A High-throughput UPLC-ESI-ITMS Measurement of

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Sep 6, 2006 - HPLC grade methanol was purchased from J. T. Baker (Phillipsburg, NJ). The ultrapure water was supplied by an in-house Millipore system ...
A High-throughput UPLC-ESI-ITMS Measurement of Phytohormone Auxin or Indole-3-Acetic Acid (IAA) a Zou ,

a Tolstikov ,

b Wakefield ,

b Dearnley

Wei Vladimir V. Michael R. Julie aMetabolomics Core, Genome Center, University of California, Davis, CA; bWaters Corporation, Dublin, CA

5. Gomez-Cadenas, A.; Tadeo, F. R.; Talon, M.; Primo-Millo, E. Plant Physiol 1996, 112, 401-408. 6. Birkemeyer, C.; Kolasa, A.; Kopka, J. J Chromatogr A 2003, 993, 89-102. 7. Schmelz, E. A.; Engelberth, J.; Alborn, H. T.; O'Donnell, P.; Sammons, M.; Toshima, H.; Tumlinson, J. H., 3rd Proc Natl Acad Sci U S A 2003, 100, 1055210557. 8. Schmelz, E. A.; Engelberth, J.; Tumlinson, J. H.; Block, A.; Alborn, H. T. Plant J 2004, 39, 790-808. 9. Muller, A.; Duchting, P.; Weiler, E. W. Planta 2002, 216, 44-56. 10. Matsuda, F.; Miyazawa, H.; Wakasa, K.; Miyagawa, H. Biosci Biotechnol Biochem 2005, 69, 778-783. 11. Durgbanshi, A.; Arbona, V.; Pozo, O.; Miersch, O.; Sancho, J. V.; GomezCadenas, A. J Agric Food Chem 2005, 53, 8437-8442. 12. Kowalczyk, M.; Sandberg, G. Plant Physiol 2001, 127, 1845-1853.

Indole3AA_060208145543

H N

90 6

70 indole

7

11

10

2

50 8

9

40

85

4

4

250

300

350

400

463.14 491.22 450

60

80

100

120

140

176.08

160

180

200

Indole3AA_060208145543 #2445 RT: 5.19 AV: 1 NL: 1.69E5 T: ITMS - c ESI Full ms2 [email protected] [ 50.00-500.00] 129.93 100 90

80

80 Relative Abundance

90

70 60 50 190.01

30

70 60 50

211.99

253.89

219.91 200

325.17

254.99

8

OH

70

1

50

H N

H N

10

45

6

3

5

O

6

indole

7

11 4

35

450

500

50

100

158.98 150

200

250

300

350

400

450

55 50 N

45

4

40

8

35 30

500

m/z

11

3

indole-3-acetonitrile

OH 1

9

7

indole-3-acetic acid

30

m/z

2

2

60

9

1

30

115.09

5

12

10 400

12

indole-3-carbinol

10

371.15

350

65

7

13

8

348.74

300

2

55

40

0

250

9

5

6

20

0 150

65 60

40

20 10

NL: 7.38E4 Base Peak m/z= 127.50-128.50+129.50-130.50+ 154.50-155.50 F: ITMS - c ESI SRM ms2 [email protected] [ 129.00-131.00] MS ICN&IAA&IOH_STD_Level5_Inj1

75

m/z

Indole3AA_060208145543 #2289 RT: 4.95 AV: 1 NL: 3.36E5 T: ITMS - c ESI Full ms [ 150.00-500.00] 174.01 100

Relative Abundance

157.99

131.09

0

500

m/z

40

80

3

70

Relative Abundance

200

10

30

Relative Abundance

0 150

85

75

10 343.16 372.79 390.62 420.36

289.06 305.13

NL: 2.22E4 Base Peak m/z= 127.50-128.50+129.50-130.50+ 154.50-155.50 F: ITMS - c ESI SRM ms2 [email protected] [ 127.00-129.00] MS ICN&IAA&IOH_STD_Level5_Inj1

95

H N

20 230.96

0.71

100

11

50 40

20 192.92 214.09

1.28

90 80

60 OH 1

NL: 3.40E5 TIC F: ITMS - c ESI SIM ms [ 154.00-156.00] MS ICN&IAA&IOH_STD_Level5_Inj1

