Summary of Study ST002977
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001853. The data can be accessed directly via it's Project DOI: 10.21228/M86B06 This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST002977 |
| Study Title | Offline Two-dimensional Liquid Chromatography-Mass Spectrometry for Deep Annotation of the Fecal Metabolome following Fecal Microbiota Transplant |
| Study Summary | In this study, we describe a novel experimental strategy using multidimensional chromatography to facilitate compound identification in untargeted metabolomics. Pooled fecal metabolite extract samples were fractionated using an offline semi-preparative liquid chromatography. The resulting fractions were analyzed by an orthogonal LC-MS/MS method, and the data were searched against commercial, public and local spectral libraries. Multidimensional chromatography yielded more than a 3-fold improvement in identified compounds compared to the typical single-dimensional LC-MS/MS approach, and successfully identified several rare and novel compounds including atypical conjugated bile acid species. Most features identified by the new approach could be matched to features that were detectable, but not identifiable, in the original single-dimensional data. An evaluation of this approach in the context of patients with recurrent Clostridioides difficile infection receiving fecal microbiota transplants is also included. Overall, our approach represents a powerful strategy for deeper annotation of the metabolome that can be implemented with common commercially-available instrumentation, and should be applicable to any dataset requiring deeper annotation of the metabolome. |
| Institute | University of Michigan |
| Department | Michigan Compound Identification Development Core |
| Last Name | Anderson |
| First Name | Brady |
| Address | 1000 Wall St, Ann Arbor, MI 48105 |
| anderbra@umich.edu | |
| Phone | 734-232-8177 |
| Submit Date | 2023-06-02 |
| Num Groups | 2 |
| Total Subjects | 8 |
| Publications | Publication to come later |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML, raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-03-11 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001853 |
| Project DOI: | doi: 10.21228/M86B06 |
| Project Title: | Offline Two-dimensional Liquid Chromatography-Mass Spectrometry for Deep Annotation of the Fecal Metabolome following Fecal Microbiota Transplant |
| Project Summary: | In this study, we describe a novel experimental strategy using multidimensional chromatography to facilitate compound identification in untargeted metabolomics. Pooled fecal metabolite extract samples were fractionated using an offline semi-preparative liquid chromatography. The resulting fractions were analyzed by an orthogonal LC-MS/MS method, and the data were searched against commercial, public and local spectral libraries. Multidimensional chromatography yielded more than a 3-fold improvement in identified compounds compared to the typical single-dimensional LC-MS/MS approach, and successfully identified several rare and novel compounds including atypical conjugated bile acid species. Most features identified by the new approach could be matched to features that were detectable, but not identifiable, in the original single-dimensional data. An evaluation of this approach in the context of patients with recurrent Clostridioides difficile infection receiving fecal microbiota transplants is also included. Overall, our approach represents a powerful strategy for deeper annotation of the metabolome that can be implemented with common commercially-available instrumentation, and should be applicable to any dataset requiring deeper annotation of the metabolome. |
