Micromass Quattro Ultima triple quadrupole mass spectrometric detector HPLC system (LC) Electrospray...
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Transcript of Micromass Quattro Ultima triple quadrupole mass spectrometric detector HPLC system (LC) Electrospray...
Micromass Quattro Ultimatriple quadrupole mass spectrometric detector
HPLC system (LC)
Electrospray ionisation source (-ve & +ve ion)
Photodiode array detector (PDA)
Fluorescencedetector
The Equipment LC-MS/MS
A little bit about triple quadrupole mass spectrometry.
Hexapoleion bridge
(I) (II)
Z-sprayion source
Extractioncone
Quadrupole(MS1)
Quadrupole(MS2)
Pre-filterPost-filter
Hexapolecollision cell
Photomultiplier
Phosphor
Ion drag
Focus ring
Conversiondynode
Whisperdetector
Electrospray or APcI ion
source
TM
A little bit about electrospray ionisation.
Desolvatongas manifold
Isolation valve
Hexapoleion bridge
Extractioncone
Ionblock
VentCleanable
baffle
Samplingcone
Measurement of protein mass by triple quadrupole MS.
Protein (pure) sample(prepared by investigator)
■ Multiple-charged ion series - deconvolution gives molecular masses
■ Intractable analysis for complex protein mixtures. Limited mass resolution.
Mass spectrometerSample infusion
Infusion pump
Electrospraysource
Parent ions
Proteinn+
Protein(n+x)+
Mass analyser-2
OFF
Photomultiplier detector
Response
020406080
100
600 1100Molecular mass (Da
(%)
Mass analyser-1
Collisioncell
Applications:Peptide mapping of haemoglobin modified by methylglyoxal
MGmin-Hb
0
20
40
60
80
100
15000 15250 15500 15750 16000 16250 16500
Molecular mass (Da)
Re
sp
on
se
(%
)
15129 (a -chain)
15870 (b -chain)
0
20
40
60
80
100
500 700 900 1100 1300 1500 1700Molecular mass (Da
(%)
Control haemoglobin
0
20
40
60
80
100
15000 15250 15500 15750 16000 16250 16500
Molecular mass (Da)
Re
sp
on
se
(%
)
15129 (a -chain)
15870 (b -chain)
Detection of protein biomarkers by LC-MS/MS:Multiple reaction monitoring (MRM)
HPLC
Mass analyser-1
Parent ion
Electrospraysource
Biomarker+
Mass analyser-2
Biomarker fragment
Collision cell
Fragment ion
+
■ High specificity
■ (LC, MS1 and MS2 resolution)
■ High sensitivity
■ Biomolecule compatible
Mass spectrometer
Enzymatic hydrolysate(prepared by investigator)
Photomultiplier detector
Response
■ Biomarker screening in 75 min per sample.
Advanced glycation endproducts
HC
CO
NH
CH
CO
NH
N N (CH2)4(CH2)4
Bis(lysyl)imidazolium crosslinks
MOLDGOLD
+HC
CO
NH
CH
CO
NH
N N (CH2)4
CH3
(CH2)4+ +
DOLD
CH
CO
NH
HC
CO
NH
N N (CH2)4(CH2)4
H2C
(CHOH)2CH2OH
ArgpyrimidinePentosidine crosslink
+
CH
CO
NH
HC
CO
NH
(CH2)3 NHN
HN N
(CH2)4 N
N
CH3
CH3
OHNHHC
CO
NH
(CH2)3
Ne-Carboxymethyl-lysine (CML) Ne-Carboxyethyl-lysine (CEL)
Monolysyl adducts
HC
CO
NH
NHCH2CO2-(CH2)4
HC
CO
NH
NH(CH2)4 C
CH3
CO2-
H
Pyrraline
HC
CO
NH
(CH2)4 N
HOCH2
HO
AGEs with intrinsic fluorescence
Hydroimidazolones
G-H1 MG-H1
HC
CO
NH
HN
NNH(CH2)3
H
H
O
HC
CO
NH
HN
NNH
CH3
H
O
(CH2)3
HN
NNH
CH2
H
O
HOCH2(CHOH)2
HC
CO
NH
(CH2)3
3DG-H1
LC-MS/MSwith stable isotope-substituted
internal standards
Detection of protein biomarkers by LC-MS/MS: Calibration, sample de-lipidification, ultrafiltration & enzymatic hydrolysis
Delipidification and AGE fractionation■ Ultrafiltration to separate protein AGE residues and free AGEs■ Ether or methanol/chloroform extraction
Analytical performance■ Limits of detection: 20 – 500 fmol.■ Recoveries: >80%; 94-100% for amino acids■ Interbatch c.v.: <10% (n = 6)
Enzymatic digestion:■ Pepsin (+ thymol)■ Pronase E (under nitrogen, penicillin and streptomycin added)■ Prolidase and aminopeptidase (under nitrogen)
Internal standardisation and calibration■ Standards and stable isotope-substituted standards
e.g. CML and [13C6]CML, MG-H1 and [15N2]MG-H1
Detection of protein biomarkers by LC-MS/MS:Retention of amino acids and AGEs and use of column switching
Hypercarb graphitic columns retain underivatised amino acids, allowing for diversion of non-volatile salts to waste.
Non-volatile salts to waste
Hypercarb column (2.1 x 250 mm)
Hypercarb column
(2.1 x 50 mm)
Sample
To MS/MS
Column switching facilitates elution of hydrophobic analytes and column washing.
