High Resolution LC-MS Data Output and Analysis

High Resolution LC-MS Data Output and Analysis

Your Sample LC/MS Result Sample Sample Efficient Separation Preparation Introduction Gradient (column) Today Data (computer) LC- MS Ions Detection Ions Separation Ionization MS Interface UV Spectra

Mass Spectrometer 1. Breaks up constituents into molecular ions and other fragments 2. The ions are separated according to their mass-to-charge ratio (m/z) 3. Measures masses

Mass Spectrometer 4. Structural information on metabolite * fragmentation pattern * accurate mass

Alternative Ionization Modes Atmospheric Pressure Ionization (API), in LC-MS Electrospray Ionisation (ESI): polar and semi-polar Atmospheric Pressure Chemical Ionization (APCI): less polar APCI ES lipids polarity of analyte molecule water

Positive or Negative Modes? The formation of positive or negative ions depends on the sign of the applied electrical field ES+: (M+H) + Good ionization of basic compounds (get proton) E.g. amino, amide, ester, aldehyde/keto functional groups (formic acid in sample solution to help ionize) ES-: (M-H) - Acidic Compounds (give proton) E.g. organic acids, containing OH (ammonium buffer in sample solution to help ionize)

Mass Analyzers Ion Cyclotron (FT-ICR-MS) Time of Flight (TOF) High Resolution Instruments Magnetic Sector Quadrupole Ion Trap Quadrupole Low Resolution Instruments

Mass Analyzers Ion Cyclotron (FT-ICR-MS) Mass Accuracy <1ppm Time of Flight (TOF) 3-1ppm Magnetic Sector 2-5ppm Quadrupole Ion Trap Quadrupole n/a n/a

LC-PDA-QTOF-MS of a Tomato Sample PDA-sample MS-reference (lock mass) injected every few scans (TIC) MS-sample - Total Ion Chromatogram (TIC)

Spectra Obtained at Each Scan PDA-sample MS-reference (lock mass) Enkephalin (556.2766 in ES+) MS-sample

Extracted chromatogram of a Specific Ion Extracted chromatogram at m/z 33 Extracted chromatogram at m/z 611 TIC

LC/UV/MS Chromatograms of Standard s Mixture (UV at 24nm, ES+ and ES-) Standard Mix MixAllStand_b1 8.e-1 6.e-1 AU 4.e-1 2.e-1 2.26 2.67 3.26 3.68 3.63 3.38 17 1.5 2. 2.5 3. 3.5 4. 4.5 5. 5.5 6. 6.5 9-Jan-26 3: Diode Array Range: 1.11 1.66. 1.5 2. 2.5 3. 3.5 4. 4.5 5. 5.5 6. 6.5 MixAllStand_b1 1 TIC 5.16 1.18e4 1.69 ILMixAllStand-c4 1 1 2.28 1 2.27 2.22 2 2 2.69 3 4 4.6 4.95 6 4 5 7 9 3 3.5 3.65 3.71 4.98 3.28 2 4 2.68 5 3.37 6 7 8 4.54 9 5 3 6 7 3.25 3.68 3.95 8 4.58 9 4.92 11 5.43 11 5.45 11 5.42 12 13 14 15 6. 6.13 5.92 12 5.93 13 14 6.2 6.7 13 12 5.99 5.91 14 15 6.1 15 6.13 6.46 UV MS (+) Time 1-Jan-26 1: TOF MS ES- TIC 8.88e3 MS (-) 6.37 6.72 6.77 1 Chlorogenic acid 2 Caffeic acid 3 Rutin 4 p-coumaric 5 Quercetin-3-glucoside 6 Ferulic acid 7 Sinapic acid 8 Benzoic acid 9 Morin (Internal Standard) 1 Tomatin 11 Quercetin 12 Cinnamic acid 13 Naringenin Chalcone 14 Naringenin 15 Kaempferol 9 1.5 2. 2.5 3. 3.5 4. 4.5 5. 5.5 6. 6.5 Time

In the Spectra: Molecular Ion -An unfragmented (parent) ion formed by loss or gain of an proton from a molecule - Has the same mass as the metabolite being analyzed (+ H or - H) The Molecular Ion

