Identification of Haemoglobinopathies by LC/ Mark Harrison; Senior Scientist, ThermoFisher Scientific Sarah Battle; Senior Biomedical Scientist, Royal Hallamshire Hospital
Introduction There are over 2 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 2
Introduction HPLC// 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 laboratory. Requires expert tuning and operation of the Data interpretation is manual, time consuming Here we present some preliminary work using HPLC// Aim for non-expert operators Use automated tuning procedures Batch analysis Chromatographic retention as additional identifier Software tools for data interpretation 3
Relative Abundance I nt ensit y x 1^6 Workflow of identifying variants J e d d a h 8 1 5 1 2 5. 8 1 5 1 4 8. 6 1 5 8 6 6. 8 IC -HPLC indicates presence of variant LC/ Intact Proteins 7 6 5 4 3 2 1 5 1 6 6. 2 1 5 1 9. 9 1 5 8 4 6. 5 Variant in alpha or beta chain. Mass Change of variant 1 5 8 8 8. 3 1 5 1 9. 8 1 5 7 8. 3 1 5 1 3 8. 2 15857 1 5 9 2. 145 9 1 2. 4 1 5 9 8. 8 15181 15 152 153 154 155 156 157 158 159 M ass, Da RT:. - 3. SM: 3B 95 9 15.27 1.3E8 Base Peak 6May_Dig ests_l8 85 8 75 7 1.76 8.75 14.55 Perform tryptic digest 65 6 55 5 45 4 1.27 12.23 12.95 17.63 15.67 Look up mass shift in tables 35 3 5.81 4.96 25.85 25.9 2 17.32 19.11 21.56 15 1.7 25.62 1 5 2.77 3.89 7.9 19.42 22.68 24.69 26.92 27.95 2 4 6 8 1 12 14 16 18 2 22 24 26 28 Time (min) Examine chromatograms of tryptic fragments Can variant be assigned unambiguously? Process / data Perform Specific / experiment Can variant be assigned unambiguously? YES YES 4
Sample Preparation Stock solution 1ul blood diluted to 5ul with water Intact analysis 2 ul Stock Solution diluted to 2ul with water Protein digests ul stock solution was denatured with 1ul 1% Formic acid and 1ul 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 37 o C for 1 hour Dilute 2ul to 2ul with water B.N. Green et. al. Rapid Identification of Hemoglobin variants by Electrospray Ionization Mass Spectrometry, Blood Cell, Molecules and Diseases (21) 27(3) 5
LC/ Methods HPLC Conditions Column BioBasic-4 *1 mm 5µm Mobile Phase A: water.1% Formic Acid Mobile Phase B: Acetonitrile.1% Formic Acid 3 min gradient Intact proteins Full scan Peakwidth.2µ FWHM 65-12 m/z Digests Full scan Peakwidth.2µ FWHM 15-145 m/z 6
Mass Spectrometry TSQ Quantum Ultra Triple Quadrupole Accela UHPLC Open Accela AS 7
Triple Stage Quadrupoles API Source Ion Optics Q2 -rf only Collision Cell Q1 Q2 Q3 rf/dc mass analysing quadrupoles Detection System 8
