LC-MS from bench to bedside. William J. Griffiths Swansea University

Transcription

1 LC-MS from bench to bedside William J. Griffiths Swansea University

2 7-Dehydrocholesterol Vitamin D3 Cholesterol xysterol Steroid hormones Bile Acids

3 Steroidomics LC MS MS LC-ESI-MS/MS

4 7-Dehydrocholesterol Vitamin D3 Cholesterol xysterol Steroid hormones Bile Acids

5 Enzyme-Assisted Derivatisation for Sterol Analysis (EADSA) Cholesterol xidase 2 GP ydrazine Specificity for 3β- Enhance sensitivity Griffiths et al, RCM Shackleton et al, Steroids Smith & ooks, J Chromatogr Girard & Sandulesco, elv Chim Acta

6 Enzyme-Assisted Derivatisation for Sterol Analysis (EADSA) SD 2 GP ydrazine Specificity for 3α- Enhance sensitivity Griffiths et al, RCM Shackleton et al, Steroids Smith & ooks, J Chromatogr Girard & Sandulesco, elv Chim Acta

7 Plasma/CSF

8 EADSA ain/csf/plasma Et 70% Et A Cholesterol oxidase (in phosphate buffer, 37 o C, 1 hr) 70% Me C 18 SPE1 GP hydrazine (in 70% Me, 5%Ac, RT, 8 hr) SPE1-Fr-1, 70% Et Dry, reconstitute in ipr B 70% Me Recycling Me 70% 17% C 18 SPE2 70% Me, 5% Ac 2 x 1 ml Me LC-MS n

9 LTQ rbitrap peration Principle 1. Ions are stored in the Linear Trap 2.. are axially ejected 3.. and trapped in the C-trap 4.. they are squeezed into a small cloud and injected into the rbitrap 5.. where they are electrostatically trapped, while rotating around the central electrode and performing axial oscillation The oscillating ions induce an image current into the two outer halves of the orbitrap, which can be detected using a differential amplifier Ions of only one mass generate a sine wave signal 9

10 ealthy Adult C Exact Mass: CTX xysterol 7α ydroxylase Deficiency

11 plasma ohchd6_091027_fr1e-fr /12/ :38:19 60% Me (1.5sample +120)0.1%FA plasma ohchd6_091027_fr1e-fr1+fr2_04 #636 RT: AV: 1 L: 1.69E3 F: ITMS + c ESI d Full ms @cid30.00 [ ] 95 MS 2 : 534 [M-79] %RA C Exact Mass: [M-107] m/z m/z

12 plasma ohchd6_091027_fr1e-fr /12/ :19:19 60% Me (1.5sample +120)0.1%FA plasma ohchd6_091027_fr1e-fr1+fr2_06 #452 RT: 7.71 AV: 1 L: 3.95E2 F: ITMS + c ESI Full ms @cid @cid35.00 [ ] MS 3 : min '*f - - '*e [M-97] %RA *b 3 -C 2 4 *b *b [M-79] + *b 3 -C 2 4 *b 1-12 *b 2 *e 325 [M-107] + *f m/z m/z

13 plasma ohchd6_091027_fr1e-fr /12/ :19:19 60% Me (1.5sample +120)0.1%FA plasma ohchd6_091027_fr1e-fr1+fr2_06 #464 RT: 7.92 AV: 1 L: 3.64E2 F: ITMS + c ESI Full ms @cid @cid35.00 [ ] MS 3 : min '*f '*h '*e [M-97] %RA [M-79] + *b 3 -C 2 4 *b 1-12 *b 2 *b 3 -C 2 4 [M-107] + *b 1-12 *h 163 *f m/z m/z

14 plasma ohchd6_091027_fr1e-fr /12/ :19:19 60% Me (1.5sample +120)0.1%FA plasma ohchd6_091027_fr1e-fr1+fr2_06 #485 RT: 8.28 AV: 1 L: 4.54E2 F: ITMS + c ESI Full ms @cid @cid35.00 [ ] MS 3 : min '*f - - '*e [M-107] *b 3 -C 2 4 %RA *b [M-79] + *b 1-12 *b 2 *b 3 -C 2 4 [M-97] *b *e 2 *f m/z m/z

15 plasma ohchd6_091027_fr1e-fr /12/ :19:19 60% Me (1.5sample +120)0.1%FA plasma ohchd6_091027_fr1e-fr1+fr2_06 #629 RT: AV: 1 L: 1.77E2 F: ITMS + c ESI Full ms @cid @cid35.00 [ ] MS 3 : min *c [M-97] %RA *b *b 3 -C *b 1-12 *b 2 *c [M-79] + *b 3 -C [M-107] m/z m/z

16 Griffiths WJ, et al. Free Radic Biol Med Jul 27.

17 7α-ydroxycholesterol & 7α-ydroxycholest-4-en-3-one Cholesterol xidase 2 GP ydrazine Both present in plasma

18 EADSA ain/csf/plasma Et 70% Et A Cholesterol oxidase (in phosphate buffer, 37 o C, 1 hr) 70% Me C 18 SPE1 GP hydrazine (in 70% Me, 5%Ac, RT, 8 hr) SPE1-Fr-1, 70% Et Dry, reconstitute in ipr B 70% Me A = 3β + 3-oxo B = 3-oxo A-B = 3β Recycling Me 70% 17% 70% Me, 5% Ac C 18 SPE2 2 x 1 ml Me LC-MS n