Chromatogram and Spectrum of Morin ES(-) [M-H] - ES(+) [M+H] + Chromatograms in ES(+) & ES(-) modes Spectra's in ES(+) & ES(-) modes

Adduct Formation in Spectra (Na) Rutin C 27 H 3 O 16 [M+Na] + C 27 H 3 O 16 Na C 27 H 31 O 16 [M+H] + Na (22)

S5 Typical Adducts formed in LCMS Adducts Ion [ M+CH 3 COO ] - Source of Adduct Acetic acid Mass of Adduct Ion M+59 [ M+Cl ] - Chlorinated solvent M+35 [ M+NH 4 ] + [ M+Na ] + [ M+K ] + Ammonia Sodium Salt Potassium Salt M+18 M+23 M+39 [ M+CH 3 CNH ] + [ M+CH Methanol M+33 3 OHH ] + Acetonitrile M+42 [ M+H 2 O] + Water M+19

Isotopes in Spectra ( 13 C) Rutin C 27 H 3 O 16 [M+Na] + C 27 H 3 O 16 Na 13 C (little from other isotopes H, O) C 27 H 31 O 16 [M+H] +

Abundance of Isotopes (in Spectra)

An example of how isotopes can aid in peak identification A (5.6) A + 2 (49.3) The ratio of peaks containing 79 Br and its isotope 81 Br (/98) confirms the presence of bromine in the compound.

Fragments of a Metabolite in Spectra MS spectrum of Rutin in ES(+) Quercetin [M+Na] + Glucose [M+H] + Rhamnose Glucose (162) Rhamnose (146)

Spectra Components Molecular Ion Fragments of Metabolite Adducts (from solvent or di-tri tri-mers) Isotopes

Spectrum of Quercetin Trisaccharide (a Rutin Derivative) loss of pentose residue 743.19 611.16 = 132.3 765.1812 [M+Na] + Rutin Quercetin 33.515 743.1995 [M+H] + 743 + sugar UV spectra of Rutin and Quercetin Trisaccharide 469.132 Rutin 391.198 611.1645 766.1797 76.834 47.1434 471.1271 612.1766 613.1899 767.1795 827.1555 828.1354 1133.4736 2 3 4 5 6 7 8 9 1 m/z

Mass Accuracy and Structure Elucidation High mass accuracy (1 mda) Low mass accuracy ( mda)

Mass Accuracy and Structure Elucidation Selected natural compounds with formula C 18 H 19 NO (monoisotopic mass 313.1314 Da)

Performing MS-MS: MS-MS of m/z 435 at different collision energies CE=3 ev CE=15 ev CE=1 ev CE=5 ev

Output Data For High Res. LC-MS Based Metabolomics Nominal Accurate

Software for comparing full-scan datasets MetAlign method Peak picking Alignment Statistics

Software for comparing full-scan datasets MarkerLynx method

Out-put from the MarkerLynx (Waters) Program : Analyses of Wild-Type Tomato Peel (Different Stages of Development; ES+) Samples Processed Markers Detected TIC of currently selected sample Response of a marker across all samples Principle Components Analysis Markers responsible for clustering (by PCA)

A Case Study in Tomato Small Green Middle Green Big Green Breaker Orange Red

Sample Preparation for Tomato Fruit Metabolome Analysis Harvesting tomato fruit at different developmental stages Separation of peel and flesh tissues Immediate freezing in liquid nitrogen Grinding to fine powder Weighing frozen samples Extraction in MeOH by vortexing and sonication Analyzing with UPLC-MS [ ES (+) and ES (-) ]

Experimental Set-Up for LC-MS Analysis: PEEL and FLESH during tomato fruit development Green stage Flesh Peel ES+ ES- ES+ ES- ES+ ES- ES+ ES- Breaker stage Flesh Peel Orange stage Flesh Peel Red stage Flesh Peel ES+ ES- ES+ ES- ES+ ES- ES+ ES-