TSQ Quantum HyperQuads Technology Why use Hyperbolic rods? Forms Pure Quadrupolar Fields Reduces Fringing Field Effects Significantly Improves Resolution Improves Transmission Improves Peak Shapes 9
Resolution Performance of HyperQuads.7u FWHM 2.6e6.1u FWHM 1.2 e6 1
Relative Abundance Use of high resolution Unit resolution.7µ FWHM High resolution.2µ FWHM 8 6 4 Normal T13 [M+2H] 2+ 69.27 8 6 4 2.41E4 EFTPPVQAAYQK +H +H 2 O: C 64 H 97 N 15 O 19 p (gss, s /p:4) Chrg 2 R:.7 Da @FWHM 689.85 Normal T13 [M+2H] 2+ 69.35 N 1 E C p R 2 8 6 4 D-Punjab T13 [M+2H] 2+ 689.78 2 8 6 4 2.41E4 QFTPPVQAAYQK +H +H 2 O: C 64 H 98 N 16 O 18 p (gss, s /p:4) Chrg 2 R:.7 Da @FWHM 689.36 689.86 69.86 691.36 D-Punjab T13 [M+2H] 2+ N 1 Q C p R 2 8 6 69.3 Mixture [M+2H] 2+ 2 8 6 2.41E4 689.85 69.37 69.87 EFTPPVQAAYQK *1.+ QFTPPVQAAYQK *1. +H +H 2 O: p (gss, s /p:4) Chrg 2 R:.7 Da @FWHM 69.36 Mixture [M+2H] 2+ N 1 E Q s R 4 4 2 2 69.86 688 69 692 694 696 m/z 691.36 691.86 692.36 689 69 691 692 693 m/z 11 Simulation of isotope patterns
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 12
Spectrum of Normal Hb 19 7May_Intacts_7 #277-282 RT: 23.32-23.74 AV: 6 SM: 3B 6.62E7 T: + p ESI Q1 [65.-12.] 2 797.13 757.27 18 841.34 95 9 85 8 75 21 721.35 17 89.78 7 65 6 55 5 45 4 35 3 25 658.66 688.54 2 19 836.14 18 882.5 17 934.37 16 946.37 16 992.63 15 9.38 15 158.84 14 2 181.42 15 1 5 862.24 898.6 955.49 147.4 1134.33 1164.55 114.7 119.71 7 75 8 85 9 95 15 1 115 12 m/z 13
Deconvoluted spectrum of Normal Hb 15125.8 15866.8 15 152 153 154 155 156 157 158 159 Mass, Da 15
Intensity x 1^9 Intensity x 1^9 Example 1 D-Punjab 2.5 15125.8 α 15126 Da Normal Haemoglobin 2. 1.5 15867 Da 15866.8 1..5. 2. 1.8 15167.1 1519.8 1578.3 15146.9 15796.5 15888.2 15176.6 D-Punjab 15 152 153 154 155 156 157 158 159 15125.8 Mass, Da α 15126 Da 1.6 1.4 1.2 1..8 β -1 Da (15867 15866 Da) 15865.9.6.4.2 1519.4 15166.2 15146.9 15888.1. 15 152 153 154 155 156 157 158 159 Mass, Da 16
Example 1 RT:. - 26.69 SM: 3B 9 8 7 Normal 8.75 1.76 14.55 15.27 NL 1. Ba 6M sts 6 5 1.27 12.23 12.95 15.67 17.63 4 3 2.9 4.96 5.81 25.85 17.32 1 9 8 7 Chain -1Da 8.75 1.76 12.19 15.18 14.46 NL 1. Ba m/z 15 145 6M sts 6 17.63 5 4 3 2 4.96 5.76 1.27 9.73 12.9 15.63 17.37 19.6 25.8 1 2 4 6 8 1 12 14 16 18 2 22 24 26 Time (min) 17
