The Equipment LC-MS/MS
Fluorescence
detector
Micromass Quattro Ultima
triple quadrupole mass
spectrometric detector
Photodiode array
detector (PDA)
HPLC system (LC)
Electrospray ionisation
source (-ve & +ve ion)
A little bit about triple quadrupole mass
spectrometry.
Hexapole
ion bridge
Electrospray
or APcI ion
(I)
(II)
Hexapole
Pre-filter
collision cell
Post-filter
Photomultiplier
source
Phosphor
Focus ring
Extraction
cone
Conversion
dynode
Z-spray
Quadrupole
Quadrupole
ion source
(MS1)
(MS2)
Ion drag
Whisper
detector
TM
A little bit about electrospray ionisation.
S
a
m
p
l
i
n
g
D
e
s
o
l
v
a
t
o
n
g
a
s
m
a
n
i
f
o
l
d
c
o
n
eC
l
e
a
n
a
b
l
e
V
e
n
t
b
a
f
f
l
e
H
e
x
a
p
o
l
e
i
o
n
b
r
i
d
g
e
I
s
o
l
a
t
i
o
n
v
a
l
v
e
I
o
n
E
x
t
r
a
c
t
i
o
n
b
l
o
c
k
c
o
n
e
Measurement of protein mass by triple quadrupole MS.
Sample
infusion
Mass spectrometer
Electrospray
source
Protein (pure)
sample
(prepared by
investigator)
n+
Protein
(%)
Infusion
pump
Mass
Mass
Collision
Photomultiplier
analyser-2
cell
detector
analyser-1
OFF
(n+x)+
Protein
100
80
60
40
20
0
600
1100
Molecular mass (Da
Response
Parent ions
■ Multiple-charged ion series deconvolution gives molecular masses
■ Intractable analysis for complex protein
mixtures. Limited mass resolution.
Applications:
(%) (%)
Response
Peptide mapping of haemoglobin modified by methylglyoxal
100
100
80
80
60
60
Control haemoglobin
15129 (a -chain)
15870 (b -chain)
40
40
20
20
0
0
15000
500
15250
700
15500
15750
16000
900
1100
1300
Molecular
mass
(Da)
Molecular
mass
(Da
16250
1500
16500
1700
Response (%)
MGmin-Hb
100
80
15129 (a -chain)
60
15870 (b -chain)
40
20
0
15000
15250
15500
15750
Molecular mass (Da)
16000
16250
16500
Detection of protein biomarkers by LC-MS/MS:
Multiple reaction monitoring (MRM)
Mass spectrometer
HPLC
Electrospray
source
Enzymatic
hydrolysate
(prepared by
investigator)
Mass
Mass
Collision
Photomultiplier
cell analyser-2
detector
analyser-1
+
+
Biomarker
Biomarker
fragment
Parent ion
Fragment ion
■ High specificity
■ (LC, MS1 and MS2 resolution)
■ High sensitivity
■ Biomolecule compatible
■ Biomarker screening in 75 min per sample.
Response
Advanced glycation endproducts
M o n o ly s y l a d d u c ts
HOCH2
CO
CO
HC
(C H 2 ) 4 N H C H 2 C O 2
HC
(C H 2 ) 4
-
CO
CH3
NH C
NH
HC
H
CO2
N
(C H 2 ) 4
NH
NH
H
O
N e -C a rb o x y m e th y l-ly s in e (C M L ) N e -C a rb o x y e th y l-ly s in e (C E L )
P y rra lin e
B is (ly s y l)im id a z o liu m c ro s s lin k s
(C H O H ) 2 C H 2 O H
CH3
CO
HC
NH
CO
H 2C
CO
CO
CO
CO
LC-MS/MS
with stable isotope-substituted
internal standards
(C H 2 ) 4
N + N
(C H 2 ) 4
CH HC
NH
(C H 2 ) 4
+ N
N
(C H 2 ) 4
CH
NH
HC
NH
(C H 2 ) 4
N +
(C H 2 ) 4 C H
NH
NH
DOLD
M OLD
GOLD
N
A G E s w ith in trin s ic flu o re s c e n c e
CO
HC
N
CH3
(C H 2 ) 3 N H
+
N
HN
NH
(C H 2 ) 4
CO
CO
(C H 2 ) 3 N H
HC
CH
N
OH
N
NH
CH3
NH
A rg p y rim id in e
P e n to s id in e c ro s s lin k
H y d ro im id a z o lo n e s
H O C H 2 (C H O H ) 2
H
CO
CO
HN
H
H C (C H 2 ) 3 N H
N
NH
G -H 1
CH3
O
HC
HN
(C H 2 ) 3
H
NH
N
NH
M G -H 1
O
CO
HC
NH
HN
(C H 2 ) 3
CH2
H
NH
N
3 D G -H 1
O
Detection of protein biomarkers by LC-MS/MS: Calibration,
sample de-lipidification, ultrafiltration & enzymatic hydrolysis
Internal standardisation and calibration
■ Standards and stable isotope-substituted standards
e.g. CML and [13C6]CML, MG-H1 and [15N2]MG-H1
Delipidification and AGE fractionation
■ Ultrafiltration to separate protein AGE residues and free AGEs
■ Ether or methanol/chloroform extraction
Enzymatic digestion:
■ Pepsin (+ thymol)
■ Pronase E (under nitrogen, penicillin and streptomycin added)
■ Prolidase and aminopeptidase (under nitrogen)
Analytical performance
■ Limits of detection: 20 – 500 fmol.
