1. Putting the following components into an empty bottle, and putting the empty bottle into a rotor to stir until the components are completely dissolved;
| Component | Consumption (100mL) |
|---|---|
| YNB for yeast culture | 1.6g |
| (NH4)2SO4 | 5g |
| H2O | 100mL |
2. The solution was filtered and sterilized by a 0.22μm filter, and then transferred to a 100 mL autoclaved empty bottle for later use.
1. Put the following ingredients into an empty bottle;
| Component | Consumption (100mL) |
|---|---|
| Peptone | 2g |
| Yeast extract | 1g |
| H2O | 100mL |
2. Autoclaving at 121℃ for 30min; 3. After autoclaving, 2g of glucose was added.
1. Put the following ingredients into an empty bottle;
| Component | Consumption (100mL) |
|---|---|
| Peptone | 2g |
| Yeast extract | 1g |
| Agar | 2g |
| H2O | 100mL |
2. autoclaving at 121 ℃ for 30 min; 3. After autoclaving, 2g of glucose, 2mg of G418(Geneticin), 100μL of Nourseothricin (clonNAT) (100μg/mL), and 100μL of Hygromycin (200μg/mL) were added.
1. Put the following ingredients into an empty bottle;
| Component | Consumption (180mL) |
|---|---|
| SC-Trp | 0.4g |
| Arg | 0.04g |
| Adenine sulfate | 0.2g |
| Glucose | 4g |
| H2O | 180mL |
To configure a solid medium: 2. Only 60mL H2O was added to the bottle containing SC-Trp, and 4g agar and 120mL water were added to another empty bottle; 3. Put the two bottles together into an autoclave for autoclaving (115 ℃, 15 min); 4. Take 20 mL of filtered and sterilized YNB solution and add it into the agar bottle after high pressure. Pouring the SC-Trp solution into an agar bottle added with YNB while the SC-Trp solution is hot, adding a rotor, and fully mixing by using a magnetic stirrer; 5. Quickly pour that mixed solution into a sterile culture dish to prepare a flat plate, Stan at room temperature for 4 hours, and refrigerating in a refrigerator at 4 ℃ for later use.
1. Put the following ingredients into an empty bottle;
| Component | Consumption (180mL) |
|---|---|
| SC-Trp-Leu | 0.4g |
| Arg | 0.04g |
| Adenine sulfate | 0.2g |
| Glucose | 4g |
| H2O | 180mL |
To configure a solid medium: 2. Only 60mL H2O was added to the bottle containing SC-Trp-Leu, and 4g agar and 120mL water were added to another empty bottle; 3. Put the two bottles together into an autoclave for autoclaving (115 ℃, 15 min); 4. Take 20 mL of filtered and sterilized YNB solution and add it into the agar bottle after high pressure. Pouring the SC-Trp-Leu solution into an agar bottle added with YNB while the SC-Trp-Leu solution is hot, adding a rotor, and fully mixing by using a magnetic stirrer; 5. Quickly pour that mixed solution into a sterile culture dish to prepare a flat plate, Stan at room temperature for 4 hours, and refrigerating in a refrigerator at 4 ℃ for later use.
1. Put the following ingredients into an empty bottle;
| Component | Consumption (180mL) |
|---|---|
| SC-Trp | 0.4g |
| Arg | 0.04g |
| Adenine sulfate | 0.2g |
| H2O | 180mL |
2. Respectively putting Xg of solid palmitic acid and Yg of lauric acid into the bottle
caps of two 50mL centrifuge
tubes, adding 150μL of absolute ethyl alcohol into each centrifuge tube to dissolve the
solid palmitic acid and the
lauric acid in a boiling water bath, and then air-drying the solid palmitic acid and the
lauric acid in a fume hood
until no liquid adheres to the surface of the centrifuge tube;
3. Transfer the air-dried palmitic acid and lauric acid to an EP tube, add 1.8mL of
water and 0.18mL of Tween-80
respectively, dissolve in a boiling water bath and stir well;
4. Take another EP tube, add ZmL of oleic acid and 0.18mL of Tween-80, heat in boiling
water bath and mix well;
5. Heat the solution in (1) and 1.8mL of glycerol in boiling water;
6. Add palmitic acid, lauric acid, oleic acid and glycerol into the bottle containing
the ingredients in (1), mix well,
and autoclave (115 ℃, 15 min);