| Institute: | University of Michigan |
| Department: | Michigan Compound Identification Development Core |
| Last Name: | Anderson |
| First Name: | Brady |
| Address: | 1000 Wall St, Ann Arbor, MI 48105 |
| Email: | anderbra@umich.edu |
| Phone: | 734-232-8177 |
| Funding Source: | NIH U2CES030164 and P41-GM108538 |
| Publications: | Publication to come later |
| Contributors: | Alexander Raskind, Rylan Hissong, Michael K. Dougherty, Sarah K. McGill, Ajay Gulati, Casey M. Theriot, Robert T. Kennedy, Charles R. Evans |
Subject:
| Subject ID: | SU003090 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Age Or Age Range: | 61.1 +/- 15.2 |
| Gender: | Male and female |
| Human Lifestyle Factors: | Unsuccessful antibiotic treatment for recurrent C.difficile infection |
| Human Medications: | Vancomycin, Metronidazole, Fidaxomicin, probiotic |
Factors:
Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Concentration | FMT | Sample type |
|---|---|---|---|---|
| SA323263 | HILIC_R4_Post_FMT_Neg | 0.5x | Post | Fecal matter |
| SA323264 | HILIC_R8_Post_FMT_Pos | 0.5x | Post | Fecal matter |
| SA323265 | HILIC_R7_Post_FMT_Neg | 0.5x | Post | Fecal matter |
| SA323266 | HILIC_R3_Post_FMT_Pos | 0.5x | Post | Fecal matter |
| SA323267 | HILIC_R14_Post_FMT_Pos | 0.5x | Post | Fecal matter |
| SA323268 | HILIC_R13_Post_FMT_Pos | 0.5x | Post | Fecal matter |
| SA323269 | HILIC_R9_Post_FMT_Pos | 0.5x | Post | Fecal matter |
| SA323270 | HILIC_R12_Post_FMT_Pos | 0.5x | Post | Fecal matter |
| SA323271 | HILIC_R3_Post_FMT_Neg | 0.5x | Post | Fecal matter |
| SA323272 | HILIC_R8_Post_FMT_Neg | 0.5x | Post | Fecal matter |
| SA323273 | HILIC_R13_Post_FMT_Neg | 0.5x | Post | Fecal matter |
| SA323274 | HILIC_R14_Post_FMT_Neg | 0.5x | Post | Fecal matter |
| SA323275 | HILIC_R12_Post_FMT_Neg | 0.5x | Post | Fecal matter |
| SA323276 | HILIC_R4_Post_FMT_Pos | 0.5x | Post | Fecal matter |
| SA323277 | HILIC_R7_Post_FMT_Pos | 0.5x | Post | Fecal matter |
| SA323278 | HILIC_R9_Post_FMT_Neg | 0.5x | Post | Fecal matter |
| SA323279 | HILIC_R12_Pre_FMT_Pos | 0.5x | Pre | Fecal matter |
| SA323280 | HILIC_R8_Pre_FMT_Pos | 0.5x | Pre | Fecal matter |
| SA323281 | HILIC_R4_Pre_FMT_Pos | 0.5x | Pre | Fecal matter |
| SA323282 | HILIC_R7_Pre_FMT_Pos | 0.5x | Pre | Fecal matter |
| SA323283 | HILIC_R9_Pre_FMT_Pos | 0.5x | Pre | Fecal matter |
| SA323284 | HILIC_R4_Pre_FMT_Neg | 0.5x | Pre | Fecal matter |
| SA323285 | HILIC_R12_Pre_FMT_Neg | 0.5x | Pre | Fecal matter |
| SA323286 | HILIC_R13_Pre_FMT_Neg | 0.5x | Pre | Fecal matter |
| SA323287 | HILIC_R14_Pre_FMT_Neg | 0.5x | Pre | Fecal matter |
| SA323288 | HILIC_R9_Pre_FMT_Neg | 0.5x | Pre | Fecal matter |
| SA323289 | HILIC_R8_Pre_FMT_Neg | 0.5x | Pre | Fecal matter |
| SA323290 | HILIC_R14_Pre_FMT_Pos | 0.5x | Pre | Fecal matter |
| SA323291 | HILIC_R3_Pre_FMT_Neg | 0.5x | Pre | Fecal matter |
| SA323292 | HILIC_R7_Pre_FMT_Neg | 0.5x | Pre | Fecal matter |
| SA323293 | HILIC_R13_Pre_FMT_Pos | 0.5x | Pre | Fecal matter |
| SA323294 | HILIC_R3_Pre_FMT_Pos | 0.5x | Pre | Fecal matter |
| SA323295 | CSH_R7_Post_FMT_Neg | 2x | Post | Fecal matter |
| SA323296 | CSH_R13_Post_FMT_Pos | 2x | Post | Fecal matter |
| SA323297 | CSH_R8_Post_FMT_Neg | 2x | Post | Fecal matter |
| SA323298 | CSH_R4_Post_FMT_Neg | 2x | Post | Fecal matter |
| SA323299 | CSH_R14_Post_FMT_Pos | 2x | Post | Fecal matter |
| SA323300 | CSH_R4_Post_FMT_Pos | 2x | Post | Fecal matter |
| SA323301 | CSH_R7_Post_FMT_Pos | 2x | Post | Fecal matter |
