Auxin (phytohormone) analysis

Jakub RolčíkJakub Rolčík

AAuxinuxin

Indole-3-acetic acid (IAA)

N

OR

O

H

R = H, Amino acids

Amounts expressed in mol:

1 mmol = 0,001 mol

1 μmol = 0,000 001 mol

1 nmol = 0,000 000 001 mol

1 pmol = 0,000 000 000 001 mol

1 fmol = 0,000 000 000 000 001 mol

1 amol = 0,000 000 000 000 000 001 mol

PhytohormonesPhytohormonesact at very low – “trace” - concentrationsact at very low – “trace” - concentrations

Amounts of phytohormones typicallyquantified in plant material

10 cm

How to imagine “trace” concentrationHow to imagine “trace” concentration? ? Comparison with Comparison with GulliverGulliver in in Brobdingnagu Brobdingnagu lags behindlags behind……

Imagine the amount of phytohormoneas an abscissa of 10 cm in length:

Then, what would be the length ofthe rest of the plant sample?

Diameter of the Earth !

Aims of trace analysis: - To reduce the diameter of the Earth to a couple of mm- To measure the length corresponding to amount of auxin

Imunization Myeloma cells

Splenocytes

Fusion

AH medium

DOTBLOT

Cloning

DOTBLOT

Cloning

in vitro in vivo

Monoclonal Abs Monoclonal Abs

Nitrogen (l)

Preparation of monoclonal antibodiesPreparation of monoclonal antibodies

- Polyclonal antibodies:

Imunization of rabbits

- Monoclonal antibodies

Immunization of mice and hybridoma technology

2: D iode A rray 268 nm

8.91

250

Tim e

7 .00 8 .00 9 .00 10.00 11.000

100

%

2-O H -3-O C H3-BAPR

2: D iode A rray 268 nm

8.89

2-O H -3-O C H 3-B A P R

IAE & DIAE & Deeririvatives ofvatives of BAP BAP

HPLC-UV chromatogramHPLC-UV chromatogramss – – mousemouse serum serum processed by processed by SPESPE

aand by combination of nd by combination of SPE & IAESPE & IAE

HPLC HPLC separation of auxinsseparation of auxins

Time5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00

AU

-1.4e-3

-1.2e-3

-1.0e-3

-8.0e-4

-6.0e-4

-4.0e-4

-2.0e-4

-9.155e-11

2.0e-4

4.0e-4

6.0e-4

8.0e-4

1.0e-3

1.2e-3

1.4e-3

1.6e-3

1.8e-3

2.0e-3

2.2e-3

2.4e-3

2.6e-3

2.8e-3

6.02

IAM

8.63

IAGlyMe

19.92

IALeuMe

16.23

IAAMe

14.27

IAGluMe11.48

IAAlaMe

10.45

IEt

12.60

IAAspMe12.28

IAN

14.77

IMe

17.45

IAValMe

19.52

IPAMe18.27

IATrpMe20.43

IAPheMe

20.93

IBAMe

UPLC (UPLC (UUltra-ltra-PPerformance erformance LLiquid iquid CChromatographyhromatography))

Novel kind of sorbent resistant to high pressure (1000 atm)

UPLC – UPLC – compared with compared with HPLC – HPLC – is significantly fasteris significantly faster

18 StdsD / 5 (25 pmol) - FS - 10 mM

m/z100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

%

0

100

06032607 - 18SD (25 pmol) FS 919 (17.454) Cn (Cen,2, 80.00, Ht); Sm (Mn, 2x0.75); Cm (915:921-(911:914+922:929)) 1: Scan ES+ 3.67e6288.98

228.06212.12198.52116.81 149.03129.07 166.86 170.93

256.90232.65

286.93275.43

310.94

306.95

348.00

326.87328.37

356.89372.92 390.71 398.68

MS spectrum MS spectrum of compoundof compound of M=of M=288 g/mol288 g/mol

Mass Spectrometry – Single QuadrupoleMass Spectrometry – Single Quadrupole

Mass Spectrometry – Triple QuadrupoleMass Spectrometry – Triple Quadrupole

-Nabitý ion je selektován prvním kvadrupólem.

-V druhém kvadrupólu (v kolizní cele) je ion bombardován atomy argonu. Vzniká směs menších iontů.

-Třetím kvadrupólem je selektován ion typický pro analyt.

RequirementsRequirements

- Deep understanding of phytohormone nature and chemistry

- Experience and tradition

- Patience and endurance (from 3 months to 3 years)

- Good instrumentation (up to € 1,000,000)

- Good people

Understandable characteristics of current Understandable characteristics of current phytohormone analysisphytohormone analysis

10 mg – amount corresponding to a quarter of peeled sunflower seed – is sufficient for analysis

Outlook: Quantification of IAA in a single root tip

Examples:-fmol per root tip-fmol per mm