7. Finally, add 20 mL of YNB solution to the bottle.
Attachment: X, Y, Z value of different components of glycerol fatty acid medium
| Glycerol fatty acid medium | X (g) | Y (g) | Z (mL) |
|---|---|---|---|
| Glycerol: oleic acid = 1:1 | 0 | 0 | 0.18 |
| Glycerol: palmitic acid = 1:1 | 0.14 | 0 | 0 |
| Glycerol: lauric acid = 1:1 | 0 | 0.12 | 0 |
| Glycerol: lauric acid: oleic acid = 1:1:1 | 0 | 0.12 | 0.18 |
| Glycerol: lauric acid: palmitic acid = 1:1:1 | 0.14 | 0.12 | 0 |
| Glycerol: palmitic acid: oleic acid = 1:1:1 | 0.14 | 0 | 0.18 |
| Glycerol: palmitic acid: oleic acid = 1:1:2 | 0.14 | 0 | 0.36 |
| Glycerol: palmitic acid: oleic acid = 1:1:3 | 0.14 | 0 | 0.54 |
| Glycerol: palmitic acid: oleic acid = 1:1:4 | 0.14 | 0 | 0.72 |
| Glycerol: palmitic acid: oleic acid = 1:1:5 | 0.14 | 0 | 0.90 |
The plated solid medium of Saccharomyces cerevisiae was placed in a constant temperature incubator at 30 ° C.
1. Taking a streaking plate of Saccharomyces cerevisiae out of an incubator and putting
the streaking plate into an
ultra-clean workbench;
2. Pour 10 mL of liquid culture medium into a 50 mL centrifuge tube;
3. Picking the monoclonal from the flat plate, putting the monoclonal into a centrifuge
tube, shaking the centrifuge
tube to fully transfer the yeast on the bacteria picker to the centrifuge tube, and
culturing the yeast on a shaker at
37 ℃ for 3 days.
1.Add 750 μL of 50% glycerol solution into the EP tube;
2. Add 750 μL of bacterial solution into each EP tube, mark it and store it in a
refrigerator at -20 ℃.
1. After burning the inoculation loop, extending the cooled inoculation loop into the
bacterial solution, and dipping a
small amount of the bacterial solution;
2. First scribing (area ①): pick up the marked plate with the left hand, open a seam of
the cover, and make a dense
zigzag or continuous scribing on the surface of area ① of the plate with the inoculation
ring dipped with the bacterial
solution, which accounts for about 1/4 of the plate area. When marking, the force should
be gentle, and the medium
should not be cut;
3. Mar for that second time (area ②): burning the inoculation re on the flame of an
alcohol lamp to kill the remaining
bacteria on the ring. After the inoculating ring is cooled, rotate the plate by about 90
degrees, extend the cooled
inoculating ring into area ①, contact with the scribing mark in area ① for 1 to 2 times,
and then scribe in area ②. When
marking, the beginning of area ② should overlap with the end of area ①, but the
subsequent marking of area ② should not
return to area ①;
4. Scribing for the third time (area ③) and the fourth time (area ④): repeating the step
(3): burning the inoculating
ring, cooling, rotating the plate by about 90 degrees again, contacting the inoculating
ring with the end of the
previous area for scribing, and scribing in new areas (area ③ and area ④).The area ④
shall be the last and most sparsely
marked area;
5. After marking, close the flat cover. Thoroughly burning and sterilizing the
inoculation ring on the flame, and
putting the inoculation ring back to the original place;
6. Turning the scratched flat plate upside down, putting the flat plate into a constant
temperature incubator at 37℃,
and culturing for 18 to 24 hours.