■ Recoveries: >80%; 94-100% for amino acids
■ Interbatch c.v.: <10% (n = 6)
Detection of protein biomarkers by LC-MS/MS:
Retention of amino acids and AGEs and use of column switching
To
MS/MS
Sample
Hypercarb
column
Hypercarb column
(2.1 x 250 mm)
(2.1 x 50 mm)
Switching
valve
Non-volatile
salts to waste
 Hypercarb graphitic columns retain underivatised amino
acids, allowing for diversion of non-volatile salts to waste.
 Column switching facilitates elution of hydrophobic
analytes and column washing.
Examples of detection by multiple reaction monitoring
(MRM): CML
N -C a rb o x ym e th yl-lys in e (C M L )
C O 2H
HC
C O 2H
NH
NH3
CH2
C O 2H
HC
+
NH3
F ra g m e n t io n
M r = 1 3 0 .1
13
CML
[ C6]CML
MRM: 204.9 > 130.1
MRM: 210.9 > 136.1
60000
40000
20000
0
5
6
7
8
Retention time (min)
9
Detector response
(counts)
Detector response
(counts)
CML
2 0 4 .9
+
60000
40000
20000
0
5
6
7
8
Retention time (min)
CML detected in plasma protein of a normal healthy human
control subject.
9
Examples of detection multiple reaction monitoring
(MRM): Methylglyoxal-derived hydroimidazolone
M e th ylg lyo x a l h yd ro im id a zo lo n e (M G -H 1 )
C O 2H
NH
NH
CH3
+
NH3
H
HC
N
NH3
M r = 1 1 4 .3
15
MG-H1
[ N2]MG-H1
150000
100000
50000
0
24
25
26
Retention time (min)
27
Detector response
(counts)
Detector response
(counts)
MRM: 229.2 > 114.3
23
H
O
M G -H 1
M r = 2 2 9 .2
22
CH3
N
O
+
NH
MRM: 231.2 > 116.3
150000
100000
50000
0
22
23
24
25
26
Retention time (min)
MG-H1 detected in rat retinal protein hydrolysate of a STZ
diabetic rat.
27
Mass spectrometric multiple reaction monitoring
detection of protein biomarkers
Analyte Rt Parent Ion Fragment ion CE Natural Fragment loss
(min) (Da)
(Da)
(eV)
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
204.9
130.1
12
NH2CH2CO2H
229.2
114.3
14
NH2CH(CO2H)CH2CH=CH2
250.4
22
NH2CH(CO2H)CH2CH2CH=CH2
CML
7.4
MG-H 23.7
Pent
16.5
379.3
Peptide
mapping
to identify
sites
of protein
modification
Peptide
mapping
to identify
glycation
sites.
Mass spectrometer
HPLC PDA
Electrospray
source
Mass
Mass
Collision
Photomultiplier
analyser-2
cell
detector
analyser-1
OFF
+
Peptides
Parent ions
100
SIR (%)
Resolution of
peptide fragments
by LC
50
0
0
Tryptic digest of
protein sample
(prepared by
investigator)
10
20
30
40
Single ion
response for
each peptide
Peptide
map
Biolynx match of
peptide M+ with
theoretical digest.
Locate modified
peptide M+ ion
Glycation of human serum albumin by methylglyoxal
Location of glycation sites by LC-MS peptide mapping




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).
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.
Response
(counts x 10 9)
150
100
50
0
0
10
20
30
Retention tim e (m in)
40
50
60
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).
Modification of arg-410
12
10
8
6
4
2
0
HSA
MGmin-HSA
Normalised .
response
Normalised.
response
Peptide(T52) FQNALLVR
MGmin-HSA
8
6
4
HSA
2
0
30
32
34
36
38
Retention time (min)
40
32
34
36
38
40
Retention time (min)
42
LC-MS/MS peptide mapping can also be used to
locate glycation, oxidation and nitration markers
Location of MG-H1 residues in human serum albumin modified minimally by methylglyoxal
Ion chromatograms for dipeptide
T52-53 (containing MG-H1-410)
12
10
8
6
4
2
0
HSA
Normalised .
response
Normalised.
response
Ion chromatograms for peptide T52
(containing R410)
MGmin-HSA
MGmin-HSA
8
6
4
HSA
2
0
30
32
34
36
38
32
40
36
38
40
42
Retention time (min)
Retention time (min)
1406.8 Da
40
Response
(counts)
34
30
20
10
0
1350
1370
1390
1410
1430
1450
Mass (Da)
Predicted mass of T52-53
peptide mass 1406.8 Da.
FQNALLVRMG-H1YTK 1406.9; found
Glycation of human serum albumin by methylglyoxal
Location of glycation sites by LC-MS peptide mapping
R186
R114
R428
R410
R218
Arg
MG-H1
(mol%)
114
36
186
25
218
31
410
89
428
25
Modification
hotspot: Arg-410
 Drug binding site 2.
 Active site of
esterase activity.