Summary of Study ST002956
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 PR001839. The data can be accessed directly via it's Project DOI: 10.21228/M80T6F 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 | ST002956 |
| Study Title | Automated preparation of plasma lipids, metabolites, and proteins for LC/MS-based analysis of a high-fat diet in mice |
| Study Type | Method Development |
| Study Summary | We designed an automated liquid-liquid extraction method with minimal contamination and human intervention. This approach enables accurate and precise collection of metabolite, lipid fractions, and protein pellet from a small volume of mice plasma for multiomic analysis. |
| Institute | Calico Life Sciences |
| Department | Department of Mass Spectrometry-Technology Lab |
| Laboratory | Metabolomics Lab |
| Last Name | Vu |
| First Name | Ngoc |
| Address | 1130 Veterans BLVD |
| ngoc@calicolabs.com | |
| Phone | 6504205430 |
| Submit Date | 2023-07-06 |
| Num Groups | 3 |
| Total Subjects | 90 |
| Num Males | 90 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-07-29 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001839 |
| Project DOI: | doi: 10.21228/M80T6F |
| Project Title: | An application of automated LLE extraction in determine the age and HFD effects in adult mice in metabolomics, lipidomics, and proteomics |
| Project Type: | Method Development |
| Project Summary: | We designed an automated liquid-liquid extraction method with minimal contamination and human intervention. This approach enables accurate and precise collection of metabolite, lipid fractions, and protein pellet from a small volume of mice plasma for multiomic analysis. |
| Institute: | Calico Life Sciences |
| Department: | Department of Mass Spectrometry-Technology Lab |
| Laboratory: | Metabolomics Lab |
| Last Name: | Vu |
| First Name: | Ngoc |
| Address: | 1130 Veterans BLVD, South San Francisco, CA 94080 |
| Email: | ngoc@calicolabs.com |
| Phone: | 6504205430 |
| Funding Source: | Calico Life Sciences |
| Publications: | https://www.jlr.org/article/S0022-2275(24)00112-3/fulltext |
Subject:
| Subject ID: | SU003069 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57BI/6 |
| Age Or Age Range: | 8wks and 32 wks |
| Gender: | Male |
| Animal Animal Supplier: | Jackson Lab |
| Animal Feed: | Ad-Lib |
| Animal Water: | Ad-Lib |
| Species Group: | Mammals |
Factors:
Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | group_mouse | Diet | time point |
|---|---|---|---|---|
| SA321966 | 1A10 | aY-M10 | Chow | 1 |
| SA321967 | 2A10 | aY-M10 | Chow | 2 |
| SA321968 | 3A10 | aY-M10 | Chow | 3 |
| SA321969 | 1A1 | aY-M1 | Chow | 1 |
| SA321970 | 2A1 | aY-M1 | Chow | 2 |
| SA321971 | 3A1 | aY-M1 | Chow | 3 |
| SA321972 | 1A2 | aY-M2 | Chow | 1 |
| SA321973 | 2A2 | aY-M2 | Chow | 2 |
| SA321974 | 3A2 | aY-M2 | Chow | 3 |
| SA321975 | 1A3 | aY-M3 | Chow | 1 |
| SA321976 | 2A3 | aY-M3 | Chow | 2 |
| SA321977 | 3A3 | aY-M3 | Chow | 3 |
| SA321978 | 1A4 | aY-M4 | Chow | 1 |
| SA321979 | 2A4 | aY-M4 | Chow | 2 |
| SA321980 | 3A4 | aY-M4 | Chow | 3 |
| SA321981 | 1A5 | aY-M5 | Chow | 1 |
| SA321982 | 2A5 | aY-M5 | Chow | 2 |
| SA321983 | 3A5 | aY-M5 | Chow | 3 |
| SA321984 | 1A6 | aY-M6 | Chow | 1 |
| SA321985 | 2A6 | aY-M6 | Chow | 2 |
| SA321986 | 3A6 | aY-M6 | Chow | 3 |
| SA321987 | 1A7 | aY-M7 | Chow | 1 |
| SA321988 | 2A7 | aY-M7 | Chow | 2 |
| SA321989 | 3A7 | aY-M7 | Chow | 3 |
| SA321990 | 1A8 | aY-M8 | Chow | 1 |
| SA321991 | 2A8 | aY-M8 | Chow | 2 |
| SA321992 | 3A8 | aY-M8 | Chow | 3 |
| SA321993 | 1A9 | aY-M9 | Chow | 1 |
| SA321994 | 2A9 | aY-M9 | Chow | 2 |
| SA321995 | 3A9 | aY-M9 | Chow | 3 |
| SA321906 | 1C10 | C-M10 | Chow | 1 |
| SA321907 | 2C10 | C-M10 | Chow | 2 |