1. Putting a solid culture medium plate into an oven for drying in advance;
2. Taking the solid culture medium into an ultra-clean bench, and pouring 5 to 6 glass
beads into the ultra-clean
bench;
3. Dripping the bacterial liquid on the glass beads, shaking the flat plate so that the
bacterial liquid is uniformly
distributed on the surface of the flat plate, and then pouring the glass beads;
4. Culture in a constant temperature incubator at 30 ℃ for about 3 days.
1. Quantification Before Transfection
OD600 in the liquid culture medium of Saccharomyces cerevisiae was measured. If OD600
= 0.3, 400 μL of bacterial
solution was added to 100 mL of YPD medium.
2. Preparation of Plasmid
(1) Taking out a plasmid dry powder tube and centrifuging;
(2) Adding sterile water into the tube according to the instructions;
(3) Take out 10 μL to EP tube, and add 32 μL sterile water for standby;
(4) Store that rest plasmid in a refrigerator at the temperature of -20 ℃.
3. Preparation of Competent Cells
(1) Put sterile water into a refrigerator at that temperature of 4 ℃ for later use, and
diluting LiAC/TE to 0.1 mol/L
for later use;
(2) Determining the OD600 of the Saccharomyces cerevisiae cultured on the previous day,
and if the OD600 is equal to
0.8, carrying out the next experiment;
(3) Take 50mL of bacterial solution, centrifuge (3500 rpm, 2min) and discard the
supernatant;
(4) Resuspend with 50 mL of cold sterile water, centrifuge (3500 rpm, 2 min), and
discarding that supernatant;
(5) 1 mL of 0.1M LiAC/TE was used for resuspension, and the supernatant was discarded
after rapid centrifugation (4000
rpm, 30 s);
(6) Resuspend with 400 μL of 0.1M LiAC/TE, mix well, and place in the refrigerator
overnight on ice.
4. Plasmid Transfection
(1) Add about 50μL of yeast competence prepared the day before to each EP tube,
centrifuge (4000rpm, 30s) and discard
the supernatant;
(2) Boile salmon sperms in boiling water for 5 min, and then quickly putting that salmon
sperms into an ice-water
mixture;
(3) 42μL of plasmid, 10μL of salmon sperm (10mg/mL), 240μL of PEG, and 36μL of LiAc (1M)
were sequentially added to the
EP tube. Repeatedly blow and mix with a 1mL pipette, and then incubate in a 30 ℃
incubator for 30 min;
(4) Add 36μL of DMSO into each EP tube, blow and mix repeatedly, and then put it into a
42℃ water bath for 20min.
5. Culture of Saccharomyces cerevisiae Aft Transfection
(1) Take out the EP tube in the water bath, centrifuge (3000rpm, 20s) and discard the
supernatant. 1mL of YPD medium was
used for resuspension and cultured in a shaker for 3h;
(2) After centrifugation (3000rpm, 20s), the supernatant was discarded and resuspended
in 100μL H2O;
(3) Dry that solid culture medium plate in an oven in advance, taking the solid culture
medium plate into an ultra-clean
table, pour 5 to 6 glass beads into the solid culture medium plate, dripping the
bacterial solution onto the glass
beads, shake the solid culture medium plate to uniformly distribute the bacterial
solution on the surface of the solid
culture medium plate, pouring the glass beads out of the solid culture medium plate, and
culturing the solid culture
medium plate in the incubator at the temperature of 30 ℃ for about 3 days.
1. Streaking Culture of Escherichia Coli
See "II-4".
2 Liquid Culture of Escherichia Coli
(1) In an ultra-clean workbench, an aseptic bamboo stick or a gun head is used for
picking Escherichia coli blocks and
adding the Escherichia coli blocks into 5 to 10 ml of LB culture medium containing
antibiotics;
(2) Unscrew that cover of the t tube or the culture bottle (to ensure ventilation),
placing the test tube or the culture
bottle into a constant-temperature shaker at 37 deg C, and violently shake and culturing
the test tube or the culture
bottle for 12 to 16 hours (overnight) at 200 to 250 rpm;
(3) The turbidity of the culture medium indicates that the bacteria have grown in large
quantities.
