Protocols

  • HILIC-MS Method (Amide12min; MetDNA)
    For polar metabolites; 12 min per injection

    Column parameter

    Waters, UPLC BEH Amide column (1.7 μm, 2.1× 100 mm)

     

    Mobile phase

    Positive mode and Negative mode

    A:100% H2O 25 mM CH3COONH4 + 25 mM NH4OH

    B:100% ACN

     

    Gradient parameter

    Time Flow rate (uL/min) A % B %
    0.00 500 5.0 95.0
    1.00 500 5.0 95.0
    7.00 500 35.0 65.0
    8.00 500 60.0 40.0
    9.00 500 60.0 40.0
    9.10 500 5.0 95.0
    12.00 500 5.0 95.0
  • RPLC-MS Method (RP12min; MetDNA)
    For nonpolar metabolites and lipids; 12 min per injection

    Column parameter

    Phenomenex, UHPLC Kinetex C18 column (2.6 μm, 2.1× 100 mm)

     

    Mobile phase

    Positive mode and Negative mode

    A:100% H2O + 0.1% Formic acid

    B:ACN:IPA = 1:1 (v/v)

     

    Gradient parameter

    Time Flow rate (uL/min) A % B %
    0.00 300 99.0 1.0
    1.00 300 99.0 1.0
    8.00 300 1.0 99.0
    9.00 300 1.0 99.0
    9.10 300 99.0 1.0
    12.00 300 99.0 1.0
  • HILIC-MS Method (pHILIC)
    For polar metabolites; 25 min per injection

    Column parameter

    Merck, SeQuant ZIC-pHILIC column (5 μm, 2.1× 100 mm)

     

    Mobile phase

    Positive mode and Negative mode

    A:100% H2O 25 mM CH3COONH4 + 25 mM NH4OH

    B:100% ACN

     

    Gradient parameter

    Time Flow rate (uL/min) A % B %
    0.00 200 20.0 80.0
    2.00 200 20.0 80.0
    17.00 200 80.0 20.0
    17.10 200 20.0 80.0
    25.00 200 20.0 80.0
  • RPLC-MS (Lipidomics; Lipid4DAnalyzer)
    For untargeted lipidomics analysis; 18 min per injection

    Column parameter

    Phenomenex, UHPLC Kinetex C18 column (1.7 μm, 2.1× 100 mm)

     

    Mobile phase

    Positive mode and Negative mode

    A:ACN: H2O = 6:4; 10mM HCOONH4

    B:ACN: IPA = 1:9; 10mM HCOONH4

     

    Gradient parameter

    Time Flow rate (uL/min) A % B %
    0.00 300 60.0 40.0
    1.50 300 60.0 40.0
    10.50 300 15.0 85.0
    14.00 300 15.0 85.0
    14.10 300 0.0 100.0
    15.00 300 0.0 100.0
    15.20 300 60.0 40.0
    18.00 300 60.0 40.0
  • RPLC-MS Method (T3_12min)
    For large-scale metabolomics; Covering polar metabolites and nonpolar lipids

    1. RPLC-MS Method for MS1 only

    Column parameter

    Waters, UPLC HSS T3 column (1.8 μm, 2.1× 100 mm)

     

    Mobile phase

    Positive mode

    A:100% H2O (0.1% Formic acid)

    B:100% ACN (0.1% Formic acid)

    Negative mode

    A:100% H2O (0.5 mM NH4F or 5 mM NH4Ac)

    B:100% ACN

     

    Gradient parameter

    Time Flow rate (uL/min) A % B %
    0.00 500 99.0 1.0
    1.00 500 99.0 1.0
    8.00 500 1.00 99.0
    10.00 500 1.00 99.0
    10.00 500 99.0 1.0
    12.00 500 99.0 1.0

    2. DDA

    Column parameter

    Waters, UPLC HSS T3 column (1.8 μm, 2.1× 100 mm)

     

    Mobile phase

    Positive mode

    A:100% H2O (0.1% Formic acid)

    B:100% ACN (0.1% Formic acid)

    Negative mode

    A:100% H2O (0.5 mM NH4F or 5 mM NH4Ac)

    B:100% ACN

     

    Gradient parameter

    Time Flow rate (uL/min) A % B %
    0.00 300 99.0 1.0
    1.50 300 99.0 1.0
    13.00 300 1.00 99.0
    16.50 300 1.00 99.0
    16.60 300 99.0 1.0
    20.00 300 99.0 1.0
  • HILIC-MS (Amide Column)
    For polar metabolites; 20 min per injection

