Derivatization and Sample Prep for (Small) Molecules Árpád Somogyi CCIC MSP OSU Summer Workshop

Why Derivatize? • To increase abundance of MH+ for better MS/MS – Improve sensitivity for detection of oligosaccharides

• To achieve selectivity in a detection scheme – Newborn screening for disease: Detection of amino acid imbalance, a marker of inherited disease

• To distinguish an analyte from an interference – Analysis of methylmalonic acid in plasma & urine (differentiated from succinic acid – same mass)

1

Why Derivatize?

• To increase abundance of MH+ for better MS/MS – Improve sensitivity for detection of oligosaccharides

• To achieve selectivity in a detection scheme – Newborn screening for disease: Detection of amino acid imbalance, a marker of inherited disease

• To distinguish an analyte from an interference – Analysis of methylmalonic acid in plasma & urine (differentiated from Succinic Acid)

Derivatization to Improve Sensitivity for Detection of Oligosaccharides • Poor ionization efficiency of free carbohydrate chains in ESI limits the utility of ESI-MS in structural carbohydrate studies • Ionization efficiency enhanced in positive-mode ESI-MS (improved 5000-fold) • 2 derivatizing agents investigated

Benzoic acid, 4-amino-, ethyl ester (ABEE)

Benzoic acid, 4-amino-, 2(diethylamino)ethyl ester (ABDEAE) Reference: Yoshino et al, 1995, 76:4028-4031

2

MS (+ ESI) maltohexaose 50 pmol

(free sugar)

5 pmol

(ABEE-derivatized)

100 fmol

(ABDEAE-derivatized)

10 fmol (ABDEAE-derivatized)

MS/MS (+ ESI) maltohexaose-ABDEAE

100 pmol/uL

1 pmol/uL

3

Why Derivatize?

• To increase abundance of MH+ for better MS/MS – Improve sensitivity for detection of oligosaccharides

• To achieve selectivity in a detection scheme – Newborn screening for disease: Detection of amino acid imbalance, a marker of inherited disease

• To distinguish an analyte from an interference – Analysis of methylmalonic acid in plasma & urine (differentiated from succinic acid)

February 2009 JMS Donald H. Chace newborn screening

Inherited metabolic diseases Phenylketonuria (PKU) Reduction in Phenylalanine to tyrosine metabolism (leads to increase in Phe levels)

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Derivatization to achieve selectivity : Newborn Screening for Disease  Blood sample from newborn collected onto dried filter paper  MS/MS is a fast, accurate, robust disease screening  method requires no chromatographic separation  extremely rapid: 2 min. per sample  methanol extraction of samples collected onto dried filter paper removes proteins and salts  Extracted sample derivatized to butyl esters  acidified butanol (3N HCl) or butanol with acetyl chloride

Product Ion Scan of Phenylalanine butyl ester shows loss of m/z 102

5

butyl ester amino acid derivatives lose 102 in MS/MS Examples: O H3N

CH

C

O

CH2

m/z 222 Phenylalanine, butyl ester

Histidine, butyl ester

Neutral Loss Spectra from Newborn Screen Control

Blood sample from Normal Newborn

Phe Tyr

Masses of deuterated internal standards are underlined Phe

Phenylketonuria

Blood sample from newborn diagnosed with phenylketonuria

Tyr Rerence: Chace, Chem. Rev. 2001, 101: 445-477

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Why Derivatize?

• To increase abundance of MH+ for better MS/MS – Improve sensitivity for detection of oligosaccharides

• To achieve selectivity in a detection scheme – Newborn screening for disease: Detection of amino acid imbalance, a marker of inherited disease

• To distinguish an analyte from an interference – Analysis of methylmalonic acid in plasma & urine (differentiated from succinic acid)

Derivatization to Distinguish an Analyte from an Impurity: Methylmalonic Acid (MMA) in Plasma & Urine (Differentiated from Succinic Acid) • Increased MMA is a specific diagnostic marker for propionate metabolism and acquired vitamin B12 deficiency • Must overcome interference from the isomer succinic acid

