Post on 12-Jan-2016
Recent advances in cytokinin Recent advances in cytokinin analysisanalysis
Karel Doležal
Acquity UPLC - Xevo TQ MSAcquity UPLC - Xevo TQ MS
Analysis of Cytokinins (~ 50–100 mg FW)
• Chromatographic separation of 21 Cks and 11 cytokinin O-glucosides and ribotides
• Determination of 57 CK metabolites after enzymatic cleavage.
• LOD for most of the cytokinins analyzed on attomolar level (100 amol)
• Calibration linearity range:
500 amol – 100pmol (R2 ~0.999)
Analysis of Cytokinins (~ 50–100 mg FW)
• Sample preparation– Extraction: Bieleski buffer– SPE purification: ion-exchange and reverse
phase chromatography followed by IAC using monoclonal antibodies
• UPLC-MS/MS analysis– 8-min linear gradient of MeOH / 15 mM
HCOONH4– column: Acquity UPLC® BEH C18 2.1x50 mm,
1.7µm – detection: MRM mode – PIC mode: MS/MS full scan data collection
Analysis of Cytokinins (~ 50–100 mg FW)
Recent projects and goals in cytokinin analysisRecent projects and goals in cytokinin analysis
1. Isolation, identification and quantification of cytokinin nucleotides by high performance liquid chromatography and capillary electrophoresis
2. Miniaturization and simplification of extraction method
- stage tips for SPE - immunoaffinity chromatography using magnetic
nanoparticles
Metabolism of cytokininsMetabolism of cytokinins
Sakakibara, H. Annu. Rev. Plant Biol. (2006).
N
NN
N
NH
O
OHOH
HH
HH
OPHO
OH
O
n
R
R n Compound name Abbreviation
CH2
CH3
CH3
1
2
3
N6-Isopentenyladenosine-5´-monophosphate
N6-Isopentenyladenosine-5´-diphosphate
N6-Isopentenyladenosine-5´-triphosphate
iPMP
iPDP
iPTP
CH2
H2C
CH3
OH
1
2
3
trans-Zeatin riboside-5´-monophosphate
trans-Zeatin riboside-5´-diphosphate
trans-Zeatin riboside-5´-triphosphate
tZRMP
tZRDP
tZRTP
CH2 H2C
CH3
OH
1
2
3
cis-Zeatin riboside-5´-monophosphate
cis-Zeatin riboside-5´-diphosphate
cis-Zeatin riboside-5´-triphosphate
cZRMP
cZRDP
cZRTP
Béres T. et al. (2010) Anal Bioanal Chem 398 2071-2080
Reconstructed SIM chromatograms of CK nucleotides used in this study under optimal chromatographic conditions.
MS/MS spectra and fragmentation patterns obtained for the putative intracellular metabolites of iPR (A) iPMP (B) iPDP (C) iPTP extracted from treated cells and the standard solutions of iPMP (D), iPDP (E) and iPTP (F).
Kowalska et al. (2010) Phytochemistry 71 1970-1978
Depletion of cytokinin phosphates from dehydrogenase reaction of (A) AtCKX1 (11 μg), (B) AtCKX3 (4 μg) and (C) AtCKX7 (12 μg). The decrease in concentration of initial 100 μM solution of each cytokinin in 50 mM MES/Tris buffer pH 5.0 in the presence of 500 μM Q0 was followed by capillary electrophoresis.
Béres T. & Gemrotová M. et al. (in preparation)
Fig. 1 Separation of six nucleotide standards a) iPMP, b) AMP, c) iPDP, d) iPTP, e) ADP, f) ATP, under optimal conditions.
Fig. 2 UV spectra based reaction
substrate/product identification.
Fig. 4 The increase of iPDP concentration in time. The enzyme was concentrated 10-fold, first time-point taken after 5 min after reaction initiation.
Fig. 3 Electrophoretograms of the AtIPT1 (20-fold concentrated) catalyzed reaction (A) and the control reaction (B) obtained by measuring the reaction products, stopped 30 min after initiation.
MiniaturarizationMiniaturarization
Main goals:Main goals:
• More efficient and simple extraction and purification
• Efficient separation – UPLC
• Higher sensitity – ESI-MS/MS
• Shorter time of analysisShorter time of analysis
StageTip purificationStageTip purification (STop-And-Go-microExtraction)(STop-And-Go-microExtraction)
Rappsilber et al. (2008) Nature Protocols 2: 1896-1906.
