1. 1 The world leader in serving science I Orbi 4 2012 Tim Stratton Optimizing the Metabolism Experiment:Small Molecule Structure ID and the Orbitrap

2. 2 General Topic Outline 1.Setup and Operation Tips 2.“True” High Resolution – What it means for you 3.Fragment based detection – applications of FISh for component detection. 4.Complex matrix analysis – tricks for single and multiple injection approaches

3. 3 Orbitrap Mass Spectrometers Benchtop Orbitraps Exactive Full scan and AIF scan only 100,000 max resolution Exactive Plus Full scan and AIF scan only (AIF with optional HCD Cell) Upgradable to Q Exactive Q Exactive Quadrupole Isolated MS2 140,000 max resolution Hybrid Orbitraps LTQ Orbitrap XL MSn 100,000 max resolution CID / HCD Orbitrap Velos Pro MSn @ 66,000 amu/sec nominal 100,000 max resolution CID / HCD Orbitrap Elite MS n @ 66,000 amu/sec nominal 240,000 max resolution CID / HCD

4. 4 Method Considerations - The Q Exactive

5. 5 The New Tune Page Actions Trays Instrument Status Real Time Scan View Instrument Status Messages Stoplight

6. 6 The New Tune Page The Tune file for benchtop Orbitraps only controls the source conditions. AGC and inject times are controlled directly in the method. Important: Remember to put source conditions for BOTH polarities in the Tune file if you want to use Polarity switching.

7. 7 Calibration – Ion Stability With correct calibration mix flowing the TIC variation should be less than 15%.

8. 8 Calibration Calibration is a one button procedure. Set MS Mass Calibration (pos) or (neg) for the current polarity and cal solution. Calibration is ~15 seconds

9. 9 Calibration Calibration performance is measured by the root mean square (RMS) deviation in value over the 20 calibration scan events. A value of 0.3/0.4 or less is ideal

10. 10 Evaluation The evaluatation procedure can check multiple performance characteristics and provide a detailed window on performance. Evaluate with calibration mix infusing.

11. 11 Critical Method Parameters – Q Exactive

12. 12 Critical Method Parameters – Q Exactive By setting “Show all properties” to True we display several more options: In source CID Microscans Number of Scan Ranges MSX Count Stepped NCE

13. 13 Critical Method Parameters – Q Exactive At the top of the method editor are a series of collapsible entries: Global Lists Tune Files External Hardware Chromatogram Scan Groups

14. 14 Critical Method Parameters – Q Exactive Global Lists Here we set the inclusion, exclusion, neutral loss, and Mass Tag lists. Neutral loss is important for one of the key Q Exactive methods – NL- triggered MS 2 Mass tags are used to trigger on stable labeled (labeled GSH, 18 O, etc) or on native isotopes (Cl, Br)

15. 15 Critical Method Parameters – Q Exactive Tune Files Here we set the tune file(s) to use during acquisition. Typically only one tune file is required. If more than one tune file is used, a swtiching time is set in the Properties. Note: Be sure to include both polarities in the Tune file if you wish to use polarity switching or have a general use Tune method

16. 16 Critical Method Parameters – Q Exactive External Hardware We can set the divert valve settings and also contact closure (for external detectors) here.

17. 17 Critical Method Parameters – Q Exactive Critical Parameters General Default charge state: Set to 1 for most small molecule work Full MS Microscans: Set to 1 for most work. Resolution: Set based on desired data use. For fine isotope work, 70-140K is best. For full scan quantitation in complex matrices, 70k is optimal for most applications unless peak width is ≤ 3 seconds, then 35k is suggested 35,000 = 7.5 Hz 70,000 = 4 Hz 140,000 = 2 Hz AGC Target (Automatic Gain Control): Desired number of ions in each scan. The higher this value is, the more injection time (IT) you will need to fill the C-Trap. Typical values for this depend on the application but generally: ID Full Scans: 5e5 – 1e6 MS 2 scans: 1e5 – 2e5 AIF scans: 5e5 – 1e6 SIM scans (quant): ~5e4 Multiplex SIM: 2e4 – 5e4

18. 18 Critical Method Parameters – Q Exactive Critical Parameters Full MS (continued) Max IT: (Maximum Inject Time) – It is best to set this value based on the resolution setting. Chosing a Max IT just shorter than the Orbitrap Time (OT) makes the most use of the ions coming in. The higher the AGC target, the higher Max IT will need to be for very low level analytes. Note however that for Multiplexed scans, the Max IT will be the value for EACH multiplexed scan – for multiplexed scans, reduce the Max IT. (i.e. a 5-plex should have a Max IT 3-5 times lower than if you were not multiplexing)

