identification of haemoglobinopathies by lc/msapps.thermoscientific.com/media/sid/europe...
TRANSCRIPT
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Identification of
Haemoglobinopathies by LC/MS
Mark Harrison;
Senior Scientist, ThermoFisher Scientific
Sarah Battle;
Senior Biomedical Scientist, Royal Hallamshire Hospital
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2
Introduction
• There are over 2000 known haemoglobin variants
• Precise identification the variant is relevant to the patient’s current clinical state or any possible inherited conditions
• Gel Based electrophoresis
• Is widely used in the diagnosis of hemoglobinopathies,
• Analyses can be done quickly and at a low cost.
• Only used as an initial screening test
• HPLC
• Ion exchange HPLC allows the detection of abnormal Hb quickly and precisely, using a small sample amount
• Quantification of Hb A2, Hb F, Hb A, Hb S, and Hb C and screening for Hb variants
• Important method for the investigation of hemoglobinopathies in routine laboratories
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Introduction
• HPLC/MS/MS
• Loop injection with SRM
• Provide rapid screening for clinically significant variants Hb S, Hb C,
D Punjab, O Arab and Hb E,
• Often samples which are shown to be abnormal but are inconclusive by existing methods are sent for investigation in a specialized MS laboratory.
• Requires expert tuning and operation of the MS
• Data interpretation is manual, time consuming
• Here we present some preliminary work using HPLC/MS/MS
• Aim for non-expert MS operators
• Use automated tuning procedures
• Batch analysis
• Chromatographic retention as additional identifier
• Software tools for data interpretation
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Workflow of identifying variants
IC -HPLC
indicates
presence of
variant
LC/MS
Intact Proteins
1 5 1 2 5 . 81 5 8 6 6 . 8
1 5 1 4 8 . 6
1 5 8 4 6 . 51 5 1 6 6 . 21 5 1 0 9 . 9
1 5 8 8 8 . 31 5 1 9 0 . 8
1 5 0 7 8 . 3
1 5 0 9 8 . 8
1 5 1 3 8 . 2 1 5 9 1 2 . 4
1 5 1 8 1
1 5 9 0 2 . 41 5 8 5 7
0
100
200
300
400
500
600
700
800
15100 15200 15300 15400 15500 15600 15700 15800 15900
J e d d a h
Intensity x 10^6
M ass, Da
Perform tryptic
digest
RT: 0.00 - 30.00 SM: 3B
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lativ
e A
bu
nd
an
ce
15.27
14.5510.76
8.75
12.23
17.63
12.95
15.6710.27
5.81 25.854.96
17.32
0.9019.11
21.56
1.07 25.62
2.77 3.89
26.9227.9519.42 22.68 24.697.90
NL:1.03E8
Base Peak MS 06May_Digests_L08
Variant in alpha or beta
chain.
Mass Change of variant
Look up mass shift in
tables
Examine chromatograms
of tryptic fragments
Can variant be
assigned
unambiguously ?
YES
Perform Specific
MS/MS
experiment
Process MS/MS
data
Can variant be
assigned
unambiguously ?
YES
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Sample Preparation
• Stock solution
• 10ul blood diluted to 500ul with water
• Intact analysis
• 20 ul Stock Solution diluted to 200ul with water
• Protein digests
• 100ul stock solution was denatured with 10ul 1% Formic acid and 10ul ACN
• Mix and stand for 5mins
• Add 6ul 1M ammonium bicarbonate and 5ul 5mg/ml TPCK treated trypsin
• Vortex mix and centrifuge for 15 secs
• Incubate at 37oC for 1 hour
• Dilute 20ul to 200ul with water
B.N. Green et. al.
Rapid Identification of Hemoglobin variants by Electrospray Ionization Mass Spectrometry,
Blood Cell, Molecules and Diseases (2001) 27(3)
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LC/MS Methods
• HPLC Conditions
• Column BioBasic-4 100*1 mm 5µm
• Mobile Phase A: water 0.1% Formic Acid
• Mobile Phase B: Acetonitrile 0.1% Formic Acid
• 30 min gradient
• Intact proteins
• Full scan MS Peakwidth 0.2µ FWHM 650-1200 m/z
• Digests
• Full scan MS Peakwidth 0.2µ FWHM 150-1450 m/z
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Mass Spectrometry
• TSQ Quantum Ultra
• Triple Quadrupole
• Accela UHPLC
• Open Accela AS
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Triple Stage Quadrupoles
API Source
rf/DC mass
analysing
quadrupoles
Ion Optics
Detection
System
Q2 -rf only
Collision Cell
Q1 Q2 Q3
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Why use Hyperbolic rods ?