Example 1 chromatogram RT: 8.1-12.5 9 8 SM: 3B Normal 8.75 1.76 12.23 7.25E7 Base Pea 6May_D sts_l8 7 6 5 9.69 1.27 4 3 2 1 9.15 11.25 9 8 7 6 5 Chain 8.75-1Da 9.73 1.27 1.76 12.19 8.57E7 Base Pea 15.- 145. 6May_D sts_l2 4 3 2 1 9.11 11.25 8.5 9. 9.5 1. 1.5 11. 11.5 12. 12.5 Time (min) 18
[M+2H] 2+ of extra peak at rt 1.6 6May_Digests_L2 #236-237 RT: 1.49-1.54 AV: 2 SM: 5B 1.82E7 T: + p ESI Q1 [15.-145.] 689.36 95 9 85 8 75 7 65 6 55 689.82.5 Da 5 45 4 35 69.36.5 Da 3 25 2 15 1 5 684 686 688 69 692 694 696 698 7 72 m/z 19
Example 1 Mass Chromatogram of 689.36 +.2µ RT:. - 3. SM: 3B 9 8 7 Normal 1.45E8 689.25-689.45 6May_Dig ests_l8 6 5 4 3 2 1 9 8 7 Chain -1Da 1.54 1.45E8 689.25-689.45 6May_Dig ests_l2 6 5 4 3 2 1 2 4 6 8 1 12 14 16 18 2 22 24 26 28 Time (min) 2
21
Table of chain -1Da shift Chain Pos T Frag Mutation Name Delta Mass Sequence / 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 / 129.5 73 9 Asp - Asn G-Accra -1 VLGAFSDGLAHLDNLK Y 557. / 835. 79 9 Asp - Asn Yaizu -1 VLGAFSDGLAHLDNLK Y 557. / 835. 9 1 Glu - Lys Agenogi -1 GTFATLSELHCDK N 412.7 / 824.5 & 34.1 / 615.3 94 1 Asp - Asn Bunbury -1 GTFATLSELHCDK N 474.2 / 71.9 99 11 Asp - Asn Kempsey -1 LHVDPENFR Y 563.3 / 1125.6 121 13 Glu-Gln D-Punjab -1 EFTPPVQAAYQK N 689.4 1.5 121 13 Glu - Lys O-Arab -1 EFTPPVQAAYQK N 625.3 1249.7 22
Intensity x 1^9 Intensity x 1^9 Example 2 G-Philadelphia 2.5 15125.8 α 15126 Da Normal Haemoglobin 2. 1.5 15867 Da 15866.8 1..5. 1.2 1.1 15167.1 1519.8 1578.3 15146.9 15796.5 15888.2 15176.6 G-Philadelphia 15 152 153 154 155 156 157 158 159 Mass, Da 15125.8 α 15126 Da 1..9.8.7 1514 Variant α 1514 Da 15867 Da 15866.8.6.5 +14 Da.4.3.2 1519.8 15166.1.1. 15178.6 15888.4 1598.1 15847.9 15 152 153 154 155 156 157 158 159 Mass, Da 23
Example 2 RT:. - 2.1 9 8 7 SM: 3B 8.75 1.76 14.55 15.27 1.3 Bas 6M ests 6 5 1.27 12.23 12.95 15.67 17.63 4 3 2 1 9 8 7 6 5 4 3 2 1 4.96 5.81 17.32.9 14.2 19.11 1.7 2.77 3.89 6.7 7.9 4.96 5.76 8.66 1.22 9.69 2 4 6 8 1 12 14 16 18 2 Time (min) 1.72 12.14 12.81 14.38 15.13 15.54 1.12 17.32 2.82 RT: 3.44 6.7 7.99 15.98 17.59 18.93 1.3 Bas 6M ests 24
Relative Abundance Example 2 RT:. - 2.44 SM: 3B 15.13 14.38 8 Hb + 14 Da 1.72 8.66 12.14 6 1.22 12.81 15.54 4 17.59 4.96 5.76 18.93 2 1.12 2.82 17.32 8 6 4 2 8 6 4 2 8 6 4 2 8 6 4 2 3.22 3.48 [M+2H] 2+ 423.25 [M+2H] 2+ 359.2 [M+3H] 3+ 771.4 T9 [M+4H] 4+ 749.86 2 4 6 8 1 12 14 16 18 2 Time (min) 14.29 17.59 19.2 RT:. - 2.11 2 9.7E7 8 359.-359.4 6 6May_Digests_L 4 4 2 SM: 3B 1.3E8 8 Normal Hb 15.27 Base Peak 6May_Digests_L 6 8.75 1.76 14.55 4 12.23 12.95 17.63 4 1.27 15.67 4.96 5.81 17.32 2.9 1.7 19.11 6.71E7 8 423.5-423.45 6 6May_Digests_L 4 4 1.15E8 8 771.2-771.6+ 1156.4-1156.8 6 6May_Digests_L 4 4 2 4.38E8 8 749.66-75.6+ 999.3-999.7 6 6May_Digests_L 4 4 2 [M+2H] 2+ 423.25 [M+2H] 2+ 359.2 [M+3H] 3+ 771.4 T9 [M+4H] 4+ 749.86 2 4 6 8 1 12 14 16 18 2 Time (min) 14.64 1.3E8 Base P 6May 8 7.E7 423.5 6May 8 1.E8 359. 6May 8 1.E8 771.2 1156.4 6May 8 4.E8 749.66 999.3 6May 8 25