MMA, MW = 118.09

MMA Succinic acid

plasma (umol/L) 0-0.4 0-32

urine (mmol/mol creatine) 0-3.6 0.5-16

succinic acid, MW = 118.09

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Methylmalonic Acid Detected in Plasma & Urine (Differentiated from Succinic Acid) • HPLC-MS/MS methods have been developed (QQQ) -MRMs – replace GC-MS methods in a high-throughput environment • extracted sample (SPE) derivatized to butyl esters – extracted from plasma or urine, eluted and derivatized with HCl in n-butanol • the method is demonstrated in this example using standards • results of plasma & urine samples are in agreement with the same samples analyzed by the standard method (GC/MS)

References: Magera et al, Clinical Chem. 2000, 46 (11): 1804-1810 Schmedes et al, Clinical Chem. 2006, 52(4): 754-757

MS/MS of Derivatized MMA and Succinic Acid succinic acid butyl ester

Loss of C4H8 butyl groups + loss of H2O (-130)

MMA butyl ester

Loss of C4H8 butyl groups (-112)

8

MRM Extracted Ion Chromatograms Selected transitions m/z = 231/119 and 234/122 (MMA-d3) 1 = succinic acid 2 = MMA-d3 3 = MMA

1 nmol succinic acid

1 nmol MMA

10 nmol succinic acid

10 nmol MMA

100 nmol succinic acid

10 nmol MMA

1000 nmol succinic acid

10 nmol MMA

Why Clean up samples? • To separate interfering species from analyte – Example: analysis of drug and metabolites in plasma  need to remove protein interferences – Off-line or in-line from MS/MS detection

• To concentrate analyte – Example: Pesticides in drinking water

• Basic principle of sample clean up involves preferential binding of analyte over interfering species or vice versa, followed by elution  to MS/MS

Separation technologies essential in sample prep

9

Types of Separation Technologies for Molecules Method Liquid-liquid Extraction

Separation based on Partitioning in one of two liquid phases

Separation done using

Further steps

Glass ware

An immiscible solvent is added to the sample which then separates into 2 distinct liquid phases. Some sample analytes will go into the bottom phase (Aqueous), some will separate into the top phase (Organic) “Trizol” – a form of liquid-liquid partitioning of RNA, DNA and protein “guanidinium thiocynatephenol-chloroform”

• • • • •

Large solvent consumption Time/labor intensive May need evaporation step >1 extraction if mixture of analytes Emulsions and contamination issues

Chomczynski P, Sacchi N. Anal Biochem. 1987 Apr;162(1):156-9

Types of Separation Technologies for Molecules Method

Separation based on

Separation done using

Liquid-liquid Extraction

Partitioning in one of two liquid phases

Glass ware

Solid-phase Extraction

Adsorption/ partitioning onto solid sorbent

Cartridges, disks, filters, plates

• • • • • • • • •

Uses chromatographic particles Packed-bed column cartridges or similar Well established commercial technology (1978) 1000s literature refs Clean extracts Good recovery for polar analytes Sample must be in liquid state Driving force: gravity, pressure, vacuum Automation

Further steps

96 well plate

disk cartridges

http://solutions.3m.com/wps/portal/3M/en_US/Empore/extraction/

10

Solid-Phase Extraction (cont’d) •

Types of Chromatography – Normal Phase • Non-polar mobile phase • Polar stationary phase – Reversed Phase Most common • Polar mobile phase • Non-polar stationary phase – Ion Exchange • Buffer/Ionic mobile phase • Cationic/Anionic exchange stationary phase

Manufacturer

Brand Name

Waters

SEP-PAK OASIS

Varian

BondElute

Baker

BakerBond

3M

Empore

Supelco

Supelclean + Many Others

Solid-Phase Extraction - common protocol •

Procedure

Sample

Prepare: Homogenize, suspend, centrifuge, etc… Load onto conditioned cartridge Wash off weakly retained interferences with weak solvent Elute product with strong solvent Analyze: HPLC, GC-MS, LC-MS/MS