2.27

70

indole-3-acetic acid

30

10

RT: 0.00 - 3.00 SM: 11G NL: 2.14E5 Base Peak m/z= 127.50-128.50+ 129.50-130.50+ 154.50-155.50 F: MS ICN&IAA&IOH_STD_Le vel5_Inj1

2.27

90

80

O

Relative Abundance

Relative Abundance

3

5

60

RT: 0.00 - 3.00 SM: 11G

90

12

80

10/5/2006 8:49:31 PM

95

Indole3AA_060208145543 #1624 RT: 2.93 AV: 1 NL: 4.38E6 T: ITMS + c ESI Full ms2 [email protected] [ 50.00-200.00] 129.98 100

13

ICN&IAA&IOH_STD_Level5_Inj1

100

9/6/2006 10:53:52 AM

Indole3AA_060208145543 #297 RT: 0.40 AV: 1 NL: 6.59E6 T: ITMS + c ESI Full ms [ 150.00-500.00] 176.03 100

Figure 2. LC-ESI-ITMS chromatograms of a standard mixture containing 8 g/L under negative SIM or SRM modes.

25 25

Indo-3-Carbinol-Scan-Infusion-2006092... For tuning

20

9/22/2006 10:03:57 AM

Indo-3-Carbinol-Scan-Infusion-20060922_060922100356 #505 RT: 0.62 AV: 1 NL: 5.43E5 T: ITMS - p ESI Full ms [ 100.00-300.00] 146.00 100 11

15

Indo-3-Carbinol-Scan-Infusion-20060922_060922100356 #684 RT: 1.05 AV: 1 NL: 2.66E5 T: ITMS - p ESI Full ms2 [email protected] [ 50.00-300.00] 128.00 100

H N

95 4

85

5

5 8

OH

1

2 6

7

60

Relative Abundance

65

60 55 50 45 40

30 25 20 144.00

15

15

140

160

180

200 m/z

5

240.91 255.09

220

240

281.18 292.82

260

280

101.91

0

300

50

118.00

100

168.64 150

200

250

300

m/z

Indo-3-Acetonitrile-Scan-Infusion-200... For tuning

1.0

1.5 Time (min)

2.0

2.5

3.0

0.0

5

N

9

1 6

85

3 2

80

8

indole-3-acetonitrile

75

70

70 214.08

65

55 157.98

45 40

0 100

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180

200 m/z

220

240

288.22

269.08

260

280

300

90 80 70

40 30 20

0.12

1.34

2.31

2.58

4.51 0.85

10

4.42 4.54 4.80

2

3

4

5

6

70

4.60 5.12

0 0

1

2

3

4

5

6

0

ioh_std_level2_inj1 #600 RT: 2.41 AV: 1 NL: 2.44E2 F: ITMS - c ESI SRM ms2 [email protected] [ 127.00-129.00] 128.02 100

80

5 0 100

119.01

120

188.04

144.08 140

214.78

230.89 249.01 264.95

160

180

200 m/z

220

240

260

293.00 280

300

30

70 60 50 40 30

30 20 10

m/z

128.5

129.0

127.5

6

40

10 128.0

5

50

10 127.5

4

60

20

0 127.0

3

70

20

0 127.0

2

IOH_STD_Level1_Inj1 #594 RT: 2.39 AV: 1 NL: 3.85E1 F: ITMS - c ESI SRM ms2 [email protected] [ 127.00-129.00] 128.08 100 90

40

1

Time (min)

80

10

3.59 4.31

10

90

50

5.71 4.48

20

80

60

4.83

30

90

171.02 187.00

2.41 1.10 1.48

40

Time (min)

70

TIC F: ITMS - c ESI SRM ms2 [email protected] [ 127.00-129.00] MS IOH_STD_Level1_In j1

80

50

0 1

NL: 1.13E2

90

60

3.24 3.76

2.14

RT: 0.00 - 6.00 SM: 11G 0.77 100

30

15 285.11

TIC F: ITMS - c ESI SRM ms2 [email protected] [ 127.00-129.00] MS ioh_std_level2_inj1