| SA323302 | CSH_R3_Post_FMT_Neg | 2x | Post | Fecal matter |
| SA323303 | CSH_R12_Post_FMT_Pos | 2x | Post | Fecal matter |
| SA323304 | CSH_R9_Post_FMT_Neg | 2x | Post | Fecal matter |
| SA323305 | CSH_R13_Post_FMT_Neg | 2x | Post | Fecal matter |
| SA323306 | CSH_R3_Post_FMT_Pos | 2x | Post | Fecal matter |
| SA323307 | CSH_R8_Post_FMT_Pos | 2x | Post | Fecal matter |
| SA323308 | CSH_R12_Post_FMT_Neg | 2x | Post | Fecal matter |
| SA323309 | CSH_R14_Post_FMT_Neg | 2x | Post | Fecal matter |
| SA323310 | CSH_R9_Post_FMT_Pos | 2x | Post | Fecal matter |
| SA323311 | CSH_R7_Pre_FMT_Pos | 2x | Pre | Fecal matter |
| SA323312 | CSH_R14_Pre_FMT_Pos | 2x | Pre | Fecal matter |
| SA323313 | CSH_R13_Pre_FMT_Pos | 2x | Pre | Fecal matter |
| SA323314 | CSH_R12_Pre_FMT_Pos | 2x | Pre | Fecal matter |
| SA323315 | CSH_R4_Pre_FMT_Pos | 2x | Pre | Fecal matter |
| SA323316 | CSH_R9_Pre_FMT_Pos | 2x | Pre | Fecal matter |
| SA323317 | CSH_R4_Pre_FMT_Neg | 2x | Pre | Fecal matter |
| SA323318 | CSH_R9_Pre_FMT_Neg | 2x | Pre | Fecal matter |
| SA323319 | CSH_R12_Pre_FMT_Neg | 2x | Pre | Fecal matter |
| SA323320 | CSH_R13_Pre_FMT_Neg | 2x | Pre | Fecal matter |
| SA323321 | CSH_R3_Pre_FMT_Pos | 2x | Pre | Fecal matter |
| SA323322 | CSH_R14_Pre_FMT_Neg | 2x | Pre | Fecal matter |
| SA323323 | CSH_R8_Pre_FMT_Neg | 2x | Pre | Fecal matter |
| SA323324 | CSH_R7_Pre_FMT_Neg | 2x | Pre | Fecal matter |
| SA323325 | CSH_R3_Pre_FMT_Neg | 2x | Pre | Fecal matter |
| SA323326 | CSH_R8_Pre_FMT_Pos | 2x | Pre | Fecal matter |
| SA323327 | HILIC_Blank_Neg_03 | N/A | N/A | Blank |
| SA323328 | HILIC_Blank_Neg_01 | N/A | N/A | Blank |
| SA323329 | HILIC_Blank_Neg_02 | N/A | N/A | Blank |
| SA323330 | HILIC_Blank_Pos_01 | N/A | N/A | Blank |
| SA323331 | CSH_Blank_Neg_03 | N/A | N/A | Blank |
| SA323332 | CSH_Blank_Neg_02 | N/A | N/A | Blank |
| SA323333 | CSH_Blank_Neg_01 | N/A | N/A | Blank |
| SA323334 | CSH_Blank_Pos_03 | N/A | N/A | Blank |
| SA323335 | HILIC_Blank_Pos_02 | N/A | N/A | Blank |
| SA323336 | CSH_Blank_Pos_02 | N/A | N/A | Blank |
| SA323337 | HILIC_Blank_Pos_03 | N/A | N/A | Blank |
| SA323338 | CSH_Blank_Pos_01 | N/A | N/A | Blank |
| Showing results 1 to 76 of 76 |
Collection:
| Collection ID: | CO003083 |
| Collection Summary: | All participants were enrolled under IRB #16-2283 at the University of North Carolina Hospital. Fecal samples were collected pre-FMT and six months post-FMT for eight patients with prior unsuccessful antibiotic treatment for C. diff. infection. Fecal samples were aliquoted into Eppendorf tubes and stored at -80 ºC until extraction. |
| Sample Type: | Feces |
| Collection Method: | Colonic flush |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR003099 |
| Treatment Summary: | Fecal samples were collected pre-FMT and six months post-FMT for eight patients with prior unsuccessful antibiotic treatment for C. diff. infection. Participants had received previous therapies including metronidazole, vancomycin short course, vancomycin taper, fidaxomicin, or a probiotic prior to FMT. |
| Treatment: | Fecal microbiota transplant (FMT) |
Sample Preparation:
| Sampleprep ID: | SP003096 |