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 26
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, can be inconclusive 27
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+ = 159.74, [M+2H+Na] 3+ = 6.8, [M+3H+Na] 4+ = 755.37 Sequence of Q-Iran T9 VADALTNAVAHVDHMPNALSALSDLHAHK [M+2H] 2+ = 159.75, [M+3H] 3+ = 6.8, [M+4H] 4+ = 755.37 28
Problem with infusion experiment SM: 5B 5.19 9 8 Normal Hb 999.85 7 6 999.51 5 4 3 2 1 99.55 992.56 993.56 996.32 111.6 1.55 2.53 9.34 112.52 113.53 999.88 9 8 Q-Iran Hb 7 999.55 6 5.22 5 4 3 2 1 1.57 99.55 2.57 7.22 9.5 112.55 992.57 111.55 996.45 993.57 114.54 99 995 5 11 115 m/z 29
Relative Abundance Extra confirmation of retention time RT: 11.75-2.1 8 6 4 2 SM: 3B Normal TIC 12.23 14.55 15.27 12.95 15.67 14.2 17.32 17.63 19.11 RT: 11.73-19.88 SM: 3B 1.3E8 Base Peak 6May_Digests_L8 8 12.19 6 12.9 Q-Iran TIC 4 14.6 2 14.42 15.13 15.58 17.59 17.28 19.2 16.7 Bas 6M 8 6 Normal T9 17.63 5.1E8 6.-6.2+ 749.8-75.+ 999.4-999.6 6May_Digests_L8 6 Normal T9 17.59 6 749 999 6M 4 4 2 14.51 2 14.33 8 6 4 Normal T8-9 17.32 2.68E8 625.8-626.+ 782.-782.2+ 8 142.1-142.3 6 6May_Digests_L8 4 Normal T8-9 17.28 625 782 14 6M 2 2 8 6 4 Normal T9 Na+ adduct 17.63 4.77E7 755.5-755.7+ 7.5-7.25 8 6May_Digests_L8 6 4 Q-Iran T9 16.7 Normal T9 Na+ 17.59 adduct 755 6M 2 2 12 13 14 15 16 17 18 19 2 Time (min) 12 13 14 15 16 17 18 19 Time (min) 3
When intact mass is inconclusive Q-Iran results in +22 Da shift in chain Sequence of Q-Iran T9 VADALTNAVAHVDHMPNALSALSDLHAHK [M+2H] 2+ = 159.75, [M+3H] 3+ = 6.8, [M+4H] 4+ = 755.37 Jeddah results in +23 Da shift in chain Sequence of Jeddah T9 VADALTHAVAHVDDMPNALSALSDLHAHK [M+2H] 2+ = 151.26, [M+3H] 3+ = 7.2, [M+4H] 4+ = 755.6 31
Problems with isotopes; simulation of [M+3H] 3+ 9 8 7 6 7.51 7.84 7.17 8.17 Jeddah T9 6.72E VAD SALS C 13 p (gs R:.2 5 4 3 8.51 2 1 8.84 9 8 7 7.17 7.51 Q-Iran T9 6.72E VAD SALS C 13 p (gs R:.2 6 7.84 5 4 3 8.18 2 1 8.51 8.85 9.52 7 8 9 11 111 112 m/z 32