35

20

215.09

NL: 1.81E2

50

ioh_std_level3_inj1 #602 RT: 2.42 AV: 1 NL: 7.86E2 F: ITMS - c ESI SRM ms2 [email protected] [ 127.00-129.00] 127.96 100

40

173.04

140

30

RT: 0.00 - 6.00 SM: 11G 2.41 100

Time (min)

25

120

3.0

60

0

45 284.13

140.96

5

40

0

25

244.13

50

50

30

203.10 216.05

60

10

55

271.13

178.04

70

20

60

130.00

118.04

2.5

0.75

65

60

173.88 196.98

TIC F: ITMS - c ESI SRM ms2 [email protected] [ 127.00-129.00] MS ioh_std_level3_inj1

80

11

4

80 75

NL: 8.09E2

90

90

85

10

2.0

10/2/2006 6:31:19 PM

RT: 0.00 - 6.00 SM: 11G 2.41 100

95

90

15

1.5 Time (min)

IOH_STD_Level1_Inj1

Indo-3-Acetonitrile-Scan-Infusion-20061005 #5353 RT: 17.97 AV: 1 NL: 3.86E6 T: ITMS - c ESI Full ms [ 100.00-300.00] 154.97 100 7

10

20

1.0

Figure 3. Methanol generated significantly less background noise than acetonitrile as the strong mobile phase B. With UPLC-ITMS, LOD of IAA could reach 64 pg per injection (5 L) or 12.8 g/L.

N

95

35

0.5

10/5/2006 4:31:20 PM

Indo-3-Acetonitrile-Scan-Infusion-20061005 #976 RT: 3.56 AV: 1 NL: 2.60E5 T: ITMS + c ESI Full ms [ 100.00-300.00] 12 155.02 100 H

50

2.29 2.59

Relative Abundance

120

145.91

213.00

0.5

Relative Abundance

0 100

10

212.09 187.00

1.62 1.76

0.27

40

25

177.91

1.65 1.92 2.15 2.57 2.90

45

35

120.00

0.73

0.28

5

2.40 2.49

50

30

5

1.80 1.85

55

35

162.00

1.39

2.97

70

65

10

0.91

0 0.0

80 75

indole-3-carbinol

70

106.00

0.57

1.37 1.58 1.82

9

75

20

0.27

1.02

0.65

10

0

85

80

1.28

90

3

0.28

15

10

95 10

90

20

0.71

Relative Abundance

4. Dachs, G. U.; Tupper, J.; Tozer, G. M. Anticancer Drugs 2005, 16, 349-359.

Figure 1. Fragmentation patterns for IAA, IOH, and ICN. (A) IAA: m/z 174 m/z 130, (B) IOH: m/z 146 m/z 128, (C) ICN: m/z 155.

Relative Abundance

3. Ostin, A.; Kowalyczk, M.; Bhalerao, R. P.; Sandberg, G. Plant Physiol 1998, 118, 285-296.

MS Optimization: With UPLC-ITMS, LODs of IAA, IOH, and ICN were 64, 320, and 1600 pg per injection (5 L), while triple-quadruple MS online with UPLC was significantly more sensitive than ion-trap MS, reached LODs of the respective compounds to 1, 5, and 50 pg per injection (10 L). The calibration curves were linear for IAA, IOH, and ICN from 0 to 500 g/L, especially within the range of 0 to 100 g/L, suitable for a variety of samples.

Relative Abundance

2. Ljung, K.; Hull, A. K.; Kowalczyk, M.; Marchant, A.; Celenza, J.; Cohen, J. D.; Sandberg, G. Plant Mol Biol 2002, 49, 249-272.

LC Optimization: Using HPLC, among the six different kinds of columns tested, Synergi Hydro and PolyLC polyhydroxyethyl columns demonstrated good sensitivity, while the PolyLC polyhydroxyethyl column generated more symmetric peaks than the Synergi Hydro column. Comparing to HPLC, UPLC and WATERS BEH RPC18 column significantly shortened analysis time and increased sensitivity, with the peaks of IAA at 0.74 min, IOH at 1.28 min, and ICN at 2.27 min in times (Figure 2). Methanol generated significantly less background noise than acetonitrile as the strong mobile phase B (Figure 3).