| Sampleprep Summary: | Fecal samples were weighed into pre-tared 2 mL Precellys (Bertin Corp.) compatible vials, and one 2.8 mm stainless steel bead was added to aid homogenization. Fecal matter was homogenized using a Precellys Evolution by two 20 s cycles separated by a 30 s break. Extraction solvent was used at a ratio of 1 mL per 5 g feces and was comprised of 1:1:1 methanol:acetonitrile:acetone containing 10 uM of D3-creatine, D10-isoleucine, D2-biotin, D5-tryptophan, D3-caffeine, D3-octanoylcarnitine, D3-palmitoylcarnitine, D4-deoxycholic acid, D4-cholic acid, and D7-arginine as internal standards. Following extraction, samples were centrifuged for 10 min at 17,000 rpm. 100 uL aliquots of supernatant were transferred to Eppendorf vials, dried under a gentle stream of nitrogen, and stored at -80 ºC. On the day of analysis, the dried extracts were reconstituted in 85:15 acetonitrile:water for HILIC analysis or 9:1 water:methanol for RPLC analysis with volumes as described below. Pooled samples were prepared by combining equal volumes of reconstituted fecal matter extracts from all subjects. |
| Processing Storage Conditions: | On ice |
| Extraction Method: | 1 g feces / 5 mL of 1:1:1 methanol:acetonitirle:acetone |
| Extract Storage: | 4℃ |
| Sample Resuspension: | 9:1 Water:Methanol for Reversed phase - CSH; 85:15 acetonitrile:water for HILIC |
Combined analysis:
| Analysis ID | AN004887 | AN004888 | AN004889 | AN004890 |
|---|---|---|---|---|
| Analysis type | MS | MS | MS | MS |
| Chromatography type | HILIC | HILIC | Reversed phase | Reversed phase |
| Chromatography system | Thermo Vanquish | Thermo Vanquish | Thermo Vanquish | Thermo Vanquish |
| Column | Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) | Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) | Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um) | Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um) |
| MS Type | ESI | ESI | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap | Orbitrap | Orbitrap |
| MS instrument name | Thermo Orbitrap ID-X tribrid | Thermo Orbitrap ID-X tribrid | Thermo Orbitrap ID-X tribrid | Thermo Orbitrap ID-X tribrid |
| Ion Mode | POSITIVE | NEGATIVE | POSITIVE | NEGATIVE |
| Units | Peak area | Peak area | Peak area | Peak area |
Chromatography:
| Chromatography ID: | CH003688 |
| Chromatography Summary: | Individual subject samples, fractionated and unfractionated pooled samples, and analytical standards were analyzed by HILIC (Waters BEH Amide, 2.1 x 100 mm, 1.7 um) and RPLC at high pH (Waters Charged-Surface Hybrid [CSH] C18, 2.1 x 100 mm, 1.7 um) in both positive and negative ion modes on a Thermo Vanquish Horizon LC coupled to an Orbitrap ID-X mass spectrometer. For HILIC separations, mobile phase A consisted of 95:5 water:acetonitrile with 10 mM ammonium formate plus 0.125 % v/v formic acid and mobile phase B was 5:95 water:acetonitrile with the same additive concentrations. HILIC separations utilized the following gradient: 0 min, 100% B; 0-0.5 min 100% B; 0.5-7 min 85% B; 7-9 min 85% B; 9-16 min 50% B; 16-16.1 min 100% B; 16.1-20 min 100% B. For CSH separations, mobile phase A consisted of water with 10 mM ammonium acetate plus 0.025% ammonium hydroxide (v/v) and mobile phase B was methanol with the same additives |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) |
| Column Temperature: | 55 |
| Flow Gradient: | 0 min, 100% B; 0-0.5 min 100% B; 0.5-7 min 85% B; 7-9 min 85% B; 9-16 min 50% B; 16-16.1 min 100% B; 16.1-20 min 100% B |
| Flow Rate: | 0.3 mL/min |
| Solvent A: | 95%water/5% acetonitrile; 10 mM ammonium formate; 0.125 % v/v formic acid |
| Solvent B: | 5% water/95% acetonitrile; 10 mM ammonium formate; 0.125 % v/v formic acid |
| Washing Buffer: | 9:1 water:methanol |
| Chromatography Type: | HILIC |
| Chromatography ID: | CH003689 |