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(KNO3)nK+ pure analyte (control)

engine oil contaminated parking lot oil

same as b) after organic matter removal by SPE

Gapeev, A. and Yinon, J. J. Forensic Sci. 2004, 49

http://www.millipore.com/techpublications/tech1/tn072

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SPE (ZipTip) of protein digest using C18 bed ZipTip C18 Prep in PBS/Urea/NaCl

40000

Counts

20000

0

Standard Prep in PBS/Urea/NaCl -20000

-40000

1000

1500

2000

2500 3000 Mass (m/z)

3500

4000

4500

labiomed.org/pdf/sample_cleanup.ppt

Types of Separation Technologies for Molecules Method

Separation based on

Separation done using

Liquid-liquid Extraction

Partitioning in one of two liquid phases

Glass ware

Solid-phase Extraction

Adsorption/ partitioning onto solid sorbent

Cartriges, disks, filters, plates

Dialysis/Ultrafiltration

Molecular weight/size

SlideAlyzer/tubing

Further steps

13

Sample loading here

Dialysis Tubing or Slide A-Lyzer Or Tube-O Dialyzer or 96 well plate format Diff MWCO ranges 0.1– 0.5 mL capacity Useful for biologicals

Spin filters polyethersulfone membrane (Vivaspin, ex) volumes from 100 μl to 20 ml, with a range of molecular weight cutoff values from Mr = 3 000 - 100 000

Types of Separation Technologies for Molecules Method

Separation based on

Separation done using

Liquid-liquid Extraction

Partitioning in one of two liquid phases

Glass ware

Solid-phase Extraction

Adsorption/ partitioning onto solid sorbent

Cartriges, disks, filters, plates

Dialysis/Ultrafiltration

Molecular weight/size

SlideAlyzer, tubing, spin filter

Distillation or Evaporation

Boiling point/vapor pressure

Destillator, He-purge

Precipitation

Solubility

Further steps

14

Precipitation  

o o

o o o

Very useful for messy/dirty protein samples TCA*, Acetone, Ethanol precipitation methods

Bring protein solution to 80% acetone using HPLC-grade acetone Incubate at –20oC overnight or in dry ice for 2-3 hours (don’t cut incubation time) Centrifuge 10 min at 4oC and carefully remove supernatant Wash pellet gently with two aliquots of 100% acetone at –20oC Dry sample briefly under vacuum and store sealed at –20oC

*TCA: trichloro acetic acid

Types of Separation Technologies for Molecules Method

Separation based on

Separation done using

Further steps

Gel (which acts like a molecular sieve) and potential

Gel Electrophoresis (1D)

Molecular mass

Gel Electrophoresis (2D)

Isoelectric point (pI; Gel, potential and IEF) & Molecular ampholytes mass

Great clean-up tool (rid of salts, detergents, etc…) Great concentration tool Biological analytes Various stains available – various detection limits USE PRECAST GELS (polymer issue) if possible Various size gels (spatial resolution) Various MW ranges Various pI ranges

In-gel digestion of proteins to peptidesLCMS/MS or MALDI-TOF-MS

 

PAGE: polyacrylamide gel electrophoresis SDS: sodium dodecyl sulphate

Protein Mixture

MW 1D SDS-PAGE pH MW

2D SDS-PAGE

15

Types of Separation Technologies for Molecules Method

Separation based on

Separation done using Gel (which acts like a molecular sieve) and potential

Gel Electrophoresis (1D)

Molecular mass

Gel Electrophoresis (2D)

Isoelectric point (pI; Gel, potential and IEF) & Molecular ampholytes mass

Reverse Phase (C18, Combination of hydrophobicity and C8 or C4) chromatography molecular weight

Further steps In-gel digestion of proteins to peptidesLCMS/MS or MALDI-TOF-MS General molecule use Protein(s)  Digest to peptides  LCMS/MS or MALDI-TOF-MS

HPLC

Protein

Peptides

Digestion is also sample prep Trypsin (R/K) Chymotrypsin (F/W/Y/L) Pepsin (indiscriminate) Others (CNBr, Formic Acid)