Relative Abundance

1. Woodward, A. W.; Bartel, B. Ann Bot (Lond) 2005, 95, 707-735.

Precursor-to-Product Ion Transition: Selection of precursor and product ions for each compound was determined by infusion of standard solutions (0.1 mg/mL in initial LC mobile phase composition). APCI was not as efficient as ESI to ionize the compounds of interest (data not shown). All full-scan MS and MS/MS selected reaction monitoring (SRM) scans were conducted in negative ESI mode (Figure 1). For IAA and IOH, the main product ions obtained after the molecular fragmentation were used as diagnostic product ions: m/z 174 m/z 130 for IAA and m/z 146 m/z 128 for IOH. Since ICN was difficult to be ionized, selective ion monitoring (SIM) of m/z 155 was used.

Relative Abundance

References

Reagents and Standards: Extra pure ammonium acetate was purchased from EMD (Gibbstown, NJ). HPLC grade acetonitrile was purchased from Burdick and Jackson (VWR International, West Chester, PA). HPLC grade methanol was purchased from J. T. Baker (Phillipsburg, NJ). The ultrapure water was supplied by an in-house Millipore system (Billerica, MA). Indole-3acetic acid, indole-3-carbinol (IOH), and 3-indoleacetonitrile (ICN) were purchased from SigmaAdrich (St. Louis, MO). A stock solution (1 mg/mL) of each reference compound was prepared freshly on each working day in a solvent system identical to the initial mobile phase composition of LC. To determine the limit of detection (LOD), a five-fold dilution series were performed with the same solvent system to generate a calibration curve for each compound. The concentrations of the diluted standards were 12.8, 64, 320, 1600, 8000, 40000, 200000 g/L. LOD, defined as the lowest amount that the peak of interest can reliably differentiate from background noise, was set at a signal-to-noise ratio (S/N) of 3. UPLC/HPLC-ITMS: This instrument was located at the Metabolomics Core Lab, University of California, Davis. The Acquity UPLC system was composed of a binary solvent manager, a sample manager, a column manager, and a TUV detector (Waters Assoc., Milford, MA). Although this configuration of UPLC hardware can reach as high as 15,000 psi, a working back pressure of 10,000 psi was targeted. Three UPLC columns from Waters had been tested for resolution and sensitivity: a BEH C18 shielded column (1.7 m, 100 x 2 mm), a HSS T3 column (2 m, 100 x 2 mm), and a BEH HILIC column (1.7 m, 50 x 2 mm). Mobile phases had been tested: A, ammonium acetate, 13 mM, pH 5.5; B, acetonitrile or methanol. All injection volumes were 5 L and column temperature was 50 C, while the flow rates were dependent on the choice of the column and mobile phases, with a typical flow rate of 0.4-0.8 mL/min. The Surveyor HPLC system was composed of a pump, an autosampler, and a photo diode array (PDA) detector (ThermoFinnigan, San Jose, CA). Six HPLC columns had been tested: a Synergi hydro-RP18 column (4 m, 150 x 3 mm; Phenomenex, Torrance, CA), an Onyx monolithic C18 column (4 m, 100 x 3 mm; Phenomenex), a ChromoLith endcapped monolithic RP18 column (4 m, 100 x 4.6 mm; Merck KGaA), a Luna phenyl-hexyl column (3 m, 150 x 3 mm; Phenomenex), a Luna NH2 column (3 m, 150 x 3 mm; Phenomenex), and a PolyLC polyhydroxyethyl column (3 m, 150 x 3 mm; PolyLC Inc., Columbia, MD). All injection volumes were 5 L and column temperature was 50 C, and a typical flow rate of 0.8-1.5 mL/min, depending on the choice of the column. The typical working back pressure was 2,000 psi. For UPLC and HPLC methods, different gradients were applied with different columns. The entire effluent from either the UPLC or HPLC column was directed into the electrospray ionization source (ESI) or atmosphere pressure chemical ionization (APCI) of a Finnigan LTQ ion trap mass spectrometer (ThermoFinnigan) operated under Xcalibur software (V1.4, ThermoFinnigan). The electrospray voltage was 5 kV. Nitrogen sheath and aux gas flow was 60 and 20 units respectively. The ion transfer capillary temperature was 350 C. Typical ion gauge pressure was 0.90 x 10–5. All scan events were acquired with one micro scan. Full scan spectra were acquired with a 200 ms maximum ionization time. Typical parameters applied in MSn scan events were an isolation width of 2 Da, an activation time of 30 ms, a normalized collision energy of 40%, and an activation Q of 0.250. UPLC-MS/MS: This instrument was located at the Waters Corporation Training Center (Dublin, CA). The Acquity UPLC system was composed of a binary solvent manager, a sample manager, and a PDA detector (Waters Assoc.). A BEH C18 shielded column (1.7 m, 100 x 1 mm) was used. The injection volumes were 10 L, the column temperature was 20 C, and the flow rate was 0.2 mL/min. Mobile phase A was 0.05% acetic acid in water and B was 0.05% acetic acid in acetonitrile. After maintaining an initial mobile phase composition of B as 10% for 0.5 min, the composition of B increased to 90% during a 1.5 min period. The entire effluent from the UPLC column was directed into the ESI of a Quattro Premier triple quadrupole mass spectrometer (Waters Assoc.) operated under MassLynx (V4.0, Waters Assoc.). Negative ions were acquired in the multiple reaction monitoring (MRM) mode using a desolvation temperature of 400 C, a source temperature of 100 C, and a capillary voltage of 3.5 kV. The collision gas was 99.995% pure argon with a pressure of 4 x 10-3 mbar. Optimized for each MRM transition used, the cone voltage and collision energy were 22 V and 13 eV for IAA, 25 V and 12 eV for IOH, and 20 V and 3 eV for ICN. Dwell times for individual transitions were adjusted to achieve approximately 15 data points across the respective UPLC chromatographic peak. In some cases, dwell times, interchannel delays, and inter-scan delays reached the intrument’s limit of 5 ms. All data were acquired at 16 points/scan.