• Forms Pure Quadrupolar Fields
• Reduces Fringing Field Effects
• Significantly Improves Resolution
• Improves Transmission
• Improves Peak Shapes
TSQ Quantum HyperQuads Technology
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Resolution Performance of HyperQuads
0.7u FWHM
2.6e6
0.1u FWHM
1.2 e6
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Use of high resolution
688 690 692 694 696
m/z
0
20
40
60
80
100
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
20
40
60
80
100
690.27
689.78
690.03
NL:2.41E4
EFTPPVQAAYQK +H +H2 O: C 64 H97 N15 O19
p (gss, s /p:40) Chrg 2R: 0.7 Da @FWHM
NL:2.41E4
QFTPPVQAAYQK +H +H2 O: C 64 H98 N16 O18
p (gss, s /p:40) Chrg 2R: 0.7 Da @FWHM
NL:2.41E4
EFTPPVQAAYQK *1.00+QFTPPVQAAYQK *1.00 +H +H2 O: p (gss, s /p:40) Chrg 2R: 0.7 Da @FWHM
Simulation of isotope patterns
Normal T13 [M+2H] 2+
689 690 691 692 693
m/z
0
20
40
60
80
100
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
20
40
60
80
100
689.85
690.35
690.86
691.36
689.36
689.86
690.37
690.87
689.85
690.36
690.86
691.36691.86 692.36
NL:1.86E4
EFTPPVQAAYQK +H +H2 O: C 64 H97 N15 O19
p (gss, s /p:40) Chrg 2R: 0.2 Da @FWHM
NL:1.86E4
QFTPPVQAAYQK +H +H2 O: C 64 H98 N16 O18
p (gss, s /p:40) Chrg 2R: 0.2 Da @FWHM
NL:1.86E4
EFTPPVQAAYQK *1.00+QFTPPVQAAYQK *1.00 +H +H2 O: p (gss, s /p:40) Chrg 2R: 0.2 Da @FWHM
D-Punjab T13 [M+2H] 2+
Mixture [M+2H] 2+
Normal T13 [M+2H] 2+
D-Punjab T13 [M+2H] 2+
Mixture [M+2H] 2+
Unit resolution 0.7µ FWHM “High” resolution 0.2µ FWHM
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Electrospray ionisation
• Electrospray can produce multiple charges
• Compounds with multiple basic centres
• Eg peptides and proteins
• Mass spectrometer
• Measures to mass / charge ratio of a compound; m/z
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Spectrum of Normal Hb07May_Intacts_07 #277-282 RT: 23.32-23.74 AV: 6 SM: 3B NL: 6.62E7T: + p ESI Q1MS [650.000-1200.000]
700 750 800 850 900 950 1000 1050 1100 1150 1200
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
797.13757.27
841.34
890.78
721.35
946.37
836.14
688.54
934.37882.50
1009.38
992.63
1058.84658.66
1081.42
1134.33
862.241164.55
898.60955.49 1047.04 1104.07 1190.71
15
14
16
17
181920
21
15
16
1718
19
20
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Deconvoluted spectrum of Normal Hb
15125.8
15866.8
15100 15200 15300 15400 15500 15600 15700 15800 15900Mass, Da
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Example 1 D-Punjab
15125.8
15866.8
15167.115109.8
15146.915176.6
15078.3 15888.215796.5
0.0
0.5
1.0
1.5
2.0
2.5
15100 15200 15300 15400 15500 15600 15700 15800 15900
Normal Haemoglobin
In
ten
sit
y x
10
^9
Mass, Da15125.8
15865.9
15166.2
15146.915109.4
15888.1
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
15100 15200 15300 15400 15500 15600 15700 15800 15900
D-Punjab
Inte
nsity
x 10
^9
Mass, Da
β ∆ -1 Da
(15867 – 15866 Da)
α 15126 Da
α 15126 Da
15867 Da
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Example 1RT: 0.00 - 26.69 SM: 3B
0 2 4 6 8 10 12 14 16 18 20 22 24 26
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
15.27
14.5510.768.75
12.23
17.6312.95
15.6710.27
5.81 25.854.9617.32
0.90
15.18
14.46
12.1910.76
8.75