19 Stable Isotope Label Analyse A and B in a single LC-MS/MS run %RA M + 5 Da m/z M + D C DC DC 2 2

20 EADSA ain/csf/plasma Et 70% Et C 18 SPE1 A SPE1-Fr-1, 70% Et Dry, reconstitute in ipr Cholesterol oxidase (in phosphate buffer, 37 o C, 1 hr) B D C DC DC 2 [ 2 5 ]GP hydrazine 70% Me [ 2 0 ]GP hydrazine 2 C 18 SPE2 C 18 SPE2 M + A = 3β + 3-oxo B = 3-oxo A-B = 3β LC-MS n %RA M + 5 Da m/z

21 PC_plasma_unknown_pclayton_Fr1a_GPd5_... 11/04/ :12:55 RT: RIC: ± 5 ppm L: 2.07E4 m/z= F: FTMS 90 + p ESI Full ms [ ] MS ICIS 80 PC_plasma_unknown_pclayton_Fr 1a_GPd5_Fr1b_GPd0_20uL_ A = _01ng/mL 60 %RA %RA RIC: ± 5 ppm C D C DC C D Exact Mass: Time (min) C Exact Mass: A = 3β + 3-oxo B = 3-oxo A-B = 3β L: 2.07E4 m/z= F: FTMS + p ESI Full ms [ ] MS ICIS PC_plasma_unknown_pclayton_Fr 1a_GPd5_Fr1b_GPd0_20uL_ _01 B = 0.00 ng/ml

22 PC_plasma_unknown_pclayton_Fr1a_GPd5_... 11/04/ :12:55 RT: RIC: ± 5 ppm L: 3.61E4 m/z= F: 90 FTMS + p ESI Full ms 80 [ ] MS PC_plasma_unknown_pclayton_F r1a_gpd5_fr1b_gpd0_20ul_ _01 60 %RA %RA RIC: ± 5 ppm Time (min) D C DC C D Exact Mass: C Exact Mass: A = ng/ml A = 3β + 3-oxo B = 3-oxo L: 3.61E4 m/z= F: A-B FTMS = + p 3β ESI Full ms [ ] MS PC_plasma_unknown_pclayton_F r1a_gpd5_fr1b_gpd0_20ul_ _01 B = ng/ml A-B = ng/ml

23 D C DC DC + Et 1) Et, 2) 2-2 * * 2-2 (GP) 2 * * Et D C DC DC * * * Et

24 differential mass tags MS 5 D C DC DC (i) (ii) 2 2 * * (iii) * * (iv) 2 2 D C DC DC * *(v) D C DC DC * * (vi) 2 2 isobaric mass tags MS 0 MS/MS: 1 differential mass tags MS 5

25 Steroid M M + M + Patient Sample + (A) %RA + Patient Sample + (B) + MS 5 Da SRM + (A) + time m/z SRM + (B) [M-Py] + MS/MS %RA [M-Py] Da M + MS/MS -79 Da %RA [M-Py] + [M-Py] Da M Da

26 differential mass tags MS 5 D C DC DC (i) (ii) 2 2 * * (iii) * * (iv) 2 2 D C DC DC * *(v) D C DC DC * * (vi) 2 2 isobaric mass tags MS 0 MS/MS: 1 differential mass tags MS 5

27 7-Dehydrocholesterol Vitamin D 3 25-ydroxyvitamin D 3 Kandutsch-Russell Cholesterol xysterol Steroid hormones Bile Acids

28 h (S) (Z) (Z) 7-Dehydrocholesterol (Z) (E) (Z) Pre-vitamin D 3 CYP (E) (E) 25-ydroxyvitamin D 3 (Z) Cholesterol oxidase (Z) (E) D C GP (S) DC C D Exact Mass:

29 MS 3 : [M + ] [M-Py- 2 ] + (E) (E) PC_Plasma_ovartis_Batch1_K083871_1of... 22/08/ :23:31 (Z) PC_Plasma_ovartis_Batch1_K083871_1of90_Fr1a-GPd5_Fr1b-GPd0_130822_02 #1844 RT: 8.73 AV: 1 L: 1.85E3 F: ITMS + c ESI Full ms @cid @cid35.00 [ ] (E) (Z) C Exact Mass: D C DC C D Exact Mass: (Z) 189 C Exact Mass: m/z 25-D 3 : 4 12 ng/ml

30 Advantages & Disadvantages Enhance solubility (RP solvent) Enhance sensitivity (ESI) Specific MS 2 (-79) (GP-specific) Informative MS 3 (differentiate isomers) Quantitative Enzyme catalysis (purity, activity) Chemical derivatisation (remove excess) Side reactions (epoxide hydrolysis) Syn/anti conformers (dilute signal) Laborious Complementary to conventional LC-MS/MS and GC-MS

31 Y Wang Thermo Scientific Stockholm EPSRC P. Crick G. Woffendin J. Sjövall BBSRC A. Meljon M. ornshaw I. Mathews UCL T. Bentley P. Clayton

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