UPLC-Q-Tof-MS Analysis of Four Stages of Tomato Fruit Development Tom_24_WTa7.8 1.5 AU 5.e-1 Tom_22_WTa6.8 1.5 AU AU 5.e-1 Tom_18_WTa4.8 1.5 AU 1.. 1. 1.. 1. 5.e-1. 2.91 2.26 2.4 3.25 5.98 4.94 3.68 4.13 5.37 2. 4. 6. 8. 3.24 5.97 2. 4. 6. 8. 2. 4. 6. 8. 9-Jan-26 3: Diode Array Range: 4.36 3: Diode Array Range: 6.418 5.e-1 4.94 1.2 2.25 2.9 3.674.21 5.36. 2. 4. 6. 8. Tom_2_WTa5 3: Diode Array.8 5.99 1.5 Range: 2.955 1.2 2.26 2.91 3.25 3.26 2.26 1.2 2.91 4.95 3.68 4.13 7.67 4.95 3.68 7.67 Red Orange Breaker 3: Diode Array Range: 3.2 Green Time Tom_24_WTa7.84.7 1.9 6.1 1.81 2.4 5.17 3.78 4.23 8.33 1. 2. 3. 4. 5. 6. 7. 8. 9. 9-Jan-26 TIC 5.e4 1. 2. 3. 4. 5. 6. 7. 8. 9. Tom_22_WTa6 TIC 5.e4.85.7 6. 5.16 1.9 1.81 2.4 4.23 3.263.78 7.71 1. 2. 3. 4. 5. 6. 7. 8. 9. Tom_2_WTa5 TIC 5.e4 5.13.85.7 5.9 1.9 3.78 4.225. 2.4 2.59 6.2 7.69 7.39 7.86 1. 2. 3. 4. 5. 6. 7. 8. 9. Tom_18_WTa4 5.13 TIC 5.e4.84.7 1.9 1.82 2.41 3.78 5. 3.28 6.13 7.38 7.69 6.51 7.94 Red Orange Breaker Green Time UV (24 nm) MS (ES(+),TIC)

Out-put from the MarkerLynx (Waters) Program : Analyses of Wild-Type Tomato Peel (Different Stages of Development; ES+)

Peel-Flesh Profiles During Fruit Development Red flesh Red peel Orange peel Component 2 Orange flesh Breaker flesh Breaker peel Green flesh Green peel Comp. 1

Wild-type Tomato Peel, Different Developmental Stages Red DOWN NO CHANGE9-Jan-26 Tom_24_WTa7 UP 9-Jan-26 Tom_24_WTa7 Tom_24_WTa7 3.27 33 578.4 42 6.1 1.e4 Area 731 Area 3.63 5.17 4.31 1 48 3 2. 4. 6. 8. Tom_22_WTa6 578.4 1.e4 Area 1. 2. 3. 4. Tom_22_WTa6 2.93 8 3.27 4 33 Area 2. 4. 6. 8. Tom_22_WTa6 6. 1158 9-Jan-26 273 2.e4 Area 273 2.e4 Area Orange Breaker 5.16 9 4.31 4 2. 4. 6. 8. Tom_2_WTa5 5.13 362 5.7 8 2. 4. 6. 8. Tom_18_WTa4 5.12 59 578.4 1.e4 Area 578.4 1.e4 Area 1. 2. 3. 4. Tom_2_WTa5 33 3.28 36 Area 2.94 7 2.93 11 1. 2. 3. 4. Tom_18_WTa4 33 3.27 35 Area 2. 4. 6. 8. Tom_2_WTa5 273 2.e4 Area 6.3 357 2. 4. 6. 8. Tom_18_WTa4 273 2.e4 Area Green 5.24 1 2. 4. 6. 8. Time 2.93 7 1. 2. 3. 4. Time 2. 4. 6. 8. Time Tomatine Rutin & Quercetin trisacharide Naringenin chalcone

Tomato (var. Ailsa Craig) and the Mutant Y, at different ripening stages Green Breaker Orange Red Wild-Type Mutant Y Tomato peel of Mutant Y and Wild-type