| SA321908 | 3C10 | C-M10 | Chow | 3 |
| SA321909 | 1C1 | C-M1 | Chow | 1 |
| SA321910 | 2C1 | C-M1 | Chow | 2 |
| SA321911 | 3C1 | C-M1 | Chow | 3 |
| SA321912 | 1C2 | C-M2 | Chow | 1 |
| SA321913 | 2C2 | C-M2 | Chow | 2 |
| SA321914 | 3C2 | C-M2 | Chow | 3 |
| SA321915 | 1C3 | C-M3 | Chow | 1 |
| SA321916 | 2C3 | C-M3 | Chow | 2 |
| SA321917 | 3C3 | C-M3 | Chow | 3 |
| SA321918 | 1C4 | C-M4 | Chow | 1 |
| SA321919 | 2C4 | C-M4 | Chow | 2 |
| SA321920 | 3C4 | C-M4 | Chow | 3 |
| SA321921 | 1C5 | C-M5 | Chow | 1 |
| SA321922 | 2C5 | C-M5 | Chow | 2 |
| SA321923 | 3C5 | C-M5 | Chow | 3 |
| SA321924 | 1C6 | C-M6 | Chow | 1 |
| SA321925 | 2C6 | C-M6 | Chow | 2 |
| SA321926 | 3C6 | C-M6 | Chow | 3 |
| SA321927 | 1C7 | C-M7 | Chow | 1 |
| SA321928 | 2C7 | C-M7 | Chow | 2 |
| SA321929 | 3C7 | C-M7 | Chow | 3 |
| SA321930 | 1C8 | C-M8 | Chow | 1 |
| SA321931 | 2C8 | C-M8 | Chow | 2 |
| SA321932 | 3C8 | C-M8 | Chow | 3 |
| SA321933 | 1C9 | C-M9 | Chow | 1 |
| SA321934 | 2C9 | C-M9 | Chow | 2 |
| SA321935 | 3C9 | C-M9 | Chow | 3 |
| SA321936 | 1B10 | HFD-M10 | HFD | 1 |
| SA321937 | 2B10 | HFD-M10 | HFD | 2 |
| SA321938 | 3B10 | HFD-M10 | HFD | 3 |
| SA321939 | 1B1 | HFD-M1 | HFD | 1 |
| SA321940 | 2B1 | HFD-M1 | HFD | 2 |
| SA321941 | 3B1 | HFD-M1 | HFD | 3 |
| SA321942 | 1B2 | HFD-M2 | HFD | 1 |
| SA321943 | 2B2 | HFD-M2 | HFD | 2 |
| SA321944 | 3B2 | HFD-M2 | HFD | 3 |
| SA321945 | 1B3 | HFD-M3 | HFD | 1 |
| SA321946 | 2B3 | HFD-M3 | HFD | 2 |
| SA321947 | 3B3 | HFD-M3 | HFD | 3 |
| SA321948 | 1B4 | HFD-M4 | HFD | 1 |
| SA321949 | 2B4 | HFD-M4 | HFD | 2 |
| SA321950 | 3B4 | HFD-M4 | HFD | 3 |
| SA321951 | 1B5 | HFD-M5 | HFD | 1 |
| SA321952 | 2B5 | HFD-M5 | HFD | 2 |
| SA321953 | 3B5 | HFD-M5 | HFD | 3 |
| SA321954 | 1B6 | HFD-M6 | HFD | 1 |
| SA321955 | 2B6 | HFD-M6 | HFD | 2 |
| SA321956 | 3B6 | HFD-M6 | HFD | 3 |
| SA321957 | 1B7 | HFD-M7 | HFD | 1 |
| SA321958 | 2B7 | HFD-M7 | HFD | 2 |
| SA321959 | 3B7 | HFD-M7 | HFD | 3 |
| SA321960 | 1B8 | HFD-M8 | HFD | 1 |
| SA321961 | 2B8 | HFD-M8 | HFD | 2 |
| SA321962 | 3B8 | HFD-M8 | HFD | 3 |
| SA321963 | 1B9 | HFD-M9 | HFD | 1 |
| SA321964 | 2B9 | HFD-M9 | HFD | 2 |
| SA321965 | 3B9 | HFD-M9 | HFD | 3 |
| Showing results 1 to 90 of 90 |
Collection:
| Collection ID: | CO003062 |
| Collection Summary: | Each mouse was fasted four hours prior to each of three retro orbital bleeds performed over three weeks at 10 AM (Time point 1- Z10), 10 PM (Time point 2- Z22), and 3 PM (Time point 3- Z15). Blood was collected into BD microtainer tubes with K2E (K2EDTA), mixed briefly, and stored on ice. All samples were centrifuged at 7000 RCF for 4 mins at 4°C to separate blood plasma. Plasma was aliquoted in 50 µL increments, immediately frozen on dry ice, and stored at -80C for later analysis. |
| Collection Protocol ID: | A00003888 |
| Collection Protocol Filename: | NIH Guide for the Care and Use of Laboratory Animals |
| Sample Type: | Blood (plasma) |
| Collection Method: | retro bleed |
| Collection Location: | Calico Life Sciences |
| Collection Frequency: | every week |
| Storage Conditions: | -80℃ |
| Collection Vials: | BD microcontainer tube with K2E |
Treatment:
| Treatment ID: | TR003078 |
| Treatment Summary: | Ten male C57BL/6 mice (Jackson Laboratory, USA) were selected for each of three groups, to minimize variance in body weight, and eliminate animals that had fighting wounded. Groups of 8-week-old and 24-week-old mice were fed a standard lab chow diet (Chow diet 5LOD, LabDiet ) and a second group of 24-week-old mice fed 10 weeks of a high-fat diet (HFD) containing 60% fat (D12492, Research Diets Inc.). |
| Treatment: | HFD |
| Animal Fasting: | 4 hours prior to sample collections |
Sample Preparation:
| Sampleprep ID: | SP003075 |
