LC ESI/MS reference measurement
procedure for HbA2
Donatella Caruso
Dept. Pharmacological and Biomolecular Sciences
Università degli Studi Milano
The determination of HbA2 is accepted as
the gold standard in the screening for βthalassemia carriers.
Highly accurate and precise measurements
are needed, also because the difference
between normal and pathological HbA2
values is slight.
Fetal hemoglobin: assessment of glyca3on and acetyla3on status by electrospray ioniza3on mass spectrometry Andrew S. Davison, Brian N. Green and Norman B. Roberts Clin Chem Lab Med 2008;46(9):1230–1238 ….Here, we describe the evaluation of ESI-MS for
measurement of glycated (GHbF) and acetylated (AcHbF)
fetal hemoglobin and the identification by mass of different
chains of fetal hemoglobin………..
AIM: To develop a reference measurement
procedure based on the quantification of intact
globin chains by LC-ESI/MS
I STEP:
CHOICE OF THE ANALYTICAL
CONDITIONS
II STEP:
VALIDATION OF THE METHOD
III STEP:
INTERLABORATORY COMPARISON
Fresh blood in EDTA Filtra>on through cellulose column Sample prepara>on Removal of WBC, platelets, plasma RBC Lysis with H2O/CCl4 Davison AS et al.
Clin Chem Lab med
2008;46:1230
(modified)
Removal of cellular stroma Hemolysate Dilu>on with acetonitrile/ formic acid Desal>ng on centrifuga>on ca>on exchange resin bead Samples for MS Hb 1 mg/mL Acetonitrile 50 % Formic acid 0.18 % Analytical conditions
Surveyor LC Pump Method -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐ Eluents A: H20-­‐CH3CN 4:1+0.05% TFA Column: Vydac C4 250x4,6mm B: H 0-­‐CH CN 2:3+0.05% TFA 2
3
Program Time (min) Flow (mL/min) A (%) B (%) -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐ -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐ -­‐-­‐-­‐-­‐-­‐ -­‐-­‐-­‐-­‐-­‐ 0.00 0.800 48.0 52.0 2.00 0.800 48.0 52.0 13.00 0.800 42.0 58.0 16.00 0.800 42.0 58.0 25.00 0.800 31.0 69.0 30.00 0.800 48.0 52.0 35.00 0.800 48.0 52.0 Instrumentation:
ESI-linear ion trap
(LTQ, Thermofisher,
USA)
TIC trace of a β-thalassemic subject hemoglobin RT: 11.00 - 27.00 SM: 7G
NL:
1.04E6
TIC MS
2THcolbis
RT: 23.55
MA: 30189496
23.48
100
95
90
85
80
75
70
RT: 15.81
MA: 18684699
Relative Abundance
65
15.71
60
ß/α=0.62
55
50
45
δ/α=0.022
40
35
24.20
30
24.42
24.62
25
24.87
RT: 17.76
MA: 676534
17.49
18.06
20
15
12.71
12.19
10
11
12
13
15.07
19.38 19.51
13.81
14
15
16
17
18
19
Time (min)
20
21.07
21
22.26
22
25.32
22.55
23
24
25
26
27
Only few words on ESI-­‐ion trap mass spectrometry…. The general term "atmospheric pressure ionization" (API)
includes the most notable technique, electrospray
ionization (ESI)
The gas phase peptide ions can be generated from the
online eluate of a LC column or from direct injection
ESI is able to produce single or multiple charged ions,
depending on several factors.
Hb_39a #1804-1953 RT: 18.86-19.79 AV: 150 NL: 4.16E5
F: ITMS + c ESI s id=10.00 Full m s [600.00-1500.00]
1081.68
100
1164.78
95
α chain
90
85
1009.66
1261.74
80
75
946.65
70
Relative Abundance
65
891.03
60
55
50
1376.32
841.63
45
40
35
30
797.40
25
20
757.60
15
10
721.60
5
636.51
0
600
898.16
688.84
700
800
900
954.31
1177.30
963.01
1215.16
1000
1100
1200
1274.93
1400.10
1316.69
1300
1446.73
1400
1500
m /z
Since MS measure the mass-to-charge (m/z) ratio, the ESI
mass spectrum for a large molecule typically contains
multiple signals corresponding to the different charge states.