17.63
12.90 15.6310.27
9.73
25.805.764.96
17.37
19.06
NL:1.03E8
Base Peak MS 06May_Digests_L08
NL:1.04E8
Base Peak m/z= 150.00-1450.00 MS 06May_Digests_L02
Normal
Chain -1Da
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Example 1 chromatogram
RT: 8.01 - 12.50 SM: 3B
8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
10.768.75
12.23
10.279.69
11.259.15
12.19
10.768.75
10.27
9.73
11.25
9.11
NL:7.25E7
Base Peak MS 06May_Digests_L08
NL:8.57E7
Base Peak m/z= 150.00-1450.00 MS 06May_Digests_L02
Normal
Chain -1Da
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[M+2H] 2+ of extra peak at rt 10.606May_Digests_L02 #236-237 RT: 10.49-10.54 AV: 2 SM: 5B NL: 1.82E7T: + p ESI Q1MS [150.000-1450.000]
684 686 688 690 692 694 696 698 700 702
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
689.36
689.82
690.36
0.5 Da
0.5 Da
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20
Example 1 Mass Chromatogram of 689.36 +0.2µ
RT: 0.00 - 30.00 SM: 3B
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
10.54
NL:1.45E8
m/z= 689.25-689.45 MS 06May_Digests_L08
NL:1.45E8
m/z= 689.25-689.45 MS 06May_Digests_L02
Normal
Chain -1Da
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21
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Table of chain -1Da shift
Chain Pos T Frag Mutation Name
Delta
Mass Sequence MS/MS Diagnostic ion(s) Ret T
6 1 Glu - Lys C -1 VHLTPEEK N 347.7 / 694.4 2.7
7 1 Glu - Lys G-Siriraj -1 VHLTPEEK Y 412.2 / 823.5
22 3 Glu -Gln D-Iran -1 VNVDEVGGEALGR Y 657.3 / 1313.7
22 3 Glu-Lys E-Saskatoon -1 VNVDEVGGEALGR Y 574.3 & 379.7/ 758.4
26 3 Glu-Gln Novel -1 VNVDEVGGEALGR Y 657.3 / 1313.7
26 3 Glu - Lys E -1 VNVDEVGGEALGR N 458.7 / 916.5 & 416.25
1.3 &
6.1
43 5 Glu - Lys Hornchurch -1 FFESFGDLSTPDAVMGNPK N 441.25 & 818.4 / 545.9
52 5 Asp - Asn Osu -1 FFESFGDLSTPDAVMGNPK Y 686.66 / 1029.5
73 9 Asp - Asn G-Accra -1 VLGAFSDGLAHLDNLK Y 557.0 / 835.0
79 9 Asp - Asn Yaizu -1 VLGAFSDGLAHLDNLK Y 557.0 / 835.0
90 10 Glu - Lys Agenogi -1 GTFATLSELHCDK N
412.7 / 824.5 & 304.1 /
615.3
94 10 Asp - Asn Bunbury -1 GTFATLSELHCDK N 474.2 / 710.9
99 11 Asp - Asn Kempsey -1 LHVDPENFR Y 563.3 / 1125.6
121 13 Glu-Gln D-Punjab -1 EFTPPVQAAYQK N 689.4 10.5
121 13 Glu - Lys O-Arab -1 EFTPPVQAAYQK N 625.3 1249.7
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Example 2 G-Philadelphia
15125.8
15866.8
15167.115109.8
15146.915176.6
15078.3 15888.215796.5
0.0
0.5
1.0
1.5
2.0
2.5
15100 15200 15300 15400 15500 15600 15700 15800 15900
Normal Haemoglobin
In
ten
sit
y x
10
^9
Mass, Da
α 15126 Da
15867 Da
15125.8
15140
15866.8
15166.1
15109.8
15178.615888.4
15098.1 15847.9
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
15100 15200 15300 15400 15500 15600 15700 15800 15900
G-Philadelphia
Inte
nsity
x 10
^9
Mass, Da
15867 Da
α 15126 Da
Variant α 15140 Da
+14 Da
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Example 2RT: 0.00 - 20.01 SM: 3B
0 2 4 6 8 10 12 14 16 18 20
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
15.27
14.5510.768.75
12.23
17.6312.95
15.6710.27
5.814.9617.32
0.90 19.1114.201.07
2.77 3.89
7.906.70
RT: 3.44
15.13
14.38
10.72
8.66
12.14
12.8115.5410.22
9.6917.59
5.764.96
18.931.12 17.32
2.82
15.987.996.70
NL:1.03E8