| Sampleprep Summary: | This method is based on the protocol Matyash et al. 2008. (Matyash et al. 2008). Briefly, lipid standards mixture (volumes as described in results) were extracted by addition of 50% methanol (pre-cooled to -20°C), vortexed for 30 sec, and incubated on ice for 10 min. The same volume of room temperature MTBE was added, and the samples were vortexed on the max setting for 30 sec before centrifuging at 3000 x g for 5 min at 4°C for a complete phase-separation (Eppendorf R5010 centrifuge with swinging bucket rotor). The maximum volume of the upper organic phase was pipetted off by hand with a Hamilton glass syringe until the meniscus of the two phases was reached. The organic phase was then transferred to a new ice-cold glass sample vial. The remaining sample volume was subjected to a secondary extraction using MTBE or other chemicals described in the results section. Then, the second organic phase was removed, combining this volume with the first organic phase extraction. All sample transfers involving MTBE-containing solutions were performed with glass syringes rinsed twice with MTBE and MeOH, between preparation steps. Following both MTBE extractions, the lower aqueous phase extract was pipetted off with the necessary care not to pick up the remaining upper phase or disturb the protein pellet. The fraction was subsequently transferred into a separate ice-cold sample vial. The phase extracts were then evaporated to dryness under nitrogen flow at 4°C and stored at -80°C. |
| Sampleprep Protocol ID: | A00003888 |
| Sampleprep Protocol Filename: | NIH Guide for the Care and Use of Laboratory Animals |
| Processing Storage Conditions: | On ice |
Combined analysis:
| Analysis ID | AN004853 | AN004854 | AN004855 | AN004856 |
|---|---|---|---|---|
| Analysis type | MS | MS | MS | MS |
| Chromatography type | Reversed phase | Reversed phase | HILIC | Ion pair |
| Chromatography system | Thermo Vanquish | Thermo Vanquish | Thermo Vanquish | Thermo Vanquish |
| Column | Thermo Accucore C30 column (250 x 2.1 mm, 2.6 µm) | Thermo Accucore C30 column (250 x 2.1 mm, 2.6 µm) | SeQuant ZIC- pHILIC (150 x 2.1mm,5um) | Agilent Zorbax Extend C18 column (150 x 2.1 mm, 1.8 µm) |
| MS Type | ESI | ESI | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Plus Orbitrap | Thermo Q Exactive Plus Orbitrap | Thermo Q Exactive Plus Orbitrap | Thermo Q Exactive Plus Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE | POSITIVE | NEGATIVE |
| Units | Log2(Top Peak Area) | Log2(Top Peak Area) | Log2(Top Peak Area) | Log2(Top Peak Area) |
Chromatography:
| Chromatography ID: | CH003664 |
| Chromatography Summary: | RPLC method for lipidomics |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Thermo Accucore C30 column (250 x 2.1 mm, 2.6 µm) |
| Column Temperature: | 35 |
| Flow Gradient: | The column was equilibrated in 30% B for 7 minutes before injection. The gradient then increased to 43% B for another 7 minutes, followed by an increase to 65% B over 5 min and then slowly increasing to 70% B over 18 min. B was then increased to 88% for 1 min, followed by another slow increase to 95% over 20 min. Over the next 2 min, B was increased to 100%, followed by isocratic flow for another 2 min. After this step, the gradient was dropped to 30% B within 0.1 min and kept on isocratic flow for the next 5 min, resulting in a total run time of 67 min. |
| Flow Rate: | 200 uL/min |
| Solvent A: | 20 mM ammonium formate in [60:40] ACN:H2O, 0.25 µM medronic acid |
| Solvent B: | 20 mM ammonium formate in 90:10 IPA:MeCN, 0.25 µM medronic acid |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH003665 |
| Chromatography Summary: | HILIC method for Metabolomics |
| Instrument Name: | Thermo Vanquish |