19. 19 Critical Method Parameters – Q Exactive Critical Parameters dd-MS2 / dd-SIM (Data dependant) Microscans – Set to 1. Usually you will not need more microscans. Loop Count and MSX Count Set MSX = 1, Loop Count = 1 – 5. Together these two values determine the “Top N” of the experiment. MSX count is the number of precursor ions that should be multiplexed together and Loop count is the number of MS/MS scans that should be performed. If you have 2 ions multiplexed together (MSX=2) and loop 3 times (Loop Count = 3) then you perform MS/MS on a total of 6 precursors, with 2 precursors fragmented together in 3 scans. To clarify, the “Top N” line displays the total number of ions fragmented. Isolation Window – isolation width of the quadrupole. Valid values are 0.4 – 20 AMU. Typically – 1.0 to 2.0 AMU.

20. 20 Critical Method Parameters – Q Exactive Critical Parameters dd-MS2 / dd-SIM (Data dependant) Fixed First Mass – Set the low end of the fragment scan range to the entered value. Useful for cutting off the low mass end of a fragment scan when doing MRM on a heavy mass fragment. NCE – Normalized collision energy. This value, as a percentage (valid values 0 – 200%) is used to determine the actual energy applied for the m/z value being fragmented. The true energy can be seen in the.raw file. Stepped NCE – This value, also a percentage, is the extra “window” that is applied around the NCE. It is determined by multiplying the NCE by the Stepped value, this percentage is added and subtracted to the NCE. (i.e. An NCE of 50%, Stepped NCE of 40% gives three scans – 30, 50, and 70%). Three discrete fragmentations are performed and all fragment ions are measured in the same Orbitrap scan.

21. 21 Critical Method Parameters – Q Exactive Critical Parameters dd-Settings (Data dependant) Underfill ratio – This can be thought of as a “floating threshold”. This value is the percentage of the desired AGC target that must be met within the Max IT time set for an ion to be considered valid for a data dependant scan. As a guideline, the “Intensity Threshold” line below the Underfill ratio displays a general intensity reference “Complete” MS 2 for ID: 5 – 10% Triggering on intense signals: 20% Dynamic Exclusion – Very important setting for unknown analysis and complex matrix ID work. Turn ON by setting to an exclusion duration time in seconds. Typically, set ≈ to average peak width.

22. 22 Critical Method Parameters – Q Exactive Critical Parameters dd-Settings (Data dependant) Appex Trigger – Optional. A set of two values (times in seconds) that are used to determine when to schedule a data dependant event to occur. When an ion meets the data dependant trigger criteria, the scan event is scheduled to occur after the first time has elapsed but before the second time has elapsed. Consider a peak eluting, rising from the baseline. As the ion meets the selection criteria, the first time delays the triggered scan until the peak elutes closer to the peak top while the second time prevents the entire peak from eluting before we get the triggered scan. First value = Normal peak width / 3 in seconds. Second value = Normal peak width minus 2-4 seconds. Example: For a run with peaks of average width 10 seconds, Set 3 to 8 sec. Exclude Isotopes – Prevents isotopes of an intense triggered mass from being fragmented allowing other important precursors to be fragmented.

23. 23 Underfill Ratio and Apex Time Triggers UR limit here. MS 2 Scan here. UR 1% Triggering 651.1541

24. 24 Low Intensity MS 2 Scan (Low AGC Target Set)

25. 25 Underfill Ratio and Apex Time Triggers UR limit later. MS 2 Scan here. UR 5% Triggering 651.1541

26. 26 Higher Intensity MS 2 Scan – Same AGC Target Set

27. 27 Underfill Ratio and Apex Triggering Underfill Ratio and Apex Trigger effect each other Raising UR delays triggering  Puts us closer to the Apex  Requires a lower Apex time (first and last) First value “Wait time” Second value “Max time”

28. 28 Apex Triggering Underfill Ratio Peak width = 11 sec. (6.48 – 6.66 min.) 2 - 4 sec. Intensity Triggers UR. First value is the “wait time” after an ion would trigger MS 2

29. 29 Apex Triggering Underfill Ratio Peak width = 11 sec. (6.48 – 6.66 min.) 2 - 4 sec. Intensity Triggers UR. Second value is the max time allowed before MS 2 must be gathered.

30. 30 Apex Triggering Underfill Ratio Peak width = 11 sec. (6.48 – 6.66 min.) 2 - 4 sec. Intensity Triggers UR. Second value – First value = Window In this case, a 2 second window is used

31. 31 Effect of Changing UR Underfill Ratio Peak width = 11 sec. (6.48 – 6.66 min.) 2 - 4 sec.

32. 32 Effect of Changing UR Underfill Ratio Peak width = 11 sec. (6.48 – 6.66 min.) 2 - 4 sec.1 - 3 sec.

