Identification of Haemoglobinopathies by LC/MS

Transcription

1 Identification of Haemoglobinopathies by LC/ Mark Harrison; Senior Scientist, ThermoFisher Scientific Sarah Battle; Senior Biomedical Scientist, Royal Hallamshire Hospital

2 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

3 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

4 Relative Abundance I nt ensit y x 1^6 Workflow of identifying variants J e d d a h IC -HPLC indicates presence of variant LC/ Intact Proteins Variant in alpha or beta chain. Mass Change of variant M ass, Da RT: SM: 3B E8 Base Peak 6May_Dig ests_l Perform tryptic digest Look up mass shift in tables 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

5 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

6 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 m/z Digests Full scan Peakwidth.2µ FWHM m/z 6

7 Mass Spectrometry TSQ Quantum Ultra Triple Quadrupole Accela UHPLC Open Accela AS 7

8 Triple Stage Quadrupoles API Source Ion Optics Q2 -rf only Collision Cell Q1 Q2 Q3 rf/dc mass analysing quadrupoles Detection System 8

9 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

10 Resolution Performance of HyperQuads.7u FWHM 2.6e6.1u FWHM 1.2 e6 1

11 Relative Abundance Use of high resolution Unit resolution.7µ FWHM High resolution.2µ FWHM Normal T13 [M+2H] E4 EFTPPVQAAYQK +H +H 2 O: C 64 H 97 N 15 O 19 p (gss, s /p:4) Chrg 2 R: Normal T13 [M+2H] N 1 E C p R D-Punjab T13 [M+2H] E4 QFTPPVQAAYQK +H +H 2 O: C 64 H 98 N 16 O 18 p (gss, s /p:4) Chrg 2 R: D-Punjab T13 [M+2H] 2+ N 1 Q C p R Mixture [M+2H] E EFTPPVQAAYQK *1.+ QFTPPVQAAYQK *1. +H +H 2 O: p (gss, s /p:4) Chrg 2 R: Mixture [M+2H] 2+ N 1 E Q s R m/z m/z 11 Simulation of isotope patterns

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 12

13 Spectrum of Normal Hb 19 7May_Intacts_7 # RT: AV: 6 SM: 3B 6.62E7 T: + p ESI Q1 [ ] m/z 13

14 Deconvoluted spectrum of Normal Hb Mass, Da 15

15 Intensity x 1^9 Intensity x 1^9 Example 1 D-Punjab α Da Normal Haemoglobin Da D-Punjab Mass, Da α Da β -1 Da ( Da) Mass, Da 16

16 Example 1 RT: SM: 3B Normal NL 1. Ba 6M sts Chain -1Da NL 1. Ba m/z M sts Time (min) 17

17 Example 1 chromatogram RT: SM: 3B Normal E7 Base Pea 6May_D sts_l Chain Da E7 Base Pea May_D sts_l Time (min) 18

18 [M+2H] 2+ of extra peak at rt 1.6 6May_Digests_L2 # RT: AV: 2 SM: 5B 1.82E7 T: + p ESI Q1 [ ] Da Da m/z 19

19 Example 1 Mass Chromatogram of µ RT: SM: 3B Normal 1.45E May_Dig ests_l Chain -1Da E May_Dig ests_l Time (min) 2

20 21

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 / Glu - Lys G-Siriraj -1 VHLTPEEK Y / Glu -Gln D-Iran -1 VNVDEVGGEALGR Y / Glu-Lys E-Saskatoon -1 VNVDEVGGEALGR Y & 379.7/ Glu-Gln Novel -1 VNVDEVGGEALGR Y / Glu - Lys E -1 VNVDEVGGEALGR N / & & Glu - Lys Hornchurch -1 FFESFGDLSTPDAVMGNPK N & / Asp - Asn Osu -1 FFESFGDLSTPDAVMGNPK Y / Asp - Asn G-Accra -1 VLGAFSDGLAHLDNLK Y 557. / Asp - Asn Yaizu -1 VLGAFSDGLAHLDNLK Y 557. / Glu - Lys Agenogi -1 GTFATLSELHCDK N / & 34.1 / Asp - Asn Bunbury -1 GTFATLSELHCDK N / Asp - Asn Kempsey -1 LHVDPENFR Y / Glu-Gln D-Punjab -1 EFTPPVQAAYQK N Glu - Lys O-Arab -1 EFTPPVQAAYQK N