3 Extraction of Escherichia Coli Plasmid (plasmid Extraction Kit)
(1) Check whether RNase A has been added to Buffer P1, whether absolute ethyl alcohol
has been added to Wash Solution,
and whether precipitation has occurred in Buffer P2 and P3;
(2) Take 3 mL of bacterial solution cultured overnight, centrifuge (8,000 × g, 2 min) to
collect the thalli, and discard
the culture medium;
(3) 250 μL of buffer P1 is added into that sediment to thoroughly suspend the
bacteria;
(4) Add 250 μL Buffer P2, immediately and gently invert the centrifuge tube for 5 to 10
times, mix well, and stand at
room temperature for 2 to 4 min;
(5) Add 350μL Buffer P3, immediately and mildly invert the centrifuge tube for 5 ~ 10
times and mix;
(6) Centrifuging (12,000 × g, 10 min), transferring the supernatant into an adsorption
column, and standing for 3 to 4
min;
(7) Centrifuge (8,000 × g, 30 s), pour out the liquid in the collection tube, add 500 μL
Buffer DW1, centrifuge (9000 ×
g 30 s), pour out the liquid in the collection tube;
(8) Add 500 μL of Wash Solution, centrifuge (9000 × g, 30s), and pour out the liquid in
the collection tube;
(9) Repeat (8) once;
(10) Empty adsorption column centrifugation (9000 × g, 1min);
(11) Put the adsorption column into a clean 1.5 mL centrifuge tube, add 50 ~ 100 μL
Elution Buffer in the center of the
adsorption membrane, stand for 1 min at room temperature, and centrifuge for 1 min. DNA
solution in storage tube.
1. Take 200 μL of yeast culture solution, centrifuge (4000 rpm, 30 s) and discard the
supernatant;
2. The cell pellet was resuspended in 100 μL LiOAc-1% SDS solution and incubated in an
oven at 70 ℃ for 5 min;
3. 300 μL of absolute ethanol was added, repeatedly blown and mixed, centrifuged (15000
rpm, 3 min), and the
supernatant was discarded;
4. Washing the precipitate with 70% ethanol solution, centrifuging, and discarding the
supernatant;
5. The precipitate was dissolved in 100 μL of water and stored in a refrigerator at -20
℃.
1. Configure a PCR reaction system according to that follow table;
| Reagent | Volume | Concentration |
|---|---|---|
| PrimeSTAR Max Premix(2X) | 25μL | 1× |
| Primer 1 | 2μL | 0.2-0.3μM |
| Primer 2 | 2μL | 0.2-0.3μM |
| Template | 5μL | |
| DNase-Free Water | To 50μL |
2.Set the PCR reaction program according to the following table;
| Temperature | Time | Number of cycles |
|---|---|---|
| 94℃ | 3 min | 1 cycle |
| 94℃ | 5 sec | 30 cycles |
| 55℃ | 15 sec | |
| 72℃ | 30 sec | |
| 72℃ | 5 min | 1 cycle |
3.Prepare 2% agarose gel according to that follow table, and dissolving by microwave heating;
| Reagent | Amount |
|---|---|
| Agarose | 12 g |
| 1×TAE | 60 mL |
| Nucleic acid dye EB | 6 μL |
4. When cooled to 50 ℃, add 6 μL of EB, shake it up, pour it into the glue preparation
tank where the comb has been
placed, lay it flat at room temperature, and wait for gel solidification;
5. Put that gel into an electrophoresis tank, and pouring a prop amount of 1 × TAE
electrophoresis buffer solution.
Electrophoresis was performed at 140 V and 100 mA after loading for 25min;
6. Observing and photographing under an ultraviolet lamp after the electrophoresis is
finished;
