Recent advances in cytokinin analysis

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Recent advances in cytokinin analysis

• Karel Doležal

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Acquity UPLC - Xevo TQ MS
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Analysis of Cytokinins ( 50100 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)

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Analysis of Cytokinins ( 50100 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

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Analysis of Cytokinins ( 50100 mg FW)
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Recent projects and goals in cytokinin analysis

• 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

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Metabolism of cytokinins
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(No Transcript)
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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).
Béres T. et al. (2010) Anal Bioanal Chem 398
2071-2080
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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.
Kowalska et al. (2010) Phytochemistry 71
1970-1978
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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.
Fig. 1 Separation of six nucleotide standards a)
iPMP, b) AMP, c) iPDP, d) iPTP, e) ADP, f) ATP,
under optimal conditions.
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. 2 UV spectra based reaction
substrate/product identification.
Béres T. Gemrotová M. et al. (in preparation)
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Miniaturarization

• Main goals
• More efficient and simple extraction and
  purification
• Efficient separation UPLC
• Higher sensitity ESI-MS/MS
• Shorter time of analysis

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StageTip purification (STop-And-Go-microExtraction
)
Rappsilber et al. (2008) Nature Protocols 2
1896-1906.
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Single 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 () n4 recovery 3H-CKs () n4 recovery 3H-CKs () n4 recovery 3H-CKs () n4 recovery 3H-CKs () n4 recovery 3H-CKs () n4
C18 C18 SDB-RPS SDB-RPS Cation-SR Cation-SR
Load Elution Load Elution Load Elution
3HcZ 98.8 3.3 0.6 0.2 1.3 0.1 96.6 1.8 1.1 0.1 100.7 3.0
3HtZR 98.8 4.4 0.6 0.2 39.2 2.2 29.8 2.2 22.4 0.6 64.7 1.1
3HiPR 102.9 3.5 0.5 0.3 4.9 0.2 95.6 1.4 4.7 0.1 97.0 2.8
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Multi StageTips
C18/SDB-RPS
C18/Cation-SR
C18/SDB-RPS/Cation-SR
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Purification 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

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Separation 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, 595 (AB) 7-16 min, 595 gt 2080
  16-24 min, 2080 gt 5050
• flow 0.25 ml min-1, column temperature 40 ºC

Compounds Precursors Products
t/cZ (R1H, R2H) 220.1 136.1
t/cZR (R1Rib, R2H) 352.2 220.1, 136.1
t/cZ3/7/9G (R1Glc, R2H) 382.2 220.1, 136.1
t/cZOG (R1H, R2Glc) 382.2 220.1, 136.1
t/cZROG (R1Rib, R2Glc) 432.2 382.2, 220.1, 136.1
t/cZRMP (R1RibMP, R2H) 514.2 220.1, 136.1
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Limits 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
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Method validation
CKs Recovery () Recovery () Recovery () 1 pmol added accuracy () precision ()
CKs 1 mg 2 mg 5 mg 1 pmol added accuracy () precision ()
tZ 80 10 63 8 21 4 0.99 0.17 17.2 0.1
tZR 72 12 46 7 8 1 0.84 0.09 10.4 16.1
tZ7G 88 6 57 4 6 1 0.91 0.13 14.6 8.6
tZ9G 59 7 31 3 8 1 0.97 0.09 9.6 2.5
tZOG 85 5 68 6 11 2 1.07 0.19 18.1 -7.1
tZROG 55 4 30 3 4 1 0.82 0.09 11.1 17.6
tZMP 35 6 11 1 5 1 0.85 0.11 13.3 14.7
cZ 75 9 65 5 24 4 0.83 0.02 2.5 17.0
cZR 81 13 44 9 8 1 0.96 0.12 12.8 4.1
cZ9G 74 12 37 5 5 1 1.18 0.13 11.3 -17.6
cZOG 89 6 66 7 9 2 1.09 0.14 13.2 -9.1
cZROG 52 6 24 2 3 1 0.89 0.11 12.9 11.2
cZMP 32 3 17 1 2 1 0.86 0.15 17.9 13.9
DHZ 77 13 61 7 20 3 0.90 0.10 10.8 9.7
DHZR 88 13 48 8 12 1 1.03 0.06 5.6 -2.9
DHZ7G 89 3 65 3 8 2 1.18 0.05 4.3 -18.2
DHZ9G 78 10 35 6 6 1 0.96 0.07 7.2 3.7
DHZOG 77 5 50 7 9 3 1.16 0.06 4.9 -15.8
DHZROG 87 8 42 5 5 1 0.90 0.12 13.3 9.8
DHZMP 37 1 12 1 3 1 0.96 0.15 15.8 3.6
iP 76 9 68 3 26 5 0.97 0.06 5.9 3.3
iPR 84 8 53 4 17 1 1.13 0.06 5.6 -12.8
iP7G 83 10 60 5 7 1 0.91 0.15 16.5 9.4
iP9G 74 8 49 8 8 2 0.97 0.08 8.0 3.1
iPMP 78 9 39 9 9 2 0.92 0.16 17.0 8.4
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isoprenoid cytokinins (1-5mg FW)
CKs cytokinin levels (pmol g-1 FW) cytokinin levels (pmol g-1 FW) cytokinin levels (pmol g-1 FW)
CKs 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
Xevo TQ MS
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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 (Zdenka 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)

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Preparation of immunoaffinity sorbent

• antibodies immobilized onto superparamagnetic
  iron oxide nanoparticles - 2 methods
• 1. one-step glutaraldehyde method (Kluchová et
  al., 2009)
• Fe3O4-NH2 OCH-(CH2)3-CHO ? Fe3O4-NCH-(CH2)3-C
  HO Ab-NH2 ? Fe3O4-NCH-(CH2)3-CHN-Ab
• 2. carbodiimide method (Aslam et al., 1998)
• 
  
  
  
  
  
  
  
  
• CH3-CH2-NCN-(CH2)3-N H-(CH3)2Cl- Ab-COOH ?
  CH3-CH2-NC-NH-(CH2)3-N H-(CH3)2 Cl-
• Ab COO- R
  AbCONH -Fe3O4
• , NHSS
  ester mg
  nanoparticles with antibody bound
• EDAC 1-ethyl-3-(3-dimethylaminopropyl)carbodimid
  hydrochlorid
• NHSS N-hydroxysulfosuccinimidu

Ab-COO
H2N - Fe3O4
R OH (NHSS)
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Purification protocol

• 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)

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
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Summary

• 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.

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Acknowlegments

• Ondrej Novák
• Jana Okleštková
• Jana Svacinová
• Tibor Béres
• Lenka Placková

• Marek Zatloukal
• René Lenobel
• Petr Tarkowski
• Miroslav Strnad