    Column parameter

    Waters, UPLC BEH Amide column (1.7 μm, 2.1× 100 mm)

     

    Mobile phase

    Positive mode and Negative mode

    A:100% H2O 25 mM CH3COONH4 + 25 mM NH4OH

    B:100% ACN

     

    Gradient parameter

    Time Flow rate (uL/min) A % B %
    0.00 300 15.0 85.0
    1.00 300 15.0 85.0
    12.00 300 35.0 65.0
    12.1 300 60.0 40.0
    15.0 300 60.0 40.0
    15.1 300 15.0 85.0
    20.0 300 15.0 85.0
  • HILIC-MS (Lunar amino column)
    For polar metabolites; 20 min per injection

    Column parameter

    Phenomenex, HPLC Luna aminopropyl column (3.0 μm, 2.0× 100 mm)

     

    Mobile phase

    Positive mode and Negative mode

    A:100% H2O 25 mM CH3COONH4 + 25 mM NH4OH

    B:100% ACN

     

    Gradient parameter

    Time Flow rate (uL/min) A % B %
    0.00 300 15.0 85.0
    2.00 300 15.0 75.0
    9.00 300 100 0.0
    14.0 300 100 0.0
    15.0 300 15.0 85.0
    20.0 300 15.0 85.0
  • HILIC-MS (Zic-HILIC)
    For polar metabolites; 20 min per injection

    Column parameter

    Merck, SeQuant ZIC-HILIC column (3.5 μm, 2.1× 100 mm)

     

    Mobile phase

    Positive mode and Negative mode

    A:95% H2O, 5% ACN, 10 mM CH3COONH4 + 10 mM NH4OH

    B:95% ACN, 5% H2O, 10 mM CH3COONH4 + 10 mM NH4OH

     

    Gradient parameter

    Time

    Flow rate(uL/min)

    A %

    B %

    0.00

    300

    1.00

    99.0

    13.0

    300

    60.0

    40.0

    15.0

    300

    60.0

    40.0

    15.1

    300

    1.00

    99.0

    20.0

    300

    1.00

    99.0

  • RPLC-MS (Lipidomics; 13min)
    For Untargeted Lipidomics Analysis

    Column parameter

    Phenomenex, UHPLC Kinetex C18 column (1.7 μm, 2.1× 100 mm)

    or Waters, UPLC HSS T3 column (1.8 μm, 2.1× 100 mm)

     

    Mobile phase

    Positive MS mode and Negative MS mode

    A:ACN: H2O = 6:4; 10mM HCOONH4

    B:ACN: IPA = 1:9; 10mM HCOONH4

     

    Gradient parameter

    Time Flow rate (uL/min) A % B %
    0.00 400 60.0 40.0
    9.00 400 0.00 100.0
    10.00 400 0.00 100.0
    10.20 400 60.0 40.0
    13.00 400 60.0 40.0
  • Lipid Extraction Protocol
    Lipid Extraction Protocol

    For plasma or serum:

    1. Add 400uL MTBE and 80uL MeOH to 200uL plasma/serum(keep MTBE:MeOH:H2O=10:2:5, v/v) and vortex for 30s
    2. Vortex for 30 s;
    3. Centrifuge 15 min at 3,000 rpm to separate organic and aqueous phases (a white precipitate will appear at the interface);
    4. The upper MTBE was taken to a new EP tube and dried(a fixed volume, such as 200 μL) and  evaporate to dryness;

    5. Reconstitute (in EP tubes) the dried extract in 100 μL of DCM:MeOH (1:1,v/v), take 5uL of each sample as polled QC sample, measure the QC sample every 8 samples

     

    For mammalian cells and bacteria cells:
    1. Add 400uL MTBE and 80uL MeOH, 200uL H2O (keep MTBE:MeOH:H2O=10:2:5, v/v) to cell pellet (recommend ~5X106 cells/sample);
    2. Vortex for 30 s and sonicate 10 min;
    3. Centrifuge 15 min at 3,000 rpm to separate phases (a white precipitate will appear at the interface);
    4. The upper MTBE was taken to a new EP tube and dried(a fixed volume, such as 200 μL) and  evaporate to dryness;

    5. Reconstitute (in EP tubes) the dried extract in 100 μL of DCM:MeOH (1:1,v/v), take 5uL of each sample as polled QC sample, measure the QC sample every 8 samples

     