22 Intensity x 1^9 Intensity x 1^9 Example 2 G-Philadelphia α Da Normal Haemoglobin Da G-Philadelphia Mass, Da α Da Variant α 1514 Da Da Da Mass, Da 23

23 Example 2 RT: SM: 3B Bas 6M ests Time (min) RT: Bas 6M ests 24

24 Relative Abundance Example 2 RT: SM: 3B Hb + 14 Da [M+2H] [M+2H] [M+3H] T9 [M+4H] Time (min) RT: E May_Digests_L SM: 3B 1.3E8 8 Normal Hb Base Peak 6May_Digests_L E May_Digests_L E May_Digests_L E May_Digests_L [M+2H] [M+2H] [M+3H] T9 [M+4H] Time (min) E8 Base P 6May 8 7.E May 8 1.E May 8 1.E May 8 4.E May 8 25

25 Example 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

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

27 Difference of 22Da Sequence of Normal T9 VADALTNAVAHVDDMPNALSALSDLHAHK [M+2H] 2+ = , [M+3H] 3+ = 999.5, [M+4H] 4+ = [M+H+Na] 2+ = , [M+2H+Na] 3+ = 6.8, [M+3H+Na] 4+ = Sequence of Q-Iran T9 VADALTNAVAHVDHMPNALSALSDLHAHK [M+2H] 2+ = , [M+3H] 3+ = 6.8, [M+4H] 4+ =

28 Problem with infusion experiment SM: 5B Normal Hb Q-Iran Hb m/z 29

29 Relative Abundance Extra confirmation of retention time RT: SM: 3B Normal TIC RT: SM: 3B 1.3E8 Base Peak 6May_Digests_L Q-Iran TIC Bas 6M 8 6 Normal T E May_Digests_L8 6 Normal T M Normal T E May_Digests_L8 4 Normal T M Normal T9 Na+ adduct E May_Digests_L8 6 4 Q-Iran T Normal T9 Na adduct 755 6M Time (min) Time (min) 3

30 When intact mass is inconclusive Q-Iran results in +22 Da shift in chain Sequence of Q-Iran T9 VADALTNAVAHVDHMPNALSALSDLHAHK [M+2H] 2+ = , [M+3H] 3+ = 6.8, [M+4H] 4+ = Jeddah results in +23 Da shift in chain Sequence of Jeddah T9 VADALTHAVAHVDDMPNALSALSDLHAHK [M+2H] 2+ = , [M+3H] 3+ = 7.2, [M+4H] 4+ =

31 Problems with isotopes; simulation of [M+3H] Jeddah T9 6.72E VAD SALS C 13 p (gs R: Q-Iran T9 6.72E VAD SALS C 13 p (gs R: m/z 32

32 Infusion SM: 5B 8 6 Normal sam 6 R.1 AV: ESI Jeddah Sam AV: ESI Q-Iran sam AV: ESI m/z 33

33 Relative Abundance Relative Abundance Extra confirmation of retention time RT: SM: 3B Normal TIC RT: SM: 3B E8 Base Peak May_Digests_L 8 Q-Iran TIC RT: SM: 3B E8 Base Peak May_Digests_ Jeddah TIC 8 L Normal T E May_Digests_L 8 4 Normal T E May_Digests_ L6 4 Normal T E May_Digests_L Q-Iran T E May_Digests_ 6 L Jeddah T Time (min) Time (min) Time (min) 34

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

35 Relative Abundance Need for / Riccarton T6 + 3Da RT: SM: 3B Normal TIC RT: SM: 3B 1.3E8 Base Peak 6May_Digests_L Normal TIC Bas 6M RT: Normal T6 1.64E Genesis 6May_Digests_L 6 8 RT: Normal T Gen 6M RT: Riccarton T6 1.64E Genesis 6May_Digests_L 8 6 RT: 12.5 RT: Riccarton T Gen 6M Time (min) Time (min) 36

36 / of multiply charged ions: confirmation of sequence 5B m/z 37

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