7. Analyze that optical density with a gel analysis system.
1. Prepare conventional R-250 staining solution and decolorizing solution respectively according to the following table:
Conventional R-250 Dyeing System
| Reagent | Amount (100 mL) |
|---|---|
| Coomassie Brilliant Blue R-250(w/v) | 0.25 g |
| Methanol | 45 mL |
| Glacial acetic acid | 10 mL |
| H2O | Make up to 100 mL (approximately 45 mL) |
Conventional decolorizing solution
| Reagent | Amount (500 mL) |
|---|---|
| Methanol | 200 mL |
| Glacial acetic acid | 50 mL |
| H2O | Make up to 500 mL |
2. Preparing SDS-Page gel according to the method in 4.2 and carrying out
electrophoresis;
3. After gel electrophoresis, take down the gel plate, carefully peel the gel from the
glass plate, and put it into the
staining box/tray; wash the gel surface with deionized water for 1 to 2 times to remove
the electrophoresis buffer
residue (gently operate to avoid tearing the gel);
4. Fixation (SDS and protein dissolution can be removed to improve the signal to noise
ratio of staining): Add
sufficient fixative (methanol/acetic acid/water, typically 45% methanol + 10% acetic
acid + 45% water) to the container
containing gel, and shake gently at room temperature for 30 min. Fixation can be
extended to 1 h or overnight at 4 ℃ to
improve the quality of staining;
5. Discarding the fixing solution, and adding newly prepared Coomassie brilliant blue
staining solution to completely
immerse the gel. Stain on a horizontal shaker at room temperature for 1 to 2 hours;
stain overnight if more sensitive
(do not exceed 24 hours to avoid background increase);
6. After dyeing, discard the dye solution, decolorize with decolorizing solution (40%
methanol, 10% acetic acid, 50%
water) for the first time, and gently shake at room temperature for 30 min ~ 1 H until
the background begins to fade
significantly;
7. Replace with new decolorizing solution, continue to decolorize on the horizontal
shaker, and replace the
decolorizing solution repeatedly for 30 ~ 60 min each time according to the background
and band color development until
the background is low enough and the band contrast is clear. Complete decolorization may
take a few hours to a day or
two (it can be left overnight and decolorized the next day); if you want to see the
bands quickly, you can decolorize
for a short time and take a picture immediately before continuing to refine the
decolorization;
8. After satisfactory decolorization, quickly rinse the gel with deionized water to
remove residual methanol/acetic
acid, then place the gel in a photographic tray and photograph/scan with a gel imaging
system or flatbed scanner; white
light transmission or reflection mode is recommended depending on the equipment, and
record the exposure parameters for
comparison;
9. Photograph/scan and measure band intensity with image software (ImageJ, etc.) For
quantification, taking care to use
the same exposure/scan settings and measure within the unsaturated range.
1. Yeast Total Protein Extraction
(1) Prepare the yeast lysate according to the following table: based on NaOH, add each
reagent in the following table
per 1mL of NaOH, and add protease inhibitors and phosphatase inhibitors according to the
specific requirements of the
experiment (optional);
| Reagent | Amount |
|---|---|
| 0.1M NaOH | 1 mL |
| 100 mM Sodium β-Glycerophosphate | 11 μL |
| 100 mM Sodium orthovanadate | 11 μL |
| 5M NaF | 11μL |
| 0.5M EDTA | 3 μL |
| 100mM PMSF | 11μL |
(2) Take 200 μL of yeast culture solution, centrifuge (4000 rpm, 30 s) and discard the
supernatant;
(3) Adding a lysis solution into the obtained thallus precipitate, and performing lysis
on ice for 10 min;
(4) Centrifuging (5000 rpm, 1 min), and discarding the supernatant;
(5) Add 80 μL of 1 × loading buffer into the precipitate, mix well, place in a metal
bath (100 ℃, 5min), and store in a
refrigerator at -20 ℃ for later use.
2. Western Blot
(1) Prepare 12% separating gel and 5% concentrated gel according to the following table.
Place the separating gel at
room temperature for 20 ~ 30 min, and place the concentrated gel at room temperature for
about 5 ~ 10 min. For loading,
add 10 μL of protein sample to each well and 3 μL of marker to one well, and be careful
not to generate bubbles.