| Chromatography Summary: | Individual subject samples, fractionated and unfractionated pooled samples, and analytical standards were analyzed by HILIC (Waters BEH Amide, 2.1 x 100 mm, 1.7 um) and RPLC at high pH (Waters Charged-Surface Hybrid [CSH] C18, 2.1 x 100 mm, 1.7 um) in both positive and negative ion modes on a Thermo Vanquish Horizon LC coupled to an Orbitrap ID-X mass spectrometer. For CSH separations, mobile phase A consisted of water with 10 mM ammonium acetate plus 0.025% ammonium hydroxide (v/v) and mobile phase B was methanol with the same additives. CSH separations utilized the following gradient: 0 min 0% B, 0-5 min 60% B; 5-13 min 99% B; 13-17 min 99% B; 17-17.1 min 0% B; 17-20 min 0% B. |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um) |
| Column Temperature: | 55 |
| Flow Gradient: | 0 min 0% B, 0-5 min 60% B; 5-13 min 99% B; 13-17 min 99% B; 17-17.1 min 0% B; 17-20 min 0% B |
| Flow Rate: | 0.45 mL/min |
| Solvent A: | 100% water; 10 mM ammonium acetate; 0.025% ammonium hydroxide (v/v) |
| Solvent B: | 100% methanol; 10 mM ammonium acetate; 0.025% ammonium hydroxide (v/v) |
| Washing Buffer: | 85:15 acetonitrile:water |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS004631 |
| Analysis ID: | AN004887 |
| Instrument Name: | Thermo Orbitrap ID-X tribrid |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Mass spectrometer settings were as follows: sheath gas, 40; aux gas, 10; sweep gas, 1; ion transfer tube temp, 325 ºC; vaporizer temp, 300 ºC; orbitrap resolution. 120000; scan range, 70-800 m/z; RF lens, 45%; normalized AGC target, 25%; maximum injection time, auto; microscans, 1; data type, profile; internal mass calibration, EASY-ICTM. Positive ion spray voltage was set to 3200 V. Instrument settings for MS1 and MS/MS methods were identical, except orbitrap resolution was decreased to maximize MS/MS spectra collection. The data-dependent MS/MS methods utilized the following settings: full scan orbitrap resolution, 60000; intensity threshold, 1.0x104; dynamic exclusion properties; exclusion duration 3 seconds (exclude after 1 time with +/- 5 ppm); isolation mode, quadrupole; isolation window, 1.2 m/z; activation type, HCD; collision energy mode, assisted; collision energies, 20, 40, and 80%; detector type, orbitrap; orbitrap resolution, 30000; normalized AGC target, 20%; maximum injection time, 54 ms; microscans, 1; data type, centroid; cycle time, 1.2 s. |
| Ion Mode: | POSITIVE |
| Capillary Temperature: | 325 |
| Collision Energy: | 20, 40, 80 |
| Collision Gas: | N2 |
| Dry Gas Flow: | Sheath: 40, Aux: 10, Sweep: 1 |
| Fragmentation Method: | Assisted |
| Ion Source Temperature: | 300 |
| Ion Spray Voltage: | +3200 |
| Ionization: | Positive |
| Mass Accuracy: | 120,000 |
| Automatic Gain Control: | 45% |
| MS ID: | MS004632 |
| Analysis ID: | AN004888 |
| Instrument Name: | Thermo Orbitrap ID-X tribrid |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Mass spectrometer settings were as follows: sheath gas, 40; aux gas, 10; sweep gas, 1; ion transfer tube temp, 325 ºC; vaporizer temp, 300 ºC; orbitrap resolution. 120000; scan range, 70-800 m/z; RF lens, 45%; normalized AGC target, 25%; maximum injection time, auto; microscans, 1; data type, profile; internal mass calibration, EASY-ICTM. Positive ion spray voltage was set to -3200 V. Instrument settings for MS1 and MS/MS methods were identical, except orbitrap resolution was decreased to maximize MS/MS spectra collection. The data-dependent MS/MS methods utilized the following settings: full scan orbitrap resolution, 60000; intensity threshold, 1.0x104; dynamic exclusion properties; exclusion duration 3 seconds (exclude after 1 time with +/- 5 ppm); isolation mode, quadrupole; isolation window, 1.2 m/z; activation type, HCD; collision energy mode, assisted; collision energies, 20, 40, and 80%; detector type, orbitrap; orbitrap resolution, 30000; normalized AGC target, 20%; maximum injection time, 54 ms; microscans, 1; data type, centroid; cycle time, 1.2 s. |