The target molecules are ionized into multiple charge
states producing a waveform spectrum that can be deconvoluted into parent peaks.
S#: 1 RT: 0.01 AV: 1 NL: 1.54E7
T: + p Full ms
1060.5
90
998.2
85
75
95
1305.0
85
90
75
893.3
60
70
1413.5
848.6
65
Relative Abundance
55
50
Z=11
45
40
808.2
1541.9
25
771.5
20
15
10
5
Z=10
30
616.2
1225.4
679.1
1315.8
1420.7
1551.7
800
900
1000
1100
1200
1300
m/z
1400
1500
1600
50
45
40
25
1706.1 1820.8
20
1888.9
1938.7
0
700
55
30
1884.2
707.3
60
35
1696.0
Z=9
35
Z=24
Relative Abundance
80
Z=12
70
65
16951.0
100
1131.1 1211.9
942.9
80
S#: 1 RT: 0.01 AV: 1 NL: 1.23E8
T: + p Full ms
Z=13
95
Z=14
Z=15
100
1700
1800
1900
2000
16995.0
15
16933.0
10
5
16784.0
16802.0
16851.0
17011.0
17031.0
16894.0
17090.0 17114.0
17142.0
0
16800
16850
16900
16950
mass
17000
17050
17100
17150
2 MULTICHARGED ION DETECTED
Hb_39a #1804-1953 RT: 18.86-19.79 AV: 150 NL: 4.16E5
F: ITMS + c ESI sid=10.00 Full ms [600.00-1500.00]
α chain
95
90
85
gamma chain m/z 1062 1138 1164.78
1081.68
100
1009.66
1261.74
80
75
946.65
70
Relative Abundance
65
891.03
60
55
50
alpha chain m/z 946 1009 1376.32
841.63
45
40
HbA2 = 3.9
Theoretical value = 4.3
35
30
797.40
25
20
757.60
15
10
721.60
5
636.51
898.16
688.84
0
600
700
800
954.31
900
1274.93
1177.30
963.01
1215.16
1000
1100
1200
1400.10
1316.69
1300
1446.73
1400
1500
m/z
RT:7.00 - 24.00
SM:13G
100
90
80
70
60
50
40
30
20
10
Hb_74b #1179-1267 RT: 13.66-14.30 AV: 89 NL: 3.10E4
F: ITMS + c ESI sid=10.00 Full ms [600.00-1500.00]
1062.85
95
1226.18
90
δ chain
85
80
996.54
75
70
1328.21
937.99
55
50
885.97
45
1448.81
40
35
30
25
839.35
697.02
636.55
20
797.48
15
731.84
10
5
0
600
908.93
965.64
1030.01
1103.39
1152.43
1252.58
1376.67
642.87
700
800
900
1000
1100
m/z
1200
1300
1400
1472.46
1500
100
90
80
70
60
50
40
30
20
10
δ
α
RT: 20.08
MA:507942
RT: 15.04
Relative Abundance
Relative Abundance
65
60
2 multichargeCalibrator
Relative Abundance
1138.70
100
MA: 13192096
7
8
9
10
11
12
13
14
15 16 17
Time (min)
18
19
20
21
22
23
24
10 MULTICHARGED IONS HAS BEEN DETECTED
RT:7.00 - 24.00
SM:13G
100
90
80
70
60
50
40
30
20
10
Relative Abundance
alpha chain m/z 757 797 841 890 946 1009 1081 1164 1262 1375 MA:63301231
10
10Multicharge
multicharged-
100
90
80
70
60
50
40
30
20
10
RT: 20.09
α
calibrator
MA: 2752548
Relative Abundance
gamma chain m/z 797 839 885 937 996 1062 1138 1225 1327 1448 HbA2 = 4.3%
Theoretical value = 4,3%
δ
RT: 14.92
δ/α = 0.043
8
10
12
14
16
Time (min)
18
20
22
24
II STEP:
VALIDATION OF THE METHOD
Samples
Human frozen hemolysates treated to remove most of the salt adducts with
proteins (no plasma, no leukocytes, no platelets).