Single StageTipsSingle StageTips
• poly-tetrafluoroethylene matrices (PTFE)
• C18 or C8 bound on silikagel or modified poly(styrene-divinylbenzen) (SDB)
• Ion-exchange sorbents - Cation-SR disk (sulfonyl groups) or Anion-SR disk (quaternary ammonium bases)
recovery 3H-CKs (%) n=4
C18 SDB-RPS Cation-SR
Load Elution Load Elution Load Elution
[3H]cZ 98.8 ± 3.3 0.6 ± 0.2 1.3 ± 0.1 96.6 ± 1.8 1.1 ± 0.1 100.7 ± 3.0
[3H]tZR 98.8 ± 4.4 0.6 ± 0.2 39.2 ± 2.2 29.8 ± 2.2 22.4 ± 0.6 64.7 ± 1.1
[3H]iPR 102.9 ± 3.5 0.5 ± 0.3 4.9 ± 0.2 95.6 ± 1.4 4.7 ± 0.1 97.0 ± 2.8
Multi StageTipsMulti StageTips
C18/SDB-RPS C18/Cation-SR
C18/SDB-RPS/Cation-SR
Purification protocolPurification protocol
• Quantification
in 1-5mg FW
• Sample volume 50 µl
• Combination of C18
and ion-exchange
chromatography
• Sorbent with high
capacity
• 1-5mg FW = lower
matrice effect, higher
purrification efficiency
Separation of CK metabolitesSeparation of CK metabolites
Acquity UPLC® BEH C18, 1.7 µm, 2.1 × 150 mm
Gradient: methanol (A) a 15 mM HCOONH4 pH 4.0 (B)
– 0-7 min, 5:95 (A:B); 7-16 min, 5:95 –> 20:80;
16-24 min, 20:80 –> 50:50;
– flow 0.25 ml min-1, column temperature: 40 ºC
v
v
vv
v
v
vv
v
Compounds Precursors Products
t/cZ (R1=H, R2=H) 220.1 136.1
t/cZR (R1=Rib, R2=H) 352.2 220.1, 136.1
t/cZ3/7/9G (R1=Glc, R2=H) 382.2 220.1, 136.1
t/cZOG (R1=H, R2=Glc) 382.2 220.1, 136.1
t/cZROG (R1=Rib, R2=Glc)
432.2 382.2, 220.1, 136.1
t/cZRMP (R1=Rib+MP, R2=H)
514.2 220.1, 136.1
Limits of detection and dynamic rangeLimits of detection and dynamic range
CKs Ret. time stability (min) LOD (fmol) Dynam. range (mol) R2
t/cZ 15.39 ± 0.04 / 16.82 ± 0.03 0.5 1x10-15-5x10-11 0.9989 / 0.9987
t/cZR 19.06 ± 0.02 / 19.72 ± 0.01 0.1 5x10-16-5x10-11 0.9993 / 0.9986
tZ7G 12.26 ± 0.02 0.1 5x10-16-1x10-11 0.9989
t/cZ9G 14.23 ± 0.02 / 15.13 ± 0.02 0.1 5x10-16-5x10-11 0.9993 / 0.9985
t/cZOG 14.83 ± 0.03 / 15.79 ± 0.03 0.5 1x10-16-1x10-11 0.9987 / 0.9988
t/cZROG 18.08 ± 0.02 / 18.77 ± 0.02 1.0 5x10-15-1x10-11 0.9992 / 0.9984
t/cZMP 13.72 ± 0.02 / 14.67 ± 0.02 5.0 1x10-14-5x10-11 0.9990 / 0.9985
DHZ 16.15 ± 0.04 0.1 5x10-16-1x10-11 0.9991
DHZR 19.61 ± 0.01 0.05 1x10-16-5x10-10 0.9989
DHZ7G 13.80 ± 0.02 / 14.13 ± 0.02 0.1 5x10-16-1x10-10 0.9994
DHZ9G 15.00 ± 0.01 0.05 1x10-16-1x10-10 0.9992
DHZOG 16.37 ± 0.03 0.1 5x10-16-5x10-12 0.9992
DHZROG 19.22 ± 0.03 1.0 5x10-15-1x10-11 0.9983