    For tissue (normalized by weight):
    1. Add 200 μL of H2O to tissue(10-50 mg), homogenize for three cycles (each cycle: 5500rpm for 20s,repeat three times) with cooled N2 gas flow from liquid N2;
    2. Then add 400uL MTBE and 80uL MeOH (keep MTBE:MeOH:H2O=10:2:5, v/v)
    3. Vortex for 30 s and sonicate 10 min;
    4. Centrifuge 15 mins at 3,000 rpm to separate phases (a white precipitate will appear at the interface);
    5. The upper MTBE was taken to a new EP tube and dried(a fixed volume, such as 200 μL) and  evaporate to dryness;
    6. Reconstitute (in EP tubes) the dried extract in 100 μL of DCM:MeOH (1:1,v/v), take 5uL of each sample as polled QC sample, measure the QC sample every 8 samples

  • Polar Metabolite Extraction Proctocol
    Polar Metabolite Extraction Proctocol

    :For mammalian cells and bacteria cells
    1.Add 1ml of MeOH:ACN:H2O (2:2:1, v/v) solvent mixture to the sample
    2.Vortex for 30 s,sonicate 10 min(4℃ water bath)
    3.1 min in liquid nitrogen;Thaw at room temperature;Sonicate 10 min (optional: in 4°C water bath).Repeat 3 times
    4.Incubate 1 h at -20°C (facilitate protein precipitation)
    5.Centrifuge 15 min at 13,000rpm and 4°C
    6.Take supernatant and evaporate to dryness at 4°C or r.t. using a vacuum concentrator
    7.Reconstitution: 100 μL of ACN:H2O (1:1, v/v)
    8.Vortex 30s , sonicate 10 min (optional: in 4°C water bath)
    9.Centrifuge 15 min at 13,000rpm and 4°C
    10.Keep supernatant in -80 °C prior to LC/MS analysis

    For plasma or serum (min. 100 uL)
    1. Add 400 uL of MeOH/ACN (1:1, v/v) solvent mixture to 100uL plasma or serum samples (keep 2:2:1 ratio, No H2O added)
    2. Vortex for 30 s
    3. Sonicate 10 min (optional: in 4°C water bath)
    4.Incubate 1 h at -20°C (facilitate protein precipitation)
    5.Centrifuge 15 min at 13,000rpm and 4°C
    6.Take supernatant and evaporate to dryness at 4°C or r.t. using a vacuum concentrator
    7.Reconstitution: 100 μL of ACN:H2O (1:1, v/v)
    8.Vortex 30s,sonicate 10 min (optional: in 4°C water bath)
    9.Centrifuge 15 min at 13,000rpm and 4°C
    10.Keep supernatant in -80 °C prior to LC/MS analysis
    Tips:
    • Frozen plasma/serum samples can be placed on ice to slowly warm up/defrost before extraction
    • 80% organic solvent is necessary to precipitate proteins in plasma, but some peptides or small proteins will be extracted

    For urine samples
    Generally, no extraction needed; centrifugation or MWCO filter to remove particulates (or proteins); Then diluted with water to 1:1 or 1:3 (v/v)
    Tips:
    • If urine samples from patients with kidney diseases, urine may contains more proteins; extraction may be necessary. And the extraction method is similar as Plasma samples to concentrate the metabolites
    • Normalization will be necessary for urine samples, e.g. creatinine

    For animal tissue
    1.Homogenize 20 mg tissue with 200 ul H2O; Add 800 uL of MeOH/ACN (1:1, v/v) solvent mixture to homogenized tissue samples
    2. Vortex for 30 s
    3. Sonicate 10 min (optional: in 4°C water bath)
    4.1 min in liquid nitrogen;Thaw at room temperature;Sonicate 10 min (optional: in 4°C water bath).Repeat 3 times
    5.Incubate 1 h at -20°C (facilitate protein precipitation)
    6.Centrifuge 15 min at 13,000rpm and 4°C
    7.Take supernatant and evaporate to dryness at 4°C or r.t. using a vacuum concentrator
    8.Reconstitution: 100 μL of ACN:H2O (1:1, v/v)
    9.Vortex 30s,sonicate 10 min (optional: in 4°C water bath)
    10.Centrifuge 15 min at 13,000rpm and 4°C
    11.Keep supernatant in -80 °C prior to LC/MS analysis
    Tips:
    • Cut tissue on top of dry ice to keep tissue frozen
    • If no homogenizer available, cut tissue into tiny pieces as small as possible
    • Avoid cross-contaminations when using hand held homogenizer
    • Most plastic homogenizer tubes have several contaminations with organic solvents, so homogenize tissue with H2O