Base Peak MS 06May_Digests_L08
NL:1.30E8
Base Peak MS 06May_Digests_L04
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Example 2RT: 0.00 - 20.11 SM: 3B
0 2 4 6 8 10 12 14 16 18 20
Time (min)
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
0
20
40
60
80
100
0
20
40
60
80
100
15.27
14.5510.768.7512.23
17.6312.95 15.6710.27
5.814.96 17.320.90 19.111.07
14.64
NL:1.30E8
Base Peak MS 06May_Digests_L08
NL:7.00E7
m/z= 423.05-423.45 MS 06May_Digests_L08
NL:1.00E8
m/z= 359.00-359.40 MS 06May_Digests_L08
NL:1.00E8
m/z= 771.20-771.60+1156.40-1156.80 MS 06May_Digests_L08
NL:4.00E8
m/z= 749.66-750.06+999.30-999.70 MS 06May_Digests_L08
[M+2H] 2+ 423.25
Normal Hb
[M+2H] 2+ 359.2
[M+3H] 3+ 771.4
T9 [M+4H] 4+ 749.86
RT: 0.00 - 20.44 SM: 3B
0 2 4 6 8 10 12 14 16 18 20
Time (min)
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
0
20
40
60
80
100
0
20
40
60
80
10015.13
14.3810.72
8.6612.14
12.81 15.5410.2217.59
5.764.9618.931.12 17.32
2.82
3.22
3.48
14.29
19.20
17.59
NL:1.30E8
Base Peak MS 06May_Digests_L04
NL:6.71E7
m/z= 423.05-423.45 MS 06May_Digests_L04
NL:9.07E7
m/z= 359.00-359.40 MS 06May_Digests_L04
NL:1.15E8
m/z= 771.20-771.60+1156.40-1156.80 MS 06May_Digests_L04
NL:4.38E8
m/z= 749.66-750.06+999.30-999.70 MS 06May_Digests_L04
[M+2H] 2+ 423.25
Hb + 14 Da
[M+2H] 2+ 359.2
[M+3H] 3+ 771.4
T9 [M+4H] 4+ 749.86
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26
Example 2
• +14 Da in Hb
• Roubaix 55 val – leu/Ile
• TYFPHFDLSHGSAQVK
• G-philadelphia 68 Asn – Lys
• VADALTNAVAHVDDMPNALS ALSDLHAHK
• (K)VADALTK & AVAHVDDMPNALS ALSDLHAHK
• Stanleyville II 78 Asn – Lys
• VADALTNAVAHVDDMPN ALS ALSDLHAHK
• VADALTNAVAHVDDMPK & ALS ALSDLHAHK
• Roanne 94 Asp – Glu
• VEPVNFK
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27
Extra confirmation of retention time
• Previous methods use infusion of total sample
• Problem when normal fragments match variant fragments
• Difference between [M+H]+ and [M+Na]+ = 22µ
• Mass shift of 1u in 15,000 can be inconclusive
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28
Difference of 22Da
• Sequence of Normal T9
• VADALTNAVAHVDDMPNALSALSDLHAHK
• [M+2H]2+ = 1498.75 , [M+3H]3+ = 999.5 , [M+4H]4+ = 749.9
• [M+H+Na]2+ = 1509.74 , [M+2H+Na]3+ = 1006.8 , [M+3H+Na]4+ = 755.37
• Sequence of Q-Iran T9
• VADALTNAVAHVDHMPNALSALSDLHAHK
• [M+2H]2+ = 1509.75 , [M+3H]3+ = 1006.8 , [M+4H]4+ = 755.37
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Problem with infusion experimentSM: 5B
990 995 1000 1005 1010 1015
m/z
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Relat
ive A
bund
ance
1005.19
999.85
999.51
1011.601001.55 1009.34 1012.52990.55
1002.53
992.56 1013.53996.32993.56
999.88
999.55
1005.22
1012.551001.57
1009.501007.22990.55 1002.57
1011.55992.57
996.45993.57 1014.54
NL: 2.35E6
Sample7_A#1 RT: 0.10 AV: 1 T: + p ESI Q1MS [150.010-1500.000]
NL: 2.30E6
sample5_a#19-29 RT: 1.87-2.04 AV: 11 T: + p ESI Q1MS [985.010-1020.000]
Normal Hb
Q-Iran Hb
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RT: 11.73 - 19.88 SM: 3B
12 13 14 15 16 17 18 19
Time (min)
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
0
20
40
60
80
10015.13
14.42
12.19
12.9015.58 17.59
17.28 19.0214.0616.70
17.59
14.33
17.28