| Ion Mode: | NEGATIVE |
| Capillary Temperature: | 325 |
| Collision Energy: | 20, 40, 80 |
| Collision Gas: | N2 |
| Dry Gas Flow: | Sheath: 40, Aux: 10, Sweep: 1 |
| Fragmentation Method: | Assisted |
| Ion Source Temperature: | 300 |
| Ion Spray Voltage: | -3200 |
| Ionization: | Negative |
| Mass Accuracy: | 120,000 |
| Automatic Gain Control: | 45% |
| MS ID: | MS004633 |
| Analysis ID: | AN004889 |
| Instrument Name: | Thermo Orbitrap ID-X tribrid |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Mass spectrometer settings were as follows: sheath gas, 40; aux gas, 10; sweep gas, 1; ion transfer tube temp, 325 ºC; vaporizer temp, 300 ºC; orbitrap resolution. 120000; scan range, 70-800 m/z; RF lens, 45%; normalized AGC target, 25%; maximum injection time, auto; microscans, 1; data type, profile; internal mass calibration, EASY-ICTM. Positive ion spray voltage was set to 3200 V. Instrument settings for MS1 and MS/MS methods were identical, except orbitrap resolution was decreased to maximize MS/MS spectra collection. The data-dependent MS/MS methods utilized the following settings: full scan orbitrap resolution, 60000; intensity threshold, 1.0x104; dynamic exclusion properties; exclusion duration 3 seconds (exclude after 1 time with +/- 5 ppm); isolation mode, quadrupole; isolation window, 1.2 m/z; activation type, HCD; collision energy mode, assisted; collision energies, 20, 40, and 80%; detector type, orbitrap; orbitrap resolution, 30000; normalized AGC target, 20%; maximum injection time, 54 ms; microscans, 1; data type, centroid; cycle time, 1.2 s. |
| Ion Mode: | POSITIVE |
| Capillary Temperature: | 325 |
| Collision Energy: | 20, 40, 80 |
| Collision Gas: | N2 |
| Dry Gas Flow: | Sheath: 40, Aux: 10, Sweep: 1 |
| Fragmentation Method: | Assisted |
| Ion Source Temperature: | 300 |
| Ion Spray Voltage: | +3200 |
| Ionization: | Positive |
| Mass Accuracy: | 120,000 |
| Automatic Gain Control: | 45% |
| MS ID: | MS004634 |
| Analysis ID: | AN004890 |
| Instrument Name: | Thermo Orbitrap ID-X tribrid |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Mass spectrometer settings were as follows: sheath gas, 40; aux gas, 10; sweep gas, 1; ion transfer tube temp, 325 ºC; vaporizer temp, 300 ºC; orbitrap resolution. 120000; scan range, 70-800 m/z; RF lens, 45%; normalized AGC target, 25%; maximum injection time, auto; microscans, 1; data type, profile; internal mass calibration, EASY-ICTM. Positive ion spray voltage was set to -3200 V. Instrument settings for MS1 and MS/MS methods were identical, except orbitrap resolution was decreased to maximize MS/MS spectra collection. The data-dependent MS/MS methods utilized the following settings: full scan orbitrap resolution, 60000; intensity threshold, 1.0x104; dynamic exclusion properties; exclusion duration 3 seconds (exclude after 1 time with +/- 5 ppm); isolation mode, quadrupole; isolation window, 1.2 m/z; activation type, HCD; collision energy mode, assisted; collision energies, 20, 40, and 80%; detector type, orbitrap; orbitrap resolution, 30000; normalized AGC target, 20%; maximum injection time, 54 ms; microscans, 1; data type, centroid; cycle time, 1.2 s. |
| Ion Mode: | NEGATIVE |
| Capillary Temperature: | 325 |
| Collision Energy: | 20, 40, 80 |
| Collision Gas: | N2 |
| Dry Gas Flow: | Sheath: 40, Aux: 10, Sweep: 1 |
| Fragmentation Method: | Assisted |
| Ion Source Temperature: | 300 |
| Ion Spray Voltage: | +3200 |
| Ionization: | Negative |
| Mass Accuracy: | 120,000 |
| Automatic Gain Control: | 45% |