Results and Discussion

Relative Abundance

Plant hormones play a crucial role in controlling plant growth and development. As a crucial plant hormone, indole-3-acetic acid (IAA), previously named as heteroauxin, modulates such diverse processes as tropic responses to light and gravity, general root and shoot architecture, organ patterning, vascular development and growth in tissue culture 1,2,3. In addition to its important physiological functions in plants, IAA has been shown to be a promising anti-cancer prodrug in synergy with the horseradish peroxidase (HRP), representing a novel gene-directed enzyme-prodrug therapy (GDEPT) 4. Preliminary results showed that human T24 bladder carcinoma cells transfected with a mammalian express vector containing the HRP cDNA were selectively sensitized to IAA in normoxic and anoxic conditions 4. In spite of numerous work in the literature, few details of IAA metabolism and its functions in plants and humans are known, due to the lack of a high-throughput sensitive and selective method measuring IAA and its intermediates/metabolites simultaneously. Previously, classical methods for hormone determination involved intensive purification steps using large amounts of tissue samples 5. Recently, gas chromatography-mass spectrometry (GC-MS) provides much higher sensitivity, but extensive purification and derivatization steps are labor-intensive, besides the possible breakdown of heatlabile compounds 6,7,8,9. The application of conventional high performance liquid chromatography-mass spectrometry (HPLC-MS) provides simpler sample preparation and larger peak capacity, with a typical sample run time of 20-60 min 10,11,12. Recently, commercially available ultra performance liquid chromatography (UPLC) offers higher resolution in much shorter analysis time than conventional HPLC. In the present study, we will discuss a high-throughput IAA measurement using UPLC coupled with either ion-trap mass spectrometry (ITMS) or triple-quadruple mass spectrometry (MS/MS).

Methods

Relative Abundance

Introduction

128.0 m/z

128.5

129.0

0 127.0

127.5

128.0

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129.0

m/z

Conclusion We have developed a high-throughput, sensitive, and accurate UPLC-MS method to determine phytohormone auxin and its key intermediate metabolites in less than 3-min run. Considering its large peak capacity, short analysis time, and high sensitivity, the current method also opens the possibility of incorporating more related hormones and metabolites into a single run. With this cutting-edge tool in hand, the scientific community can conduct innovative research on metabolic profiling or metabolomics in hormone signal transduction, functional food nutrition, and natural drug discovery.