16.70
17.59
NL: 1.38E8
Base Peak MS 06May_Digests_L06
NL: 5.96E8
m/z= 600.00-600.20+749.80-750.00+999.40-999.60 MS 06May_Digests_L06
NL: 3.23E8
m/z= 625.80-626.00+782.00-782.20+1042.10-1042.30 MS 06May_Digests_L06
NL: 7.62E7
m/z= 755.40-755.60+1007.05-1007.25 MS 06May_Digests_L06
Extra confirmation of retention timeRT: 11.75 - 20.01 SM: 3B
12 13 14 15 16 17 18 19 20
Time (min)
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
0
20
40
60
80
10015.27
14.55
12.2317.6312.95 15.67
17.32 19.1114.20
17.63
14.51
17.32
17.63
NL: 1.03E8
Base Peak MS 06May_Digests_L08
NL: 5.10E8
m/z= 600.00-600.20+749.80-750.00+999.40-999.60 MS 06May_Digests_L08
NL: 2.68E8
m/z= 625.80-626.00+782.00-782.20+1042.10-1042.30 MS 06May_Digests_L08
NL: 4.77E7
m/z= 755.50-755.70+1007.05-1007.25 MS 06May_Digests_L08
Normal TIC Q-Iran TIC
Normal T9
Normal T8-9
Normal T9
Na+ adduct
Normal T9
Normal T8-9
Normal T9
Na+ adductQ-Iran T9
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31
When intact mass is inconclusive
• Q-Iran results in +22 Da shift in chain
• Sequence of Q-Iran T9
• VADALTNAVAHVDHMPNALSALSDLHAHK
• [M+2H]2+ = 1509.75 , [M+3H]3+ = 1006.8 , [M+4H]4+ = 755.37
• Jeddah results in +23 Da shift in chain
• Sequence of Jeddah T9
• VADALTHAVAHVDDMPNALSALSDLHAHK
• [M+2H]2+ = 1510.26 , [M+3H]3+ = 1007.2 , [M+4H]4+ = 755.6
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Problems with isotopes; simulation of [M+3H] 3+
1007 1008 1009 1010 1011 1012
m/z
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Relat
ive A
bund
ance
1007.51
1007.84
1007.17
1008.17
1008.51
1008.84
1007.51
1007.84
1007.17
1008.18
1008.51
1008.851009.52
NL:6.72E3
VADALTHAVAHVDDMPNALSALSDLHAHK +H +H2 O: C 130 H210 N39 O42 S 1
p (gss, s /p:40) Chrg 3R: 0.2 Da @FWHM
NL:6.72E3
VADALTNAVAHVDHMPNALSALSDLHAHK +H +H2 O: C 130 H211 N40 O41 S 1
p (gss, s /p:40) Chrg 3R: 0.2 Da @FWHM
Jeddah T9
Q-Iran T9
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Infusion
SM: 5B
995 1000 1005 1010 1015
m/z
0
20
40
60
80
100
0
20
40
60
80
100
Relat
ive A
bund
ance
0
20
40
60
80
1001005.19
1005.53999.85
999.51
1011.601009.431001.56
1012.521013.53996.32 998.25 1007.55
999.89 1005.221012.91
1007.54
999.55
1001.55
1002.53
1011.58996.511014.53
997.53
999.88
999.55
1005.22
1012.551001.571009.501007.22
1002.541011.55
996.45 1013.54
NL:2.34E6
sample7_a#1-6 RT: 0.10-0.61 AV: 6 F: + p ESI Q1MS
NL:1.24E6
Sample2_A#16-20 RT: 4.34-4.44 AV: 5 F: + p ESI Q1MS
NL:2.27E6
sample5_a#19-34 RT: 1.87-2.12 AV: 16 F: + p ESI Q1MS
Jeddah
Q-Iran
Normal
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Extra confirmation of retention timeRT: 11.70 - 20.03 SM: 3B
12 13 14 15 16 17 18 19 20
Time (min)
0
20
40
60
80
100
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
0
20
40
60
80
10015.13
14.42
12.19
12.9015.58 17.59
17.28 19.0214.0616.70
17.59
14.33
16.70
17.59
NL:1.38E8
Base Peak MS 06May_Digests_L06
NL:5.96E8
m/z= 600.00-600.20+749.80-750.00+999.40-999.60 MS 06May_Digests_L06
NL:7.62E7
m/z= 755.40-755.60+1007.05-1007.25 MS 06May_Digests_L06
Q-Iran TIC
Normal T9
Q-Iran T9
RT: 11.73 - 19.88 SM: 3B
12 13 14 15 16 17 18 19
Time (min)
0
20
40
60
80
100
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
0
20
40
60
80
10015.18