33. 33 Setting UR and Apex. Underfill Ratio values (assuming an AGC of 5e5) 5% is good if you want to trigger on everything, including noise and baseline 10% is a robust average value for both clean in vitro and “dirty” in vivo samples. Very little will be missed but we will still trigger on some noise. 20% will limit triggering to higher intensity components but small metabolite peaks will still be triggered. Smaller peaks will trigger late and require a lower Apex start time. Apex Values For most applications Max time = Peak width / 2 Wait time = Max time / 2 When a wide range of peak intensities is expected (large and small metabolites) and UR ≥ 10%, widen the window by: HPLC conditions – Take 2 seconds off Wait time (minimum 1 sec) UHPLC Conditions – Take 1 second off Wait time (minimum 1 sec)

34. 34 Isolation Width and Transfer Efficiency

35. 35 Isolation Width and Transfer Efficiency

36. 36 Backing up Master Calibration Files – Exactives The files we need (plus some extra backup copies in this case) C:/Xcalibur/System/Exactive/Instrument/msx_instrument_files

37. 37 Backing up Master Calibration Files – Exactives Original Config file Three “original” master cal files Default tune files Copy the Contents (all files) of the msx_instrument_files folder to the new PC in the same location.

38. 38 Backing up Master Calibration Files - Exactives When to Back-up your Master Cal Files When upgrading Xcalibur / Foundation software Every 6 months (disaster prevention) When to use Master Cal File Back-ups Installing on a new PC – the default factory Cal files installed with a fresh installation of Exactive Family Drivers may no longer be a suitable starting point for an instrument that has been in the field. Your back-up Master Cal files are better. Install Foundation Install Xcalibur Install Exactive Family Drivers Copy over your backup Cal files Calibrate the instrument in positive and negative. Perform an evaluation.

39. 39 Method Considerations – Hybrid Orbitraps

40. 40 Critical Parameters - Hybrids

41. 41 Critical Parameters - Hybrids Data Dependant Scan Settings

42. 42 Critical Parameters - Hybrids Dynamic Exclusion One of the most important settings – always have it on Exceptions – When only doing targetted applications – Quan, Known screening, etc.

43. 43 Critical Parameters – Dynamic Exclusion Dynamic Exclusion The ability to acquire MS 2 / MS n data on as many things as possible allows us to find metabolites during processing, however Peaks co-elute. MS n What we want.

44. 44 Critical Parameters – Dynamic Exclusion Dynamic Exclusion Dynamic exclusion lets you drill down to trigger MS/MS on lower intensity peaks without having to build a targeted inclusion list. Without it, you just gather scan after scan on the most intense things. Endogenous Adjusted to the same vertical scale. MS n What we’d get.

45. 45 Critical Parameters - Hybrids Targeted, Untargeted, and Combined Triggering Even if you are using an inclusion list, when doing ID work it’s good to gather fragmentation data on multiple targets to help uncover unexpected metabolites.

46. 46 “Very High Resolution” – What can we do with true high resolution data

47. 47 What Can We Do with True High Resolution Fine Isotopic Structure Atom counting Requirements for Fine Structure Resolution

48. 48 Why Do We Need True High Resolution? Ziprasidone Metabolism (60 min, Human Liver S9, 0.2 µM) Orbitrap Elite – 240,000 Resolution Full Scan Analysis

49. 49 Why Do We Need True High Resolution? Ziprasidone Metabolism (60 min, Human Liver S9, 0.2 µM) Extremely minor oxidative metabolite (t R =4.98 min) Less than 1% of Ziprasidone peak

50. 50 Why Do We Need True High Resolution? Looking into the Isotopic Pattern Clearly we see the Chlorine isotope pattern. But what more can we see with high resolution?

51. 51 Why Do We Need True High Resolution? Resolving Chlorine and Carbon Fine isotopic structure: True confirmation of elemental composition only possible with true high resolution. 37 Cl 2 X 13 C

52. 52 Considerations for Fine Structure - Elite

53. 53 Considerations for Fine Structure - Elite

54. 54 Considerations for Fine Structure - Elite

55. 55 Real World Application - Confirmation Human urine sample post dosing with omeprazole Analysis at 70,000 resolution (FWHM @ m/z 200)

56. 56 Confirmation with Fine Isotope Structure Human Urine 0-3 hours Post Dose (Raw data)

57. 57 Confirmation of Metabolite Suspected metabolite

58. 58 Resolution and Identification – Fine Isotope Structure C 16 H 18 N 3 O 2 S (-CH 2 O) -0.43 ppm 34 S 2X 13 C

59. 59 Using High Resolution: Counting Atoms Splitting fine isotopes allows for: Confirmation of elemental composition Confirmation of a component being a metabolite (same fine pattern as parent) What about isotope ratios? Can we split elements and count the atoms?

