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MB Sample ID: SA323298
Local Sample ID: | CSH_R4_Post_FMT_Neg |
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 |
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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:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
CSH_R4_Post_FMT_Neg | SA323298 | FL038623 | 2x | Concentration |
CSH_R4_Post_FMT_Neg | SA323298 | FL038623 | Post | FMT |
CSH_R4_Post_FMT_Neg | SA323298 | FL038623 | Fecal matter | Sample type |
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% |