Switching valve
Examples of detection by multiple reaction monitoring (MRM): CML
N-Carboxymethyl-lysine (CML)
CO2H
HC
NH3+
NH CH2 CO2H
CO2H
HC
NH3+
Fragment ionMr = 130.1
CML204.9
CML detected in plasma protein of a normal healthy human control subject.
0
20000
40000
60000
5 6 7 8 9Retention time (min)
Det
ecto
r re
spo
nse
(c
ou
nts
)
CMLMRM: 204.9 > 130.1
0
20000
40000
60000
5 6 7 8 9Retention time (min)
Dete
cto
r re
sp
on
se
(co
un
ts)
[13C6]CMLMRM: 210.9 > 136.1
Examples of detection multiple reaction monitoring (MRM): Methylglyoxal-derived hydroimidazolone
Methylglyoxal hydroimidazolone (MG-H1)
CO2H
HC
NH3+
NH
N
NH CH3
O
H NH3
N
NH CH3
O
H+
MG-H1Mr = 229.2
Mr = 114.3
MG-H1 detected in rat retinal protein hydrolysate of a STZ diabetic rat.
0
50000
100000
150000
22 23 24 25 26 27
Retention time (min)
Dete
cto
r re
sp
on
se
(co
un
ts)
MG-H1MRM: 229.2 > 114.3
0
50000
100000
150000
22 23 24 25 26 27
Retention time (min)
Dete
cto
r re
sp
on
se
(co
un
ts)
[15N2]MG-H1MRM: 231.2 > 116.3
Arg 14.2 175.2 70.3 15 H2CO2, NH2C(=NH)NH2
Lys 6.0 147.1 84.3 15 H2CO2, NH3
Met 9.2 150.0 104.2 11 H2CO2
MetSO 7.5 166.1 102.2 14 CH3SOH
CML 7.4 204.9 130.1 12 NH2CH2CO2H
MG-H 23.7 229.2 114.3 14 NH2CH(CO2H)CH2CH=CH2
Pent 16.5 379.3 250.4 22 NH2CH(CO2H)CH2CH2CH=CH2
Analyte Rt Parent Ion Fragment ion CE Natural Fragment loss
(min) (Da) (Da) (eV)
Mass spectrometric multiple reaction monitoring detection of protein biomarkers
Peptide mapping to identify sites of protein modification
Mass spectrometer
Mass analyser-1
Collisioncell
Electrospraysource
Parent ions
Peptides+
Tryptic digest of protein sample(prepared by investigator)
HPLC PDA
Resolution of peptide fragments by LC
Mass analyser-2
OFF
Photomultiplier detector
0
50
100
0 10 20 30 40
SIR
(%)
Single ion response for each peptide
Peptide map
Biolynx match of peptide M+ with theoretical digest.Locate modified peptide M+ ion
Peptide mapping to identify glycation sites.
Glycation of human serum albumin by methylglyoxal
Location of glycation sites by LC-MS peptide mapping
MS detection of peptide fragments by LC-MS and quantitation of the MS response
Peptides are partially resolved by HPLC with ODS chromatography and detected by positive ion electrospray MS.
Limited proteolysis of MGmin-HSA and HSA control Reduction of disulphide bonds with dithiothreitol. S-Alkylation of cysteine thiols by iodoacetamide. Digestion with trypsin (and independently with Glu-C for
corroboration).
0
50
100
150
0 10 20 30 40 50 60
Retention time (min)
Res
po
nse
(co
un
ts x
10
9 )
MS detection of peptide fragments by LC-MS and quantitation of the MS response
Peptide responses are normalised to the C-terminal peptide (LVAASQAALGL).
Loss of peptides in MGmin-HSA digest was quantified by the mean normalised peptide response for MGmin-HSA, relative to HSA control (mean c.v. = 11%). This is assumed due to glycation
The glycated peptides were also detected as modified dipeptides (resistant to proteolysis in tryptic maps).
02
46
810
12
30 32 34 36 38 40Retention time (min)
No
rmali
sed.
resp
on
se
HSA
MGmin-HSA
Modification of arg-410
Peptide(T52) FQNALLVR
0
2
4
6
8
32 34 36 38 40 42Retention time (min)
No
rmal
ised
.
resp
on
se
MGmin-HSA
HSA
LC-MS/MS peptide mapping can also be used to locate glycation, oxidation and nitration markersLocation of MG-H1 residues in human serum albumin modified minimally by methylglyoxal
0
2
4
6
8
10
12
30 32 34 36 38 40
Retention time (min)
No
rmal
ised
.
resp
on
se
HSA
MGmin-HSA
Ion chromatograms for peptide T52 (containing R410)
0
2
4
6
8
32 34 36 38 40 42
Retention time (min)
No
rmal
ised
.re
spo
nse
MGmin-HSA
HSA
Ion chromatograms for dipeptide T52-53 (containing MG-H1-410)
0
10
20
30
40
1350 1370 1390 1410 1430 1450
Mass (Da)
Resp
on
se
(co
un
ts)
1406.8 Da
Predicted mass of T52-53 FQNALLVRMG-H1YTK 1406.9; found peptide mass 1406.8 Da.
Glycation of human serum albumin by methylglyoxalLocation of glycation sites by LC-MS peptide mapping
R410R218
R114R186
R428 Arg MG-H1
(mol%)
114 36
186 25
218 31
410 89428 25
Modification hotspot: Arg-410
Drug binding site 2. Active site of
esterase activity.