DHZMP 14.34 ± 0.01 1.0 5x10-15-1x10-11 0.9992
iP 23.21 ± 0.01 0.1 5x10-16-1x10-11 0.9991
iPR 23.88 ± 0.01 0.05 1x10-16-1x10-11 0.9989
iP7G 18.70 ± 0.01 0.05 1x10-16-1x10-11 0.9988
iP9G 21.50 ± 0.01 0.5 1x10-15-1x10-11 0.9992
iPMP 21.31 ± 0.02 5.0 1x10-14-5x10-11 0.9993
Method validationMethod validation
CKs
Recovery (%)
1 pmol addedaccuracy
(%)precision
(%)1 mg 2 mg 5 mg
tZ 80 ± 10 63 ± 8 21 ± 4 0.99 ± 0.17 17.2 0.1tZR 72 ± 12 46 ± 7 8 ± 1 0.84 ± 0.09 10.4 16.1tZ7G 88 ± 6 57 ± 4 6 ± 1 0.91 ± 0.13 14.6 8.6tZ9G 59 ± 7 31 ± 3 8 ± 1 0.97 ± 0.09 9.6 2.5tZOG 85 ± 5 68 ± 6 11 ± 2 1.07 ± 0.19 18.1 -7.1tZROG 55 ± 4 30 ± 3 4 ± 1 0.82 ± 0.09 11.1 17.6tZMP 35 ± 6 11 ± 1 5 ± 1 0.85 ± 0.11 13.3 14.7cZ 75 ± 9 65 ± 5 24 ± 4 0.83 ± 0.02 2.5 17.0cZR 81 ± 13 44 ± 9 8 ± 1 0.96 ± 0.12 12.8 4.1cZ9G 74 ± 12 37 ± 5 5 ± 1 1.18 ± 0.13 11.3 -17.6cZOG 89 ± 6 66 ± 7 9 ± 2 1.09 ± 0.14 13.2 -9.1cZROG 52 ± 6 24 ± 2 3 ± 1 0.89 ± 0.11 12.9 11.2cZMP 32 ± 3 17 ± 1 2 ± 1 0.86 ± 0.15 17.9 13.9DHZ 77 ± 13 61 ± 7 20 ± 3 0.90 ± 0.10 10.8 9.7DHZR 88 ± 13 48 ± 8 12 ± 1 1.03 ± 0.06 5.6 -2.9DHZ7G 89 ± 3 65 ± 3 8 ± 2 1.18 ± 0.05 4.3 -18.2DHZ9G 78 ± 10 35 ± 6 6 ± 1 0.96 ± 0.07 7.2 3.7DHZOG 77 ± 5 50 ± 7 9 ± 3 1.16 ± 0.06 4.9 -15.8DHZROG 87 ± 8 42 ± 5 5 ± 1 0.90 ± 0.12 13.3 9.8DHZMP 37 ± 1 12 ± 1 3 ± 1 0.96 ± 0.15 15.8 3.6iP 76 ± 9 68 ± 3 26 ± 5 0.97 ± 0.06 5.9 3.3iPR 84 ± 8 53 ± 4 17 ± 1 1.13 ± 0.06 5.6 -12.8iP7G 83 ± 10 60 ± 5 7 ± 1 0.91 ± 0.15 16.5 9.4iP9G 74 ± 8 49 ± 8 8 ± 2 0.97 ± 0.08 8.0 3.1iPMP 78 ± 9 39 ± 9 9 ± 2 0.92 ± 0.16 17.0 8.4
isoprenoid cytokininsisoprenoid cytokinins (1-5mg FW) (1-5mg FW)
Xevo TQ MS
CKscytokinin levels (pmol g-1 FW)
Seedlings Shoots Roots
tZ n.d. n.d. 0.96 ± 0.12
tZR 2.23 ± 0.37 0.88 ± 0.32 3.27 ± 0.52
tZ7G 37.99 ± 2.81 23.49 ± 3.07 6.54 ± 0.75
tZ9G 3.88 ± 1.22 4.32 ± 1.31 2.08 ± 0.52
tZOG 9.25 ± 2.77 9.42 ± 1.75 7.10 ± 1.33
cZR 0.80 ± 0.16 1.14 ± 0.37 3.71 ± 0.79
cZ9G n.d. n.d. 2.02 ± 0.47
cZOG 0.90 ± 0.27 1.04 ± 0.33 2.89 ± 0.66
DHZR 0.64 ± 0.19 0.94 ± 0.27 0.97 ± 0.28
DHZ7G 5.48 ± 1.28 5.78 ± 1.51 2.07 ± 0.42
DHZ9G n.d. 0.71 ± 0.22 0.29 ± 0.07
DHZOG 0.46 ± 0.13 0.28 ± 0.07 0.15 ± 0.05
iP 0.24 ± 0.10 0.15 ± 0.04 0.56 ± 0.17
iPR 1.96 ± 0.28 1.24 ± 0.26 2.18 ± 0.46
iP7G 53.87 ± 2.86 65.76 ± 12.47 30.16 ± 4.57
Magnetic nanoparticles
• Magnetite - iron(II,III) oxide, Fe3O4 – particle diameter 20 – 50 nm
