Title: Recent advances in cytokinin analysis
1Recent advances in cytokinin analysis
2Acquity UPLC - Xevo TQ MS
3Analysis 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)
4Analysis 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
5Analysis of Cytokinins ( 50100 mg FW)
6Recent 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
7Metabolism of cytokinins
8(No Transcript)
9Reconstructed 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
10Depletion 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
11Fig. 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)
12Miniaturarization
- Main goals
- More efficient and simple extraction and
purification - Efficient separation UPLC
- Higher sensitity ESI-MS/MS
- Shorter time of analysis
13StageTip purification (STop-And-Go-microExtraction
)
Rappsilber et al. (2008) Nature Protocols 2
1896-1906.
14Single 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
15Multi StageTips
C18/SDB-RPS
C18/Cation-SR
C18/SDB-RPS/Cation-SR
16Purification 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
17Separation 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
18Limits 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
19Method 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
20isoprenoid 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
21Magnetic 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) -
22Preparation 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)
23Purification 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
24Summary
- 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.
25Acknowlegments
- Ondrej Novák
- Jana Okleštková
- Jana Svacinová
- Tibor Béres
- Lenka Placková
- Marek Zatloukal
- René Lenobel
- Petr Tarkowski
- Miroslav Strnad