MS 5

Time (min)

Separates based on combination of hydrophobicity and molecular weight

65

Abundance

HPLC

m/z Abundance Abundance Abundance

Enzyme

UV Absorbance

Most often for proteomics, chromatography clean-up is essential if not mandatory (ie: LC-MS/MS)

MS/MS

16

HPLC - hardware (often nano)

http://www.eksigent.com/ http://www.michrom.com http://www.waters.com

http://www.lcpackings.com/ http://www.chem.agilent.com

http://www.microlc.com/

HPLC Column Configurations and Applications Column Type

ID (mm)

Length (mm)

Particle Size (m)

Flow Rate Ranges

Applications

Sensitivity Increase

Nano

0.1-0.075

150

3.5

100-600 nL/min

Proteomics, Sample Limited PTM Characterization

20003700

Capillary

0.3, 0.5

35-250

3.5, 5

1-10 L/min

Peptide Mapping LC/MS

100

Micro Bore

1.0

30-150

3.5, 5

30-60 L/min

High Sensitivity LC/MS

20

Narrow Bore

2.1

15-150

3.5, 5

0.1-0.3 mL/min

Sample Limited. 5 LC/MS

Analytical

4.6

15-250

3.5, 5

1-4 mL/min

Analytica;

1

Semi-prep

9.4

50-250

5

4-10 mL/min

Small Scale protein purification

--

Preparative

21.2

50-250

5, 7

20-60 mL/min

CombiChem purification

--

17

Protein

Peptides

Abundance Abundance Abundance

HPLC MS/MS

Enzyme

m/z

HOW IMPORTANT IS REPRODUCIBLE and HIGH QUALITY HPLC SEPARATION OF PEPTIDES ? IT ALL DEPENDS ON GOALS/NEEDS/RESOURCES

707 34.22

100 95 90

713 34.51

85 737 35.73

80 75

Poor chromatography

70 65 60 55 50 555 26.96

45

773 37.47

Relative Abundance

40 561 27.22

35

781 37.88

549 26.68

30

787 38.17

657 31.75

503 24.53

25

20 15

21 0.95

141 6.88

5

927 45.24

395 19.39

371 18.25

49 2.26

10

809 39.24

457 22.35

11 0.48

937 45.75

1031 50.75

189 9.24

1201 60.14

1185 59.33

1321 66.75

1343 67.96

1589 83.09

1509 78.21

1679 88.44

1951 105.00

1815 96.63

2091 113.80

0 0

10

20

30

40

50

60

70

80

90

100

110

Time (min)

877 44.85

100

95 90

85 80 819 41.85

75

1005 51.55

70 549 28.15

65

Decent chromatography

1207 62.22

669 34.19

60

999 51.23

55 50

765 39.07

45

907 46.41

561 28.77

40 Relative Abundance

543 27.86

1009 51.76

911 46.62

615 31.43

35

1017 52.18

507 26.02

30

499 25.59

20

489 25.08

399 20.67

15

10 5

303 15.87

139 7.22

13 0.57

1295 66.94

1145 58.94

1067 54.83

25

349 18.20

1309 67.66

1357 70.54

1441 76.14

0 0

10

20

30

40

50

60

70

1523 81.61

1653 90.46

80

1687 92.75

1817 101.40

90

1861 104.36

1945 109.90

100

2069 118.18

110

Time (min)

2345 73.52

100

95 90

Nice chromatography

85 80

169 5.24

75

201 6.22 1633 51.91

1449 46.33

70 65

1769 56.06

60 217 6.70

55

2049 64.75

50 1937 61.34

45

2509 78.31

Relative Abundance

40

35

1417 45.35

30 2937 90.80

1485 47.43

25 1385 44.38

20

3321 101.91

2065 65.29

15 2181 68.69

269 8.32

10

369 11.49

5

493 15.46

637 20.16

717 22.77

1077 34.45

873 27.87

2841 87.97

2621 81.60

1153 36.90

3265 100.25

3121 96.10

3345 102.60

0 0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

105

110

Time (min)