60. 60 Real World Example – Melamine in Albumin Melamine analysis in pharmaceutical excipients was a hot topic following contamination events in China N=6 Melamine N=5 Ammeline N=4 Ammelide N=3 Cyanuric Acid

61. 61 Melamine Injection – Real Data Scan at Peak Apex Clear split of 15 N and 13 C in the A 1 isotope peak of Melamine. Does the isotopic ratio match? Can we count the number of N in a structure? A1A1

62. 62 Using High Resolution: Counting Nitrogen To count nitrogen we need to measure the isotope ratio of the 15 N signal in A 1 [A 1,N ] and the 13 C signal in A 1 [A 1,C ] As the number of nitrogen atoms (#N) increases the value of the ratio [A 1,N ] / [A 1,C ] increases If we know #C, then we can calculate #N We can get a reasonable estimate of #C from: Relative Abundance 13 C A 1,C A 0,C / = # C (3 for our case) Note: This formula provides an estimate only, however it breaks down for as MW increases (>400 MW) and molecules where carbon makes up less than ~65% of the non-hydrogen atoms.

63. 63 Using High Resolution: Counting Nitrogen Estimating the value of #N can be performed by recognizing that the ratio of 15 N to 13 C in A 1 is reflected by: Which gives an equation for #N: Our observed 15 N/ 13 C ratio calculated a value of 6 nitrogens. A 1,N A 1,C = #N X Relative Abundance 15 N #C X Relative Abundance 13 C #N = A 1,N A 1,C Relative Abundance 15 N #C X Relative Abundance 13 C X

64. 64 Isotopic Ratio of 15 N / 13 C A1 Across the Peak Theoretical Ratio (For 5 Nitrogens in the structure) 0.6821 Observed Ratio (mean)0.6419 Std. Dev. Ratio0.0835 Number of Scans36 Good ratio in the fine isotopic pattern across the entire peak. Only scans well into the tail / baseline go outside 1.5 SD.

65. 65 Finding and Identifying Metabolites - FISh

66. 66 FISh Considerations (HRAM) Predict fragments from parent structure – use only those observed in the parent structure to start the search. Start small with observed fragments + simple modifications Identify the structure of metabolites, add in unique new fragments to the search filter and re-FISh

67. 67 The FISh Search Window

68. 68 The FISh Search Window Fragments Section: This determines what fragments the FISh search will use and where they are taken from. There are four ways to populate the search 1)Generated from Structure – the user selects a structure (.mol file) which will be fragmented 2)Fragments – the user has already generated fragments and adds them directly. This can be done by picking a Fragment window from the “Available Sources” and clicking “Add” or by copying and pasting fragment(s) into the Fragments using the “Edit” button 3)Library – An entry in a Mass Frontier library is used as the source of search fragments. All entries in the selected library and their fragments will be used. 4)Reference Spectra – Instead of using known fragments, the user chooses a representative fragment spectra to search with.

69. 69 The FISh Search Window Modifications: Select whether or not the FISh search uses metabolic modifications of the provided fragments. Select “Edit” to choose the specific modifications used. In the Edit window the user can create custom modifications and custom lists of modifications

70. 70 The FISh Search Window FISh Filter Options Filtering Target: For metabolite finding, the “Mark Peaks” option should always be selected. “Remove Peaks” will remove any fragment / mass that cannot be explained (The “Max eliminated abundance” option at the bottom is only available when “Remove Peaks” is selected and determines the abundance limit for removed ions) When performing FISh, “Detect Components” should be checked and “Joint Component Detection” selected for LC data.

71. 71 The FISh Search Window Filtering Targets: “Filter Scans” should be selected to perform FISh on all fragmentation scans. If “Filter Precursors” is selected, FISh will not find components. The user can do a “targetted” full scan search by using “Filter Precursors” and then selecting “Filter Top Stage Only”. Fragments will be ignored but fill scan peaks will be checked for “expected metabolite” masses (Parent + modifications) “Apply to Top Stage Only” used in combination with “Filter Scans” performs a similar “expected metabolites” scan – only full scan masses matching parent + modifications are found, fragments are not used. Neutral Mass Loss – adds searching for neutral losses from the provided fragments. This is optional but can help when finding as many metabolites as possible is the goal. Mark Precursors – Important especially when using AIF only data. This setting helps Mass Frontier determine how sensitive to be in the Full Scan when fragment ions are found. Increasing the sensitivity will find extremely minor metabolites. A setting of ~65-75% is usually ideal.