• Magnetic nanoparticles prepared at Regional Centre of Advanced Technologies and Materials, Palacky University (Zdeňka Marková)
• surface coated with chitosan – free aminogroups to protect aggregation and to introduce functional groups.
• Another possibility - nanoparticles modified by TEOS/APTES
TEOS = tetraethoxysilan APTES = 3-aminopropyltriethoxysilan• Monoclonal antibody 1G6 bound on aminogroup (of chitosan)
Preparation of immunoaffinity sorbent
• antibodies immobilized onto superparamagnetic iron oxide nanoparticles - 2 methods
1. one-step glutaraldehyde method (Kluchová et al., 2009)
Fe3O4-NH2 + O=CH-(CH2)3-CH=O → Fe3O4-N=CH-(CH2)3-CH=O + Ab-NH2 → Fe3O4-N=CH-(CH2)3-CH=N-Ab
2. carbodiimide method (Aslam et al., 1998)
CH3-CH2-N=C=N-(CH2)3-N + H-(CH3)2Cl- + Ab-COOH → CH3-CH2-N=C-NH-(CH2)3-N + H-(CH3)2 Cl-
Ab –COO- R Ab–CONH -Fe3O4 , NHSS ester mg nanoparticles with antibody bound
EDAC = 1-ethyl-3-(3-dimethylaminopropyl)carbodimid hydrochlorid
NHSS = N-hydroxysulfosuccinimidu
R –OH (NHSS)
H2N - Fe3O4
Ab-COO
• Sample: 100 µl mg particles in PBS + 10 µl sample CK + 90 µl PBS – no column!• Incubation: 60 min at room temperature• Wash: 200 µl PBS → 60 s vortex → particles concetrated using mg rack → supernatant
tranferred (2x)
200 µl H2O → 60 s vortex → particles concetrated using mg rack → supernatant tranferred
• Elution: 100 µl MeOH → 60 s vortex → particles concetrated using mg rack → supernatant tranferred (2x)
Purification protocol
CK (1 pmol)
Elution(%)
iP 5,1
iPR 37,1
oTR 128
cZR 30,1
tZ 37,8
cZ 23,0
tZR 45,1
oT 115
SummarySummary
• First report of using StageTip mikroSPE and magnetic nanoparticles
for plant hormone isolation.
• Quantification in 1-5mg FW possible
• Lower matrix effect, higher sensitivity (LOD 50 pmol).
• Separation of all cytokinin metabolites (including intact O-glucosides
and nucleotides) in one chromatographic run in 24.5 min
• Intact mono- , di- and triphosphorylated Z and iP-type nucleotides can be determined by an RP-HPLC method with single/tandem MS detection
• Capillary zone electrophoresis is a suitable analytical technique to assay the in vitro reaction catalyzed by the recombinant AtIPT1 and this approach may bring a new light into the earlysteps of CK biosynthesis.
AcknowlegmentsAcknowlegments
• Ondřej Novák
• Jana Oklešťková
• Jana Svačinová
• Tibor Béres
• Lenka Plačková
• Marek Zatloukal
• René Lenobel
• Petr Tarkowski
• Miroslav Strnad