| 12% separating gel | |
|---|---|
| Reagent | Amount |
| ddH2O | 1.6 mL |
| 30% Polyacrylamide | 2 mL |
| 1.5M Tris-HCl (pH8.8) | 1.3 mL |
| 10%SDS | 50 μL |
| 10%APS | 50 μL |
| TEMED | 5 μL |
| 5% concentrated gel | |
| Reagent | Amount |
| ddH2O | 2.1 mL |
| 30% Polyacrylamide | 0.5 mL |
| 1.0M Tris-HCl (pH8.8) | 0.38 mL |
| 10%SDS | 30 μL |
| 10%APS | 30 μL |
| TEMED | 3 μL |
(2) Electrophoresis: electrophoresis conditions: 75 V, 30 min for the upper concentrated
gel, 110 V, 60 ~ 80 min for the
lower separating gel;
(3) Film transfer: when the electrophoresis is completely finished, the rubber plate is
taken down, the rubber plate is
lightly gummed, and the corresponding PVDF film and filter paper are cut according to
the size; The PVDF membrane was
activated by methanol for about 60 s before membrane transfer. Carry out film transfer
in a mixture of ice and water,
wherein that film transfer voltage is 100 V, and the time is 30 to 50 minute;
(4) Milk blocking and hybridization: Take out the PVDF membrane, wash it with TSBT
solution on a horizontal shaker for 3
times, 10 min each time, block it with 5% skim milk, and block it on a horizontal shaker
at room temperature for 1 H;
(5) After blocking for 1 h, wash with TBST for three times, 5 min each time, and then
incubate with the diluted primary
antibody, and dilute with TBST solution according to the specific instructions of the
antibody; After the primary
antibody was added, it was placed in a refrigerator at 4 ℃ overnight, and the next day
it was placed on a shaker and
washed with TBST for three times, 10 min each time, and then incubated with the
secondary antibody for 1 h, and finally
washed with TBST for three times, 10 min each time;
(6) ECL color development: Add solution a and solution B according to the ECL color
development instructions (Thermo
Fisher ECL), with a volume ratio of 1:1. The gel image processing system was used to
take pictures after the two liquids
were mixed and kept away from light for 1 ~ 2 min at room temperature, and the gray
value of each band was scanned by
its image processing software, and finally the statistical analysis was carried out.
1. Extraction of Target Protein from Engineered Strains
(1) Inoculate the selected engineered strains onto selective solid medium. Pick
well-grown single colonies and transfer
them into liquid medium for cultivation;
(2)After ~20 h of incubation, when the OD600 reaches between 1.8 and 2.0, transfer 1 mL
of the culture into fresh liquid
medium and continue cultivation until the OD600 reaches 1.8 ~ 2.0 again;
(3) Harvest 15 mL of the culture by centrifugation at 10,000 rpm for 2 min, and collect
the cell pellet;
(4) Wash the cell pellet once with 1 mL PBS, centrifuge at 10,000 rpm for 2 min, and
resuspend the pellet in 1.5 mL PBS;
(5) Disrupt the yeast cells using an ultrasonic cell disruptor at 400–700 W, with 6 s
on/5 s off cycles, for a total
sonication time of 20 min;
(6) After disruption, the cell suspension becomes noticeably clarified. The resulting
lysate represents the crude total
protein extract, which can be used directly for enzymatic reactions.
2. HPLC-Based Assay of Glycosyltransferase Activity
(1)Experimental group: Add methanol-dissolved Zingibroside R1 and Chikusetsusaponin IVa
to the crude enzyme extract from
disrupted engineered yeast cells, together with UDP-glucose (UDPG);
Control group: Add methanol-dissolved Zingibroside R1 and ChikusetsusaponinIVa to the
crude enzyme extract from
disrupted non-recombinant Saccharomyces cerevisiae cells, together with UDPG.
(2) Incubate both groups at 20 °C for 20 h;
(3) After 20 h, inactivate the enzymes by heating at 99 °C;
(4) Centrifuge the reaction mixtures at 12,000 rpm for 2 min, and extract the products
with ethyl acetate;
(5) Evaporate the ethyl acetate to dryness in a 50 °C water bath;
(6) Redissolve the residue in 300 μL methanol, filter through a 0.45 μm organic
membrane, and analyze product conversion
using high-performance liquid chromatography (HPLC).
Attachment: Glycosyltransferase Reaction System (Total Volume: 300 μL)
| Reagent | Amount(300μL) |
|---|---|
| Tween-20 (undiluted) | 3μL |
| 50 mM UDP