14.38
12.14
12.8115.58
17.63
14.02 19.0615.94
17.32
17.63
14.33
14.02
15.94
17.63
NL:1.11E8
Base Peak MS 06May_Digests_L03
NL:3.77E8
m/z= 600.00-600.20+749.80-750.00+999.40-999.60 MS 06May_Digests_L03
NL:6.82E7
m/z= 755.40-755.60+1007.05-1007.25 MS 06May_Digests_L03
Jeddah TIC
Normal T9
Jeddah T9
RT: 11.89 - 19.97 SM: 3B
12 13 14 15 16 17 18 19
Time (min)
0
20
40
60
80
100
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
0
20
40
60
80
10015.27
14.55
12.2317.63
12.95 15.67
17.3219.1114.20
17.63
14.51
17.63
NL:1.03E8
Base Peak MS 06May_Digests_L08
NL:2.03E9
m/z= 599.60-600.60+749.40-750.40+999.00-1000.00 MS 06May_Digests_L08
NL:1.56E8
m/z= 755.10-756.10+1006.70-1007.70 MS 06May_Digests_L08
Normal TIC
Normal T9
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Need for MS/MS
• Sequence of Normal T9
• VADALTNAVAHVDDMPNALSALSDLHAHK
• Sequence of Q-India T9
• VAHALTNAVAHVDDMPNALSALSDLHAHK
• Sequence of Q-Thailand T9
• VADALTNAVAHVHDMPNALSALSDLHAHK
• Sequence of Q-Iran T9
• VADALTNAVAHVDHMPNALSALSDLHAHK
• Sequence of possible variant T9
• VADALTNAVAHVDDMPNALSALSHLHAHK
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Need for MS/MS Riccarton T6 + 30DaRT: 9.78 - 20.67 SM: 3B
10 11 12 13 14 15 16 17 18 19 20
Time (min)
0
20
40
60
80
100
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
0
20
40
60
80
10015.27
14.5510.76
12.2317.63
12.95 15.6710.27
11.25 17.3219.1114.20
RT: 12.95
RT: 12.23
17.32
NL: 1.03E8
Base Peak MS 06May_Digests_L08
NL: 1.64E9
m/z= 458.73-459.73+611.48-612.48+916.95-917.95 MS Genesis 06May_Digests_L08
NL: 1.64E8
m/z= 621.45-622.45+931.95-932.95 MS Genesis 06May_Digests_L08
RT: 9.33 - 20.15 SM: 3B
10 11 12 13 14 15 16 17 18 19 20
Time (min)
0
20
40
60
80
100
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
0
20
40
60
80
10015.09
10.6314.29
12.05
9.5517.50
15.54
12.77 13.9310.13
17.1911.16 18.93
19.2415.94
RT: 12.72
RT: 12.68
RT: 12.05
17.19
NL: 9.57E7
Base Peak MS 06May_Digests_L07
NL: 1.42E9
m/z= 458.73-459.73+611.48-612.48+916.95-917.95 MS Genesis 06May_Digests_L07
NL: 2.00E8
m/z= 621.45-622.45+931.95-932.95 MS Genesis 06May_Digests_L07
Riccarton T6
Normal TIC
Normal T6
Riccarton T6
Normal TIC
Normal T6
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MS/MS of multiply charged ions: confirmation of sequence
SM: 5B
100 200 300 400 500 600 700 800 900
m/z
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Rela
tive
Abun
danc
e
237.0
265.0711.8612.1
524.0442.6
474.9 663.2
785.2589.2
110.0 363.5
237.1
622.5726.7
265.1
533.9
678.2452.6
800.9484.8
588.2120.0 378.7810.2
NL: 7.16E6
06May_Digests_MSMS07#359-369 RT: 12.68-13.04 AV: 6 F: + p ESI Full ms2 612.300 [50.000-1400.000]
NL: 1.97E6
06May_Digests_MSMS07#359-366 RT: 12.72-12.93 AV: 4 F: + p ESI Full ms2 622.300 [50.000-1400.000]
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38
Conclusions
• Ability to unequivocally identify Hb variants
• No “special” MS tuning knowledge needed
• Having LC retention gives extra confidence
• May eliminate the need for MS/MS in some cases
• Future work
• Investigate use of metabolomics software tools
• Automatically identify changing peaks
• Run further pilot studies
• Implement at Sheffield Northern General