Biosynthesis of Naringenin Chalcone in the Flavonoid Pathway Phenylalanine Cinnamate Coumarate Coumaroyl - CoA Chalcone synthase (CHS) Naringenin chalcone Naringenin Lignins, Coumarins, Chlorogenic-acid Anthocyanines Flavonols Condensed tannins

Experimental Set-Up for LC-MS Analysis: PEEL and FLESH during Y fruit development Y Green stage Flesh Peel ES+ ES- ES+ ES- ES+ ES- ES+ ES- Y Breaker stage Flesh Peel Y Orange stage Flesh Peel Y Red stage Flesh Peel ES+ ES- ES+ ES- ES+ ES- ES+ ES-

HPLC-QTOF QTOF-MS (Ultima-PRI) - Wild Type and Y Peels 1 st principal component (X axis) - developmental stage (47.1) 2 nd principal component (Y axis) mutation (9.4) WT-Orange All breaker WT- Red Y -Orange All green Y -Red

UV and MS Chromatograms of Red Tomato Peel (Wild Type and Mutant Y ) Tom_56_WTa15.84.89.7 1.9 1.81 Mutant Y 5.18 2.4 3.78 4.24 3.64 4.98 2.6 4.86 5.27 5.75 7.95 8.2 8.31 9-Jan-26 TIC 2.11e4 Tom_56_WTa15.8 2.5 2. 1.5 1. AU Mutant Y 3.25 IS 9-Jan-26 3: Diode Array Range: 2.916 1. 2. 3. 4. 5. 6. 7. 8. 9. Tom_8_WTa3.85 TIC 2.12e4.7 Wild Type 1.9 1.81 5.16 6.2 5.e-1. 1. 2. 3. 4. 5. 6. 7. 8. 9. Tom_8_WTa3 3: Diode Array.8 Range: 3.22 2.5 2. 2.91 1.9 2.4 2.2 3.68 Wild Type 4.95 2.41 3.28 3.62 2.94 3.77 4.23 4.98 1. 2. 3. 4. 5. 6. 7. 8. 9. 5.43 6.46 7.63 7.9 8.32 Time AU 1.5 1. 5.e-1. 1.2 2.3 2.33 2.9 3.25 4.94 3.68 4.13 5.37 5.99 1. 2. 3. 4. 5. 6. 7. 8. 9. Time UV (24 nm) MS (ES(+),TIC)

The Yellow Pigment Absent in the Y Mutant Fruit Peel is Naringenin Chalcone Tom_8_WTa3 13 6.2 TIC 1.37e4 273.797 2.35e4 5.75 Tomato peel 274.554 Tomato peel 5.7 5.8 5.9 6. 6.1 6.2 6.3 6.4 MixAllStand_b1 6.2 13 14 TIC 6.1 8.16e3 6.15 5.69 12 5.93 15 6.416.46 147.5 275.623 357.263 583.1375 855.1465 629.711 987.4974 1151.2333 273.789 7.9e3 Standard s mixture 274.617 Naringenin Chalcone Standard 5.7 5.8 5.9 6. 6.1 6.2 6.3 6.4 Time 153.22 357.297 583.132 651.2269 855.1419 111.27516.111 m/z 2 3 4 5 6 7 8 9 1 12 Cinnamic acid 13 Naringenin Chalcone 14 Naringenin 15 Kaempferol

Unknown Mass peak (RT=3.51 min) Detected Only in Wild Type Tom_72_WTa19 2. 4. 6. 8. 9-Jan-26 621.1 3 7.44 8.39 2. 4. 6. 8. Tom_56_WTa15 621.1 3 5.47.74 3.8 5.7 5.75 7.69 7.868.4 2. 4. 6. 8. Tom_4_WTa11 621.1 3.51 3 Tom_24_WTa7 Tom_8_WTa3 2.35 5.7 5.47 7.37 2. 4. 6. 8. 621.1 3 3.51 3.64 5.48 7.5 8.39 2. 4. 6. 8. 3.51 621.1 3.76 2.34 7.54 Time Mutant Y Mutant Y Wild Type Wild Type Wild Type Extracted mass chromatogram at m/z 621.1 Da