72. 72 The FISh Search Window Isotopes: There are three options for Mass Frontier to utilize isotope data. Use Monoisotopic Peaks Only – This is the best option to use generally. Detected components are all considered A0 isotope. No attempt is made to identify isotopes or group them together. This is the fastest processing setting Mark Isotopic Pattern When Available – Based on the value of the detected ion, isotopes detected are color coded. Use Full Isotopic Pattern Only – The entire isotopic pattern is scrutinized and must be present for a detected potential FISh peak to remain. This forces isotopes detected as separate peaks to be dropped. This may also cause the “Tree Branching” option for peak detection not to work properly (Discussed later)

73. 73 The FISh Search Window Click Select “Options” to set the Peak detection options for the selected algorithm (Joint Component Detection for LC data)

74. 74 The FISh Search Window Component Detection Options: The “Wizard” view of the component detection options simplifies setup. Selecting “Details” will show all the individual values instead. Mass Merge Power – This value is used to collect close signals together as a single ion (scan-to-scan variations, oscillating ions, ringing effects, etc) and prevents them from erroneously being considered separate peaks. A value of 80-95% is suitable. Too low a value may result in false positives. Average Peak Width – Automatic is best, the detected peaks in a run are used to educate the algorithm about the typical peak shape. In special cases where very minor peaks in the presence of major peaks are desired, it may be required to set a low manual value. Slider Bars Baseline Correction – degree to which minor full scan signals are removed as noise. Smoothing power – degree of smoothing applied before peak detection Overlapping sensitivity – attempts to split close peaks apart or avoid splitting a “jagged” signal. Intensity – Absolute minimum intensity for a component. Tree Branching – at what level branching is considered. The option at left is best for Hybrid / ion trap data and attempts to merge source fragmentation to eliminate false positives. The option at the right is best for AIF-only data. The displayed values here are “good” for typical HPLC/UHPLC peaks with 10-30 minute gradients and sensitive detection.

75. 75 FISh Processing of Urine Samples Raw chromatogram, Urine 0-3 hours

76. 76 FISh Processing of Urine Samples FISh TIC

77. 77 FISh Processing of Urine Samples

78. 78 FISh Processing of Urine Samples

79. 79 FISh Results – m/z 362.11164 m/z 362.1164 MS 2 FISh Components Identical fragment to parent Fragment from parent shifted by metabolism

80. 80 Structural Identification – m/z 362.11164 m/z 362.1164 MS 2 FISh Components

81. 81 Structural Identification – m/z 362.11164 m/z 362.1164 MS 2 FISh Components Modification: +O

82. 82 Structural Identification – m/z 362.11164 m/z 362.1164 MS 2 FISh Components Omeprazole oxidation (-1.38 ppm)

83. 83 Complex Matrix Analysis – Tips and Tricks

84. 84 Outline Complex matrix analysis – tricks for single and multiple injection approaches Using AIF Processing AIF only fragmentation data (can Exactive Plus do ID?) AIF as a “safety net” Using AIF as a triggering scan – the NL-triggered scan on the Q Exactive Alternative triggering – Mass tags / Isotope patterns Using differential analysis as a “targeting” tool When to use MS 2 vs MS n – Stealing the TOP“n” HCD scan from Proteomics Getting diagnostic fragmentation – Multi-MS 3 and NL-MS 3

85. 85 Steps to Apply FISh to All Ion Fragmentation 1. First FISh Using a large number of theoretical fragments (Rules+Library, 3-5 steps) 2. Parent Target FISh Use the observed fragments and modifications. 3. Refined FISh (Optional) Add in fragments observed for metabolites. 4. Modification FISh (Target FISh) Specific for a metabolite using theoretical fragments and limited modifications.. 5. Confirmation FISh Large theoretical fragment list with specific modifications for one peak.

86. 86 MDPV – Raw Data – Urine Sample, 50k

87. 87 First FISh – Parent Detected

88. 88 MDPV – Fragments Observed XIC’s of 6 largest fragments – They match MDPV so they can be used for our FISh Search. Tip: Avoid using low m/z fragments – these can lead to false positives.

89. 89 Parent FISh – MDPV and Metabolites FISh performed using the 6 most intense observed MDPV fragments and adding in a list of potential modifications. Tip: Avoid FISh’ing with too many fragments. It’s often better to start small and grow.

