identification of haemoglobinopathies by lc/msapps.thermoscientific.com/media/sid/europe...

Identification of

Haemoglobinopathies by LC/MS

Mark Harrison;

Senior Scientist, ThermoFisher Scientific

Sarah Battle;

Senior Biomedical Scientist, Royal Hallamshire Hospital

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

3

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

4

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

5

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)

6

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

7

Mass Spectrometry

• TSQ Quantum Ultra

• Triple Quadrupole

• Accela UHPLC

• Open Accela AS

../../../Documents and Settings/sarah.robinson/Local Settings/Temporary Internet Files/My Documents/Strumenti e Applicazioni/CLINICA TURBO FLOW/TF Method development/Cohesive/TurboFlow Demo.exe

8

Triple Stage Quadrupoles

API Source

rf/DC mass

analysing

quadrupoles

Ion Optics

Detection

System

Q2 -rf only

Collision Cell

Q1 Q2 Q3

9

Why use Hyperbolic rods ?

• Forms Pure Quadrupolar Fields

• Reduces Fringing Field Effects

• Significantly Improves Resolution

• Improves Transmission

• Improves Peak Shapes

TSQ Quantum HyperQuads Technology

10

Resolution Performance of HyperQuads

0.7u FWHM

2.6e6

0.1u FWHM

1.2 e6

11

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


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


NL:1.86E4

QFTPPVQAAYQK +H +H2 O: C 64 H98 N16 O18


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

12

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

13

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

15

Deconvoluted spectrum of Normal Hb

15125.8

15866.8

15100 15200 15300 15400 15500 15600 15700 15800 15900Mass, Da

16

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

17

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


NL:1.04E8

Base Peak m/z= 150.00-1450.00 MS 06May_Digests_L02

Normal

Chain -1Da

18

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


NL:8.57E7

Base Peak m/z= 150.00-1450.00 MS 06May_Digests_L02

Normal

Chain -1Da

19

[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

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


Normal

Chain -1Da

22

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

23

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

24

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


NL:1.30E8


25

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


NL:7.00E7


NL:1.00E8


NL:1.00E8

m/z= 771.20-771.60+1156.40-1156.80 MS 06May_Digests_L08

NL:4.00E8


[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


NL:6.71E7


NL:9.07E7


NL:1.15E8


NL:4.38E8


[M+2H] 2+ 423.25

Hb + 14 Da

[M+2H] 2+ 359.2

[M+3H] 3+ 771.4

T9 [M+4H] 4+ 749.86

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

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

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

29

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

30

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


NL: 5.96E8

m/z= 600.00-600.20+749.80-750.00+999.40-999.60 MS 06May_Digests_L06

NL: 3.23E8


NL: 7.62E7


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


NL: 5.10E8


NL: 2.68E8


NL: 4.77E7


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

31

When intact mass is inconclusive

• Q-Iran results in +22 Da shift in chain



• [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

32

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


NL:6.72E3

VADALTNAVAHVDHMPNALSALSDLHAHK +H +H2 O: C 130 H211 N40 O41 S 1


Jeddah T9

Q-Iran T9

33

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

34

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


NL:5.96E8


NL:7.62E7


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


NL:3.77E8


NL:6.82E7


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


NL:2.03E9


NL:1.56E8


Normal TIC

Normal T9

35

Need for MS/MS

• Sequence of Normal T9

• VADALTNAVAHVDDMPNALSALSDLHAHK

• Sequence of Q-India T9

• VAHALTNAVAHVDDMPNALSALSDLHAHK

• Sequence of Q-Thailand T9

• VADALTNAVAHVHDMPNALSALSDLHAHK



• Sequence of possible variant T9

• VADALTNAVAHVDDMPNALSALSHLHAHK

36

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


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


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

37

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]

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