced current
concerns
m/z range
resolution
scanning or not , scanning speed
17
Time of flight 飛行時間式 ,TOF
20
getting the
same kinetic
energy
m/z = 2eV(t / L)2techniques for reducing
space & energy spread
5-30 kV
flight tube : 1 - 2 m
reflectron , electrostatic reflector
correcting the energy dispersion
longer flight distance
21
Quadrupole vs TOF
22
Quadrupole Time of Flight
mass range < 4,000 ; usually < 2,000 > 100,000
resolution usually set for unit resolution linear: ~3000
reflector: > 30,000 ; usually ~15,000
mass accuracy 50-100 ppm 2-50 ppm
scan speed < 4,000 Th/sec 106 Th/sec
kinetic energy of ion low high
accelerating voltage ~ 5-20 V ~ 5-30 kV
operating pressure < 10-3 Torr < 10-6 Torr
other features continuous pulse
compact size large size
$ $($$)
Tandem MS 串聯質譜
why tandem MS ??
single or multiple analytes
different operating modes
precursor ion product ion
occur during hard ionization
collision-induce dissociation, CID
precursor ion inert gas (N2, Ar,…)
photo dissociation & other techniques
24
?
MS/MS operating modes
25
操作模式 目的 操作方法
子離子(產物離子)product ion
獲得母離子的結構資訊
MS1 選擇單一母離子MS2 掃描記錄所有子離子
母離子(前驅物離子)precursor ion
尋找會產生相同子離子的母離子
MS1掃描所有母離子MS2選擇單一子離子
中性丟失neutral loss
尋找會產生相同中性碎片的母離子
MS1和MS2維持相同質荷比差,同時掃描
選擇反應監測selected reaction
monitoring
監測特定反應MS1選擇單一母離子MS2選擇單一子離子
Quadrupole-time-of-flight, Qq-TOF (Q-TOF)
30
collision gas
MS1
(Q)
ion guide
precursor ion product ions
collision chamber
(AC only q)
ion guide
MS2
(TOF)
detector
reflectron
QqQ & Q-TOF
Why there is a quadrupole in the collision chamber ?
comparison
operating modes : QqQ – all 4 modes
TOF can not be a filter Q-TOF – product ion mode
product ion sensitivity , resolution , mass accuracy : Q-TOF
simple & easy : QqQ
other MS/MS ? ion traps , TOF-TOF , TOF-Q
31
not for LC/MS
pressure , ion guide , energy
MS (even tandem MS) is not enough
sample complexity
multiple analytes have to be analyzed
number , variety , concentration of analytes & matrixes
most samples, especially “real world” samples, vary widely in
complexity
sample – MS compatibility
matrixes/impurities , concentration
more information about a complex analyte
macro biomolecules : protein, DNA, polysaccharide
35
Principle of chromatography
36
mobile
phase
stationary
phase
flow of mobile phase
A
B
detecting
time
A B
chromatograph
層析圖
B
B
B
A
A
Gas vs liquid chromatography
GC
volatile molecules : low molecular weight , low-polarity
+ thermal stable
easy to couple with various ion sources of MS
LC (HPLC , HP = high performance or high pressure)
difficult to couple with MS
nonpolar to ionic , small to Marco molecules
various mechanism of separation alternatives
37
most versatile
technique !!
HPLC
38
PUMP column detector
sample
injector -
manual or
automatic
eluent 沖提液
(mobile phase)
separation according to :
polarity – normal or reverse phase
ion interaction – ion exchange
size of molecule
40
LC/MS/MS
time
TIC (total ion counts)
chromatograph
m/z
precursor ion
spectra
m/z
product ion
spectrainformation
of molecule
quantitation
analyzer
Considerations
41
sample
column
& eluent
ion
source
flow rate
solvent of eluent
additive in eluent
separation efficiency
amount of sample
recovery
molecular weight
ionization efficiency
soft or hard ionization
number of charge
Part III
Application Examples
melamine 三聚氰胺 additive in food
protein identification
peptide mass fingerprinting 胜肽質量指紋
peptide sequence tag 胜肽序列標籤
42
Separating melamine from foods
foods : complex , variety can
to be analysis by MS directly
melamine
polar , slightly dissolved in water
using reverse phase HPLC + ESI
44
45
food sample (eg milk powder)
+ (water + CAN + 2.5% formic acid)
removing insolubles , proteins
HPLC separation
MS/MS analysis
hydrophilic
interaction
operate manually
on line analysis
46
– H2NCN – NH3
precursor ion
m/z = 127
product ion
m/z = 85
product ion
m/z = 68
selected reaction monitoring : m/z 127 85
quantification
Amino acid peptide protein
amino acid
20 essential a-amino acids with different side groups (R)
average molecular weight ~110 Da
peptide
50 or fewer amino acids linked together by peptide bonds
47
protein
> 50 amino acids
function
may have a few modifications on side group of amino acid
structure
primary structure – sequence of amino acid
▲ sequencing, identification, quantification…
secondary , tertiary , quaternary structures
48
MS
Protein identification
MS analysis
Why MS data from
peptides
is usually useful than
that
from intact protein ?
49
protein
peptides
Peptide mass fingerprinting , PMF
50
protease
(蛋白酶)
database search ,
identification
purified protein
AB
D
C
EF
mixture of peptides
A
B D
CE
F
MS
mass fingerprint
…….……………….….……
m/z
separation is not required!!
Peptide analyzed by MS/MS
53
x3 y3 z3 x2 y2 z2 x2 y2 z2
a1 b1 c1 a2 b2 c2 a3 b3 c3
peptide : SIMAETLK
precursor ion ([M+H]+)
monoisotopic mass
= 892.5
product ionscollision condition !!
54
protein mixture
A BC
DE
a
b g d
23
45
1
6
peptide mixture
A
B
C
D
E
a
b
g
d
2
3
4
5
1
6
MS/MS
LC separation
time
chromatograph
m/z
spectra of peptide
precursor ions
m/z
spectra of
product ions
database search
identify
multiple
proteins
protease
(蛋白酶)
vs PMF
proteins do NOT need to be separated
spectrum of product ions
sequence of peptide
find an unique peptide the protein can be identified
▲ multiple peptide is needed in PMF
type & position of modification
55