18

For protein identification using LC-MS/MS, chromatography may not be an issue because one can rely on mass spectrum resolving power of co-eluting peptides

For biomarker discovery, post translational modification (PTM) characterization and label free peptide quantitation, reproducible chromatography is very important

Contaminants To Avoid for LCMS/MS Applications • Ideal Salt and buffer concentrations are < 10 mM, there are various ways to clean-up the samples • Desalting very important, especially with glycoproteins, oligonucleotides, and higher mass proteins – i.e. - less peak broadening, less overall interference, less interference with matrix crystal formulation (MALDI MS applications)

• Preferred Solvents are H2O and ACN, avoid DMSO, DMF and other large polar solvents • Storing samples in glass vials (Na & K contamination) – Store samples in Sarstedt vials only (minimizes polymer contamination)

• Detergents, all types (a big no) • Protease inhibitors (remove these before sample submission) Keller et al, Interferences and contaminants encountered • Glycerol

in modern mass spectrometry. Analytica Acta 2008, 627, 71-81

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Appendix Material • Keller et al, Interferences and contaminants encountered in modern mass spectrometry. Analytica Acta 2008, 627, 71-81

Types of Separation Technologies for Molecules Method

Separation based on

Separation done using Gel (which acts like a molecular sieve) and potential

Gel Electrophoresis (1D)

Molecular mass

Gel Electrophoresis (2D)

Isoelectric point (pI; Gel, potential and IEF) & Molecular ampholytes mass

Reverse Phase (C18, Combination of hydrophobicity and C8 or C4) chromatography molecular weight

HPLC

Gel Filtration

Molecular Weight

HPLC

Ion Exchange

Cation or Anion affinity

FPLC

Affinity Chromatography “pull down”

DNA,RNA, Antibody, peptides etc

Further steps In-gel digestion of proteins to peptidesLCMS/MS or MALDI-TOF-MS

Protein(s)  Digest to peptides  LCMS/MS or MALDI-TOF-MS

HPLC

20

Affinity Chromatography 1

anti -

Identification of components

Protein complex

data mining algorithms

excise bands digest

1

MALDI -TOF MS or LC-MS/MS

SDS-PAGE

immuno precipitate proteolysis peptide mixture

LC-MS-MS

Identification of components

data mining algorithms

MS-MS spectra

Types of Separation Technologies for Molecules Method

Separation based on

Separation done using

Gel Electrophoresis (1D)

Molecular mass

Gel (which acts like a molecular sieve) and potential

Gel Electrophoresis (2D)

Isoelectric point (pI; IEF) & Molecular mass

Gel, potential and ampholytes

Reverse Phase (C8 or C4) chromatography

Combination of hydrophobicity and molecular weight

HPLC

Gel Filtration

Molecular Weight

HPLC

Ion Exchange

Cation or Anion affinity

FPLC

Affinity Chromatography

DNA,RNA, Anti-body, peptides etc

MudPIT (Multidimensional Protein Identification Technology

Cation Exchange & hydrophobicity (used for peptides; not for proteins)

Further steps In-gel digestion of proteins to peptidesLCMS/MS or MALDITOF-MS

Protein(s)  Digest to peptides  LCMS/MS or MALDITOF-MS

HPLC

HPLC

Online MS/MS analysis

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Multidimensional Protein Identification Technology (MudPIT)

SCX

RP

HPLC

1.8 kV

MudPIT

In chromatographic theory, theoretical plates of orthogonal separation columns back-to-back are multiplied rather than summed – that’s why it works

Load peptide mixture To MS

SCX

RP

Salt Bump To MS

SCX

RP

Reverse Phase Gradient To MS

SCX

RP

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Sample Clean-up/Prep Conclusions (for proteomic applications) •There are many different ways to get from protein/peptide to tandem mass spec •What you use depends on what you are trying to find out e.g. identification, structural characterization, quantitation •Choosing the best tool for the job can be very difficult and may require a combination of approaches

http://www.gbiosciences.com/

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http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-us/service-and-support/handbooks