Infusion SM: 5B 8 6 Normal 999.51 999.85 5.19 5.53 2.34 sam 6 R.1 AV: ESI 4 2 1.56 996.32 998.25 7.55 9.43 111.6 112.52 113.53 8 6 4 Jeddah 999.89 5.22 999.55 1.55 2.53 7.54 112.91 1.24 Sam 6-2 4.34 AV: ESI 2 996.51 997.53 111.58 114.53 8 6 Q-Iran 999.55 999.88 5.22 2.27 sam 9-34 1.87 AV: ESI 4 2 1.57 112.55 7.22 9.5 2.54 111.55 996.45 113.54 995 5 11 115 m/z 33
Relative Abundance Relative Abundance Extra confirmation of retention time RT: 11.89-19.97 SM: 3B 15.27 8 6 Normal 14.55 TIC 12.23 12.95 15.67 17.63 RT: 11.7-2.3 SM: 3B 15.13 1.3E8 Base Peak 14.42 6May_Digests_L 8 Q-Iran TIC 8 12.19 6 12.9 15.58 17.59 RT: 11.73-19.88 SM: 3B 15.18 1.38E8 Base Peak 14.38 6May_Digests_ Jeddah TIC 8 L6 12.14 6 12.81 15.58 4 4 4 17.63 2 14.2 17.32 19.11 2 14.6 17.28 19.2 16.7 2 14.2 19.6 15.94 17.32 8 6 4 Normal T9 17.63 2.3E9 599.6-6.6+ 8 749.4-75.4+ 999.-. 6 6May_Digests_L 8 4 Normal T9 17.59 5.96E8 6.-6.2+ 8 749.8-75.+ 999.4-999.6 6 6May_Digests_ L6 4 Normal T9 17.63 2 14.51 2 14.33 2 14.2 14.33 8 6 4 17.63 1.56E8 755.1-756.1+ 8 6.7-7.7 6May_Digests_L 6 8 4 Q-Iran T9 16.7 17.59 7.62E7 755.4-755.6+ 8 7.5-7.25 6May_Digests_ 6 L6 4 15.94 Jeddah T9 17.63 2 2 2 12 13 14 15 16 17 18 19 Time (min) 12 13 14 15 16 17 18 19 2 Time (min) 12 13 14 15 16 17 18 19 Time (min) 34
Need for / 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 35
Relative Abundance Need for / Riccarton T6 + 3Da RT: 9.78-2.67 SM: 3B 15.27 8 6 4 2 1.76 14.55 12.23 12.95 1.27 15.67 Normal TIC 17.63 11.25 17.32 14.2 19.11 RT: 9.33-2.15 SM: 3B 1.3E8 Base Peak 6May_Digests_L 1.63 8 8 6 4 2 9.55 1.13 12.5 12.77 13.93 14.29 15.9 15.54 17.5 11.16 17.19 18.93 15.94 Normal TIC 19.24 Bas 6M 7 8 6 RT: 12.95 Normal T6 1.64E9 458.73-459.73+ 611.48-612.48+ 8 916.95-917.95 Genesis 6May_Digests_L 6 8 RT: 12.72 Normal T6 458. 611. 916. Gen 6M 7 4 4 2 2 8 6 RT: 12.23 Riccarton T6 1.64E8 621.45-622.45+ 931.95-932.95 8 Genesis 6May_Digests_L 8 6 RT: 12.5 RT: 12.68 Riccarton T6 621. 931. Gen 6M 7 4 2 17.32 4 2 17.19 1 11 12 13 14 15 16 17 18 19 2 Time (min) 1 11 12 13 14 15 16 17 18 19 2 Time (min) 36
/ of multiply charged ions: confirmation of sequence 5B 237. 265. 612.1 711.8 442.6 524. 11. 363.5 474.9 589.2 663.2 785.2 237.1 622.5 726.7 265.1 533.9 452.6 484.8 678.2 8.9 12. 378.7 588.2 81.2 2 3 4 5 6 7 8 9 m/z 37
Conclusions Ability to unequivocally identify Hb variants No special tuning knowledge needed Having LC retention gives extra confidence May eliminate the need for / in some cases Future work Investigate use of metabolomics software tools Automatically identify changing peaks Run further pilot studies Implement at Sheffield Northern General 38