Unknown Mass peak Detected in Higher Levels in Y Mut. Y Mut. Y Wild-type Wild type Wild type Tom_72_WTa19 1.8 1.85 1.9 1.95 2. 2.5 2.1 2.15 Tom_56_WTa15 2.1 1 1.8 1.85 1.9 1.95 2. 2.5 2.1 2.15 9-Jan-26 265.1 2.e3 Area 1.8 1.85 1.9 1.95 2. 2.5 2.1 2.15 Tom_4_WTa11 265.1 2.e3 Area 2.1 11 1.8 1.85 1.9 1.95 2. 2.5 2.1 2.15 Tom_24_WTa7 265.1 2.e3 Area 2.9 11 1.8 1.85 1.9 1.95 2. 2.5 2.1 2.15 Tom_8_WTa3 265.1 2.e3 Area 2.9 7 2.1 67 265.1 2.e3 Area Time Extracted mass chromatogram at m/z 265.1 Da

In-depth Analysis of Red Tomato Peel (ES+) Wild-type vs. the Y Mutant MarkerLynx Differential Markers - Filtering out non-replicated data - Identifying molecular ions, isotopes, adducts, fragments and passing from Markers to Metabolites - Analysis of differential metabolites and identification

What are the Differentially Expressed Metabolites between WT and Y? Peak assignment by: - Accurate mass and elemental composition (MassLynx and natural products database) - MS-MS fragmentation - UV spectrum - Literature - UV & MS spectra databases (e.g. for tomato in PRI)

Differential Metabolites between the "Y" and Wild Type Peel UPLC-QTOF-MS in ES- Mode Differential metabolites (ES-) R only O only Both R & O Total WT > "Y" 1 12 26 48 "Y" > WT 11 16 4 31

Differential Metabolites between the "Y" and Wild Type Peel UPLC-QTOF-MS in ES+ Mode Differential metabolites (ES+) R only O only Both R & O Total WT >"Y" 3 16 19 "Y" > WT 2 2 8 12

What are the Differentially Expressed Metabolites between WT and Y? Most mass peaks showing higher levels in WT than in the Y mutant are derivatives of Naringenin Chalcone or Naringenin.

Biosynthesis of Naringenin Chalcone in the Flavonoid Pathway Phenylalanine Isomers (identical mass) Cinnamate Coumarate Coumaroyl - CoA Chalcone synthase (CHS) Naringenin chalcone Naringenin Lignins, Coumarins, Chlorogenic-acid Anthocyanines Flavonols Condensed tannins

Hydroxylated Naringenin & Naringenin- Chalcone Ilana wt-tom rp 23 M5983 Y HPLC-QTOF 289.7.5Da 875 M5982 26.52 127.537 27.5 3. 32.5 35. 37.5 4. 42.5 WT 37.5 289.717 35.79 289.79 289.7.5Da 875 28.34 58.7736 29.95 949.259 42.64 273.382 27.5 3. 32.5 35. 37.5 4. 42.5 Time

UV Spectra of Differential Peaks (35.7 min. and 37.5 min.) AU 1.e-2 8.e-3 6.e-3 4.e-3 225.6 287.6 Naringenin 1.126e-2 AU 6.e-1 4.e-1 2.e-1 211.574 366.574 6.238e-1 Naringenin Chalcone 2.e-3.. M5982 2135 (35.667) Cm (2131:2138-2149:216) 286.6 2.e-3 1.5e-3 1.e-3 5.e-4 AU 3: Diode Array 2.195e-3 Peak at 35.7 min M5982 229 (36.9) Cm (229-(2223+2196)) 211.574 376.574 AU 4.e-3 2.e-3 3: Diode Array 5.444e-3 Peak at 37.5 min. 225 25 275 3 325 35 375 4 425 45 475 nm. nm 25 3 35 4 45 5 55 6

Spectra of differential compounds at RT=35.79 min and 37.5 min RT=35.79 min RT=37.5 min

Elemental Composition (289.712) Elemental composition: C 15 H 12 O 6 for neutral compound Elemental composition of Naringenin: C 15 H 12 O 5 Difference: OH group instead of H