90. 90 Parent FISh – MDPV and Metabolites FISh Trace

91. 91 MDPV – XIC Values for Major FISh’d Metabolites

92. 92 Modification FISh Example – m/z 310 (+H 2 O 2 ) Predicted Change = +H 2 O 2 Observed m/z 310.1649 (0.03ppm)

93. 93 Modification FISh (Target FISh) Example Set up the FISh filter targeting for m/z 310. Start with MDPV parent structure and predict theoretical fragments based on rules and libraries (Both), 4 steps. Use only +H 2, +O, +O 2, +H 2 O, and +H 2 O 2 as possible modifications

94. 94 Modification FISh – Increasing the Diagnostic Fragments XIC’s of the top 10 observed fragments. Note that many of them are shared by other metabolites but all 10 elute as peaks under the target metabolite 310. Re-FISh’d fragments

95. 95 Comparison – Parent FISh vs Target FISh for m/z 310 Parent FISh Fragments (These helped to find 310) Target FISh Fragments (These will help ID 310)

96. 96 Interpreting Target FISh – Structure ID for m/z 310 +O 2 -H 2 O We know we have at least 2 aliphatic alcohols. -H 2 O (One possible structure)

97. 97 Using FISh with Proposed Structures As a final step we can feed a FISh filter a proposed structure and NO modifications. If our proposed structure is good, we should see multiple/extensive coverage of observed fragments. For this example we used the proposed structure for 310 and allowed extensive fragment prediction. Multiple diagnostic ions were observed including one (87.0044) that supported the proposal.

98. 98 Final Step – FISh with Proposed Structure Modification FISh results (FISh using MDPV and H 2 O 2 based modifications) “Confirmation” FISh (FISh using proposed structure of 310)

99. 99 Comparison of Processed Blank to Sample MDPV-001-blank-urine-50k-ms2 MDPV-002-sample-urine-50k-ms2

100. 100 Using AIF – The Modern Day NL-Triggering Experiment Neutral loss (NL) scanning is a useful technique on QQQ instruments. Limited by the number of NL’s you can scan Nominal mass Q1 Scans First Mass Fragmentation Q3 Scanned Offset by NL Mass

101. 101 Using AIF – The Modern Day NL-Triggering Experiment Q Exactive AIF-NL Triggering Full ScanAIF Scan Precursor Selected MS 2 Check for any accurate mass NL in any pair of ions from Full Scan and AIF scan. Trigger

102. 102 Omeprazole Fragmentation Observed fragments of Omeprazole (HCD, nCE = 35%, 25% spread)

103. 103 Neutral Loss Triggered MS 2 NL = 166.0202

104. 104 Neutral Loss Triggered MS 2 Precursor ion selected MS 2 of m/z 378 NL 166

105. 105 Scan Speed and Sensitivity 4.81 to 4.90 (5.4 sec.) 4 peaks <1.5% 4 triggered MS 2 scans

106. 106 Setting up the AIF-NL Experiment.

107. 107 Setting up the AIF-NL Experiment. AIF Scan settings: Same or 1 level lower Resolution and AGC Max m/z equal to Full MS Scan Remember, Default Charge state = 1

108. 108 Setting up the AIF-NL Experiment. MS 2 and Data Dependant (dd) Settings Set loop count to 1 or 2. 1 – good for very fast chromatography (3-5 min runs) 2 – good for HPLC / UHPLC ID work (15-60 min runs) Isolation window – 2 to 4 AMU NCE – Compound class specific. Using 35-45% with Stepped NCE of 20-30% is usually suitable. Typically, use the same for AIF and MS 2 Turn Dynamic exclusion ON. Underfill Ratio – 10% typically suitable for sensitivity in complex samples.

109. 109 Setting up the AIF-NL Experiment. Build the NL List No charge state Build the list from known parent fragments (4-8 NL’s usually suitable) Include Phase II conjugate NL’s expected in the sample matrix (GSH, Gluc, Sulfates, etc)

110. 110 AIF-NL Acquisition Points Number of NL’s Usually 4-6 NL’s from the parent standard injection are sufficient Consider the mechanism of fragmentation – If all NL’s come from the same side of the molecule, add more or selectively add NL’s that cover the other side of the molecule Consider polarity Fragmentation is often different between positive and negative. For molecules that light up in both polarities, this can further increase triggering efficiency. Addition of Phase II NL’s For hepatocytes, Phase II supplemented microsomes/S9/Cytosol, or for in vivo samples, add in the appropriate phase II NL’s Glucuronides (as 176.0321) Sulfate (as 79.9568) GSH (as polarity appropriate common NL’s) Less common conjugates – Glucosides, glycine, acetylcysteine, etc.