Suggested Reading List Protein ID from Gels Wilm, M., Shevchenko, A., Houthaeve, T., Breit, S., Schweigerer, L., Fotsis, T., and Mann, M. . Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. (1996)Nature 379, 466–469. 2D gel review Gorg A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R, Weiss W. The current state of two-dimensional electrophoresis with immobilized pH gradients. (2000) Electrophoresis. 21(6):1037-53. Comparative 2D gels B.Cooper , D. Eckert, N.L. Andon, J. R. Yates III and P. A. Haynes: “Investigative Proteomics: identification of an unknown plant virus from infected plants using mass spectrometry” – (2003), J Am. Soc. Mass Spectrom., Vol 14, no. 7, 736-741.

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Mudpit Link A.J, Eng J, Schieltz D.M, Carmack E, Mize G.J, Morris D.R, Garvik B.M, Yates J.R, Direct analysis of protein complexes using mass spectrometry. (1999) Nature Biotechnology, 17, 676-682. Washburn MP, Wolters D, Yates JR III Large-scale analysis of the yeast proteome by multidimensional protein identification technology (2001) Nature Biotech. 19:242-247. TAP Tagging Rigaut G, Shevchenko A, Rutz B, Wilm M, Mann M, Seraphin B. A generic protein purification method for protein complex characterization and proteome exploration. (1999) Nat Biotechnol. Oct; 17(10): 1030-2. Gavin, A. C., Bosche, M., Krause, R., Grandi, P., Marzioch, M., Bauer, et al. Functional organization of the yeast proteome by systematic analysis of protein complexes. (2002) Nature 415, 141-147. Ho, Y., Gruhler, A., Heilbut, A., Bader, G. D., Moore, L., Adams, S. L., Millar, et al. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. (2002) Nature 415, 180-183.

Protein Modification Mapping MacCoss M.J., McDonald W.H., Saraf A., Sadygov R., Clark J.M., Tasto J.J., Gould K.L., Wolters D., Washburn M., Weiss A., Clark J.I., Yates J.R. III. Shotgun identification of protein modifications from protein complexes and lens tissue. Proc Natl Acad Sci USA. 2002 99(12):7900-7905. DIGE Unlu M, Morgan ME, Minden JS (1997). Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18:2071-2077. Somiari RI, Sullivan A, Russell S, Somiari S, Hu H, Jordan R, George A, Katenhusen R, Buchowiecka A, Arciero C, Brzeski H, Hooke J, Shriver C. (2003) High-throughput proteomic analysis of human infiltrating ductal carcinoma of the breast. Proteomics. 3:1863-73

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Carbohydrate MS/MS Lattova E, Snovida, S., Perreault, H, and Krokhin O. Influence of the labeling group on ionization and fragmentation of carbohydrates in mass spectrometry J Am Soc Mass Spectrom. 2005 May;16(5):683-96. Mass spectrometry of oligosaccharides. Zaia J., Mass Spectrom Rev. 2004 23(3):161-227. Structural characterization of NETNES, a novel glycoconjugate in Trypanosoma cruzi epimastigotes. Macrae JI, Acosta-Serrano A, Morrice NA, Mehlert A, Ferguson MAJ, J Biol Chem. 2005 Apr 1;280(13):12201-11. Epub 2005 Jan 13. Lipid and glycolipid MS/MS Tong Y, Arking D, Ye S, Reinhold B, Reinhold V, Stein DC. Neisseria gonorrhoeae strain PID2 simultaneously expresses six chemically related lipooligosaccharide structures. Glycobiology. 2002 Sep;12(9):523-33. Mycobacterial lipid II is composed of a complex mixture of modified muramyl and peptide moieties linked to decaprenyl phosphate, Mahapatra S, Yagi T, Belisle JT, Espinosa BJ, Hill PJ, McNeil MR, Brennan PJ, Crick DC. J Bacteriol. 2005 187(8):2747-57

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