Suggested Metabolite MS-MS Fragmentation of m/z 289.7 289.717 274 163.419 153.219 29.741 Eriodictyol (C 15 H 12 O 6 ) Main fragmentations in reference: I. J. M. S. 247 (25) 93 179.395 271.564 125 15 175 2 225 25 275 3 325 MS-MS fragmentation of m/z 289.717 m/z

Naringenin and Naringenin Chalcone Glycosides HPLC-QTOF Ilana Y-tom rp 55 M5983 435.12.1Da 1.41e3 28. 3. 32. 34. M5982 28.72 435.1274 28.72 929.2811 29.22 565.762 3.53 517.1318 32.8 457.238 32.79 457.286 33.42 528.761 Y WT 435.12.1Da 1.41e3 28. 3. 32. 34. Time

UV Spectra of Peak at 32.8 min. and Naringenin Chalcone M5982 2544 (42.483) Cm (2544-(2562+2526)) 366.6 AU 6.e-1 211.6 4.e-1 3: Diode Array 6.774e-1 Peak at 32.8 min 2.e-1. M5982 1954 (32.65) Cm (1954-(1965+1944)) 366.6 211.6 3: Diode Array 3.933e-3 AU 3.e-3 2.e-3 Naringenin Chalcone 1.e-3. 555.6 nm 25 3 35 4 45 5 55 6

MS spectra at RT=28, 3 32 and 33 min RT=28.7 min For MS-MS RT=3.5 min Exact mass of 435.1273 shows elemental composition of C 21 H 22 O 1 RT=32.8 min RT=33.4 min

MS-MS Fragmentation of m/z 435.12 for Peak at RT=32.8 Peak at 32.8 M67 47 (33.469) Cm (45:411) Naringenin Chalcone 273.735 Naringenin Chalcone-hexose 2: TOF MSMS 435.13ES+ 1.59e3 435.126 Hexose (m/z 162) 153.214 154.275 274.769 275.669 436.1255 437.1289 15 2 25 3 35 4 45 m/z

Mass Peak at RT=43.4min (>WT) MS spectra at RT=43.4min Selected Mass Chromatogram m/z 33.8 Da TIC chromatogram Mut Mut Mut WT WT WT

Methyl ether of Hydroxylated Naringenin and Naringenin Chalcone Peaks at 43.4 min and 44.6 min - Calculated formula is C 16 H 14 O 6 based on exact mass - Proposed structure for the peak at 43.4 min: Hesperetin or a B-ring positional isomer

MS-MS Fragmentation of m/z 33.8 (43.4 min.) Ilana wt-tom rp 23 msms M67 55 (43.325) Cm (55:56) 153.225 15. 2: TOF MSMS 33.8ES+ 33.883 369 [M+H] + 177.581 145.342 [M+H-H 2 O] + 154.266 179.368 34.926 117.441 35.851 18.453 285.826 12 14 16 18 2 22 24 26 28 3 32 m/z 153 179 177 Hesperetin

Proposed Scheme of Naringenin & Naringenin Chalcone Hydroxylation, Methylation and Glycosylation Glycosylation OH HO OH OH O Naringenin Chalcone HO O B A C OH O Naringenin OH OH OH OH O OH O OH OH Glycosylation e.g. Naringenin glucoside O O OH OH OH OH Glycosylation HO OH HO O Glycosylation OH O Hydroxylated Naringenin Chalcone OH O Hydroxylated Naringenin e.g. Eriodyctyol or a B-ring isomer OH OMe OH OMe Glycosylation HO OH HO O Glycosylation OH O OH O Methyl ether of Hydroxylated Naringenin Chalcone Methyl ether of Hydroxylated Naringenin e.g.hesperetin or a B-ring isomer

Biosynthesis of Naringenin Chalcone in the Flavonoid Pathway Phenylalanine Cinnamate Coumarate Coumaroyl - CoA Chalcone synthase (CHS) Naringenin chalcone Naringenin Lignins, Coumarins, Chlorogenic-acid Anthocyanines Flavonols Condensed tannins