First batch: samples 1-16
4 calibrators (HbA2 concentration determined by Tosoh G8 WHO calibrated; 2.5,
3.4, 5.6 and 6.2%)
12 samples to be analyzed in triplicate
Second batch: sample 17-38
4 calibrators (HbA2 concentration determined by Tosoh G8 WHO calibrated ; 2.5,
3.6, 4.9 and 6.3%)
18 samples to be analyzed in triplicate
Third batch: samples 39-78
4 calibrators (HbA2 concentration determined by Tosoh G8 WHO calibrated ; 2.4,
3.3, 5.1 and 6.1%)
35 samples to be analyzed in triplicate
15
0,050
0,045
0,040
The calibra>on curve parameters y = 0,0063x + 0,0052
R² = 0,99647
0,035
0,030
I batch 0,025
0,020
0,015
0,010
0,005
0,000
0
0,050
0,045
0,040
0,035
0,030
0,025
0,020
0,015
0,010
0,005
0,000
1
2
3
4
5
6
7
Batch 1 2 3 y = 0,0068x + 0,0014
R² = 0,99892
II batch 0
1
2
3
4
5
6
7
0,040
0,035
y = 0,0061x - 0,0005
R² = 0,99933
0,030
0,025
mean SD 0,020
0,015
0,010
CV% III batch
0,005
0,000
0
1
2
3
4
5 2 (%)6
HbA
HbA2 (%)
7
r2 0,997 0,999 0,998 slope (x103) 6,5 6,3 6,1 0,998 0,001 6,300 0,200 0,10 3,17 RT: 7.00 - 24.00 SM:13G
100
90
80
NL: 2.14E5
m/z=
756.80-757.80+796.60-797.60+840.80-841.80+
890.20-891.20+945.80-946.80+1008.80-1009.80+
1080.80-1081.80+1163.90-1164.90+1261.00-1262.00+
1375.30-1376.30 F: ITMS + c ESI sid=10.00 Full ms
[600.00-1500.00] MS Hb_051010_10b
RT: 20.48
MA: 10252087
10 Multicharge - sample
α
Relative Abundance
70
60
50
40
30
20
10
7.73
12.89 13.51
9.37 10.34
15.08 15.76 17.44
22.29
19.38
NL: 2.05E4
m/z=
796.70-797.70+838.60-839.60+885.20-886.20+
937.10-938.10+995.60-996.60+1062.00-1063.00+
1137.80-1138.80+1225.20-1226.20+1327.20-1328.20+
1447.80-1448.80 F: ITMS + c ESI sid=10.00 Full ms
[600.00-1500.00] MS Hb_051010_10b
20.55
100
90
80
Relative Abundance
RT: 15.18
MA: 396872
70
60
δ
50
40
30
13.53
20
8.84 10.22 12.26
10
8
10
13.01
12
21.74 22.55
16.33 16.84 19.59
14
16
Time (min)
18
20
22
24
1st batch
MS precision profile 3rd batch
2nd batch
Rela>onship between HbA2 measured by the MS and HPLC techniques III STEP:
INTERLABORATORY COMPARISON
TAKE-­‐HOME MESSAGE home
The possibility to perform the quan3ta3ve determina3on of HbA2 without protein diges3on, has been explored. ESI-­‐MS of the intact Hb chains appears to be a viable method for determining the δ-­‐chain and hence detec3ng β-­‐thalassemia trait in blood samples. acknowledgment Renata Paleari
Federico Abbiati
Andrea Mosca
Flavio Giavarini
all the HbA2 working group