| Column Name: | SeQuant ZIC- pHILIC (150 x 2.1mm,5um) |
| Column Temperature: | 35 |
| Flow Gradient: | t = −6, 80% B; t = 0, 80% B; t = 2.5, 73% B; t = 5, 65% B, t = 7.5, 57% B; t = 10, 50% B; t = 15, 35% B; t = 20; 20% B; t = 22, 15% B; t = 22.5, 80% B; t = 24; 80% B. |
| Flow Rate: | 150uL/min |
| Solvent A: | 20 mM ammonium carbonate in water (pH 9.2) |
| Solvent B: | 100% MeCN |
| Chromatography Type: | HILIC |
| Chromatography ID: | CH003666 |
| Chromatography Summary: | RPLC method for Metabolomics |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Agilent Zorbax Extend C18 column (150 x 2.1 mm, 1.8 µm) |
| Column Temperature: | 30 |
| Flow Gradient: | The gradient eluted isocratically in 0% B for 2.5 min, increased to 20% B over 2.5 min, held at 20% B for 2.5 min, increased to 55% B over 5.5 min, increased to 95% B over 2.5 minutes, maintained at 95% B for 3 min, then decreased to 0% B over 0.5 min, where it was held for 3 min, resulting in a total run time of 26 min. |
| Flow Rate: | 200 uL/min |
| Solvent A: | 10 mM tributylamine and 15 mM acetic acid in [97:3] H2O:MeOH at pH 4.95 with 0.25 µM medronic acid |
| Solvent B: | 100% MeOH |
| Chromatography Type: | Ion pair |
MS:
| MS ID: | MS004598 |
| Analysis ID: | AN004853 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Set parameters on MS1 were 140,000 resolution, AGC target of 3e6 at max. IT of 100 ms with scan range of 200 to 2000 m/z. MS2 parameters consisted of 17,500 resolution at loop count 8, an AGC target of 3e6 at max. IT of 150 ms with isolation window set to 1 m/z and underfill ratio at 1%. Dynamic exclusion was set at 15 sec with an apex trigger from 5 to 30 sec. Stepped collision energies were set to 20, 30 and 40% NCE. Raw files were converted to mzML using MsConverter, and search against in-house libraries for feature identification using Maven 2. |
| Ion Mode: | POSITIVE |
| MS ID: | MS004599 |
| Analysis ID: | AN004854 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Set parameters on MS1 were 140,000 resolution, AGC target of 3e6 at max. IT of 100 ms with scan range of 200 to 2000 m/z. MS2 parameters consisted of 17,500 resolution at loop count 8, an AGC target of 3e6 at max. IT of 150 ms with isolation window set to 1 m/z and underfill ratio at 1%. Dynamic exclusion was set at 15 sec with an apex trigger from 5 to 30 sec. Stepped collision energies were set to 20, 30 and 40% NCE. Raw files were converted to mzML using MsConverter, and search against in-house libraries for feature identification using Maven 2. |
| Ion Mode: | NEGATIVE |
| MS ID: | MS004600 |
| Analysis ID: | AN004855 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The mass spectrometer was operated in positive ion mode using data-dependent acquisition (DDA) mode with the following parameters: resolution = 70,000, AGC target = 3.00E + 06, maximum IT (ms) = 100, scan range = 70–1050. The MS2 parameters were as follows: resolution = 17,500, AGC target = 1.00E + 05, maximum IT (ms) = 50, loop count = 6, isolation window (m/z) = 1, (N)CE = 20, 40, 80; underfill ratio = 1.00%, Apex trigger(s) = 3–10, dynamic exclusion(s) = 25 |
| Ion Mode: | POSITIVE |
| MS ID: | MS004601 |
| Analysis ID: | AN004856 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The mass spectrometer was operated in negative ion mode using data-dependent acquisition (DDA) mode with the following parameters: resolution = 70,000, AGC target = 1.00E + 06, maximum IT (ms) = 100, scan range = 70–1050. The MS2 parameters were as follows: resolution = 17,500, AGC target = 1.00E + 05, maximum IT (ms) = 50, loop count = 6, isolation window (m/z) = 1, (N)CE = 20, 50, 100; underfill ratio = 1.0 0%, Apex trigger(s) = 3–12, dynamic exclusion(s) = 20 |
| Ion Mode: | NEGATIVE |