111. 111 Mass Tags and Isotope Triggering Mass Tags Inclusion of stable label isotopic pattern through metabolism or synthesis Isotope Triggering Using common natural abundances to trigger acquisition (Cl and Br) These two approaches are very similar and have two major components Mass shift – the difference from A 0 to the expected heavier signal Ratio – The expected level of the heavier signal compared to A 0

112. 112 Mass Tag Example – Labeled GSH Incubation with GSH/iso-GSH (1:0.8) Generic data-acquisition and data-processing methods 10 µM Parent Compound Ruan, et al. “A Novel Methodology for Screening Reactive Metabolites Using Isotope Pattern Filtering of High Resolution Mass Spectrometry Data” ASMS Conference on Mass Spectrometry. June, 2008, Denver, CO MS/MS spectra of GSH adducts MS/MS data set Isotope-dependant MS 2 Orbitrap LC/MS Isotope Search MH + of GSH adducts HRAM Full Scan MS data set Full scan Data Acquisition Data Mining Reactive Metabolites

113. 113 Mass Tag Example – Labeled GSH Artificial isotopic pattern induced by incubation labeling with 15 N,2 13 C-GSH. 3.0047

114. 114 Mass Tag Example – Labeled GSH The ratio of the actual measured MS signal is a combination of the incubation mixture ratio (± pipette error) and the contribution from the light partners isotope pattern (± MS error) A 0,Light A 1,Light A 2,Light A 0,Heavy + A 3,Light A 1,Heavy A 2,Heavy ~0.8 * A 0

115. 115 Mass Tag Triggering m/z 603.1879 – the 65 th most intense ion in the scan… BUT 3.004 XIC 603.1879

116. 116 We Still Triggered MS2 NL 273, consistent with a GSH conjugate. Weak MS 2, but still diagnostic. AGC = 2e5, mIT = 50ms

117. 117 Mass Tag – Hybrids Mass Tag triggering Global  Mass tags Set the mass change and expected ratio range.

118. 118 Mass Tag – Hybrids Mass Tag triggering Scan Event  Mass Tags Set the mass tag partner to fragment (usually Low Mass)

119. 119 Mass Tag – Q Exactive Set up a ddMS 2 method.

120. 120 Mass Tag – Q Exactive Critical General / Full MS Settings Default Charge State: 1 Tags: On – turns on mass tag triggering

121. 121 Mass Tag – Q Exactive Critical ddMS 2 and dd Settings Loop Count – 1-5, depending on run time, peak width, coeluting peaks, etc. Dynamic Exclusion – On, set to estimated peak width If idle…: Do not pick others, if you only want MS 2 triggered on labeled peaks, Pick Others, if you want more fragmentation data.

122. 122 Mass Tag – Q Exactive Build a mass tag table. Accurate mass setting of up to 5 decimal places.

123. 123 Mass Tag – Q Exactive Set all other parameters (AGC, Resolution, UR, Max IT, etc) as normal for an MS2 experiment.

124. 124 Isotope Triggering Similar to Mass Tags, define the mass difference from A 0 to the isotope peak and the ratio (natural abundance)

125. 125 What instrument is best? Q Exactive Has the speed to use a “TOP5” HCD approach. Can perform AIF as a “catch” for non- triggered peaks. Elite Has the speed to use a TOP5 HCD approach Can perform MS m to generate AIF-like data. Orbitrap Velos Pro / LTQ Orbitrap XL Should limit to TOP3 if performing UHPLC Can perform MS m to generate AIF-like data when the HCD option is installed with correct firmware. Data Acquisition Workflow – Multi-injection Identification Acquisition Orbi MS n Survey Acquisition TOP5 HCD (Q E / Elite) AIF / MS m Note: MS m HCD fragmentation on the LTQ Orbitrap XL requires the HCD cell and firmware upgraded to v2.5.5 Metabolite Finding with FISh in Mass Frontier 7.0

126. 126 What application is it best for? Complex biological samples (feces, bile) Hybrids. The survey HCD MS 2 makes full use of FISh to identify metabolites so we can fully utilize MS n level fragmentation on related components.. Data Acquisition Workflow – Multi-injection Survey Acquisition TOP5 HCD (Q E / Elite) AIF / MS m Metabolite Finding with FISh in Mass Frontier 7.0 Identification Acquisition Orbi MS n

127. 127 Data Acquisition Workflow – Multi-injection TopN HCD Approach Survey Acquisition – 15-60 minute Metabolite ID Run Full scan (35-120k depending on instrument) TOP-N MS 2 using HCD (preferred, if available) or CID. dd Threshold – >1e4, Max IT MSn = 125-250 N = 3 for XL/Velos Pro, 5 for Elite and Q Exactive, can raise N for wider peaks (HPLC) HCD fragmentation gives a wider range of fragments for FISh-based detection. Dynamic exclusion should be ON (set for 3-6 sec. for UHPLC, 8-12 sec. for HPLC, or appropriate to your chromatography) Identification Acquisition – Same LC conditions as the Survey Full scan (35-120 k depending on instrument) Targeted MS 2 on the list of related components. FTMS, 7,500-15,000 resolution, CID 3 X MS 3 scans on the MS2 (this increases chance for fragmenting a diagnostic MS 2 ion) FTMS, 7,500 – 35,000 resolution, CID or HCD OR NL-MS 3 based on parent MS 2 fragments (multiple) Dynamic exclusion should be ON (set as for the Survey) Time bound inclusion is optional. Best used if there is significant peak overlap.

