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