128. 128 Data Acquisition Workflow – Multi-injection AIF / MS m Approach Survey Acquisition – 15-60 minute Metabolite ID Run Full scan (35-120k depending on instrument) AIF or MS m scan (35-70k) Identification Acquisition – Same LC conditions as the Survey Full scan (35-120 k depending on instrument) Targeted MS 2 on the list of related components. FTMS, 7,500-15,000 resolution, CID 3 X MS 3 scans on the MS2 (this increases chance for fragmenting a diagnostic MS 2 ion) FTMS, 7,500 – 35,000 resolution, CID or HCD OR NL-MS 3 based on parent MS 2 fragments (multiple) Dynamic exclusion should be ON (set as for the Survey) Time bound inclusion is optional. Best used if there is significant peak overlap.

129. 129 Getting Diagnostic Fragmentation – MS 3 Fragmentation isn’t what’s important – Diagnostic fragmentation is. Getting sequential fragmentation on fragments identical to parent gets you identically nothing useful. Parent MS 2 Met. MS 2 Parent MS 3 Met. MS 3 = We want this one.

130. 130 Getting Diagnostic Fragmentation – MS 3 Two approaches Multi-MS 3 – Simple “Shotgun” approach. Have 3-4 MS 3 events to increase our chances of triggering on a diagnostic MS2 fragment ion NL-directed MS 3 – Target our MS 3 scans on fragment ions that have the same NL as parent (meaning they are likely fragments bearing the metabolic shift)

131. 131 Multi – MS 3 Approach Go to MS 3, use multiple (3-4) events to increase the chance to trigger on a diagnostic MS 2 fragment ion. Met. MS 2 Met. MS 3

132. 132 NL Directed MS 3 Diagnostic fragments are diagnostic because they bear the metabolic change This means they are mass shifted (don’t match parent product ions) What about the Neutral Loss portion? Parent MS 2 Met MS 2

133. 133 NL Directed MS 3 Met. MS 2 NL Matches Parent Met. MS 3

134. 134 Setting up a NL Triggered MS 3 Method Data Dependant Settings > Global Neutral Loss – Set “By Intensity” typically. Focuses on most intense MS 2 fragments. Set the mass width narrow if the MS2 scan is in the Orbitrap (0.01) or wider if it is in the Ion Trap (Hybrid, 0.25-0.5)

135. 135 Setting up a NL Triggered MS 3 Method Data Dependant Settings > Segment Neutral Loss – Include a list of Neutral Loss values. These can be taken from the fragment scan of an injection of standard. Increase the “Within Top N” value to 5 if you wish to trigger on larger numbers or less intense NL MS 2 fragments.

136. 136 Setting up a NL Triggered MS 3 Method Data Dependant Settings > Scan Event Current Scan Event – In the current scan event (for 1 to n data dependant MS3 events) set the “Mass determined from scan event” to the MS 2 event. Set the triggering to “From neutral loss list”

137. 137 Structure Based Triggering – Q Exactive AIF – NL What instrument is best? First injection – Q Exactive Target Acquisition – Hybrid / Ion trap First Acquisition All Ion Fragment NL triggered MS 2 Target Acquisition of AIF-only metabolites Orbi MS n Metabolite Finding with FISh in Mass Frontier 7.0

138. 138 Structure Based Triggering – Q Exactive AIF – NL What application is it best for? Complex matrix analysis Compounds which fragment extensively Phase II screening First Acquisition All Ion Fragment NL triggered MS 2 Target Acquisition of AIF-only metabolites Orbi MS n Metabolite Finding with FISh in Mass Frontier 7.0

139. 139 Data Acquisition Workflow – AIF–NL Survey Acquisition – 15-60 minute Metabolite ID Run Full scan (35-70k depending on instrument) AIF-NL scan. Full Scan – AGC 2e5/5e5, Max IT 80-120 msec AIF Scan – AGC 1e5/2e5, Max IT 60-80 msec Use 4-6 neutral loss fragments + Phase II losses (if appropriate) MS2 scan – 17.5/35k, AGC 5e4/1e5, Max IT 60 msec Identification Acquisition – Same LC conditions as the Survey Full scan (35-120 k depending on instrument) Targeted MS 2 on the list of related components. FTMS, 7,500-15,000 resolution, CID 3 X MS 3 scans on the MS2 (this increases chance for fragmenting a diagnostic MS 2 ion) FTMS, 7,500 – 35,000 resolution, CID or HCD OR NL-MS 3 based on parent MS 2 fragments (multiple) Dynamic exclusion should be ON (set as for the Survey) Time bound inclusion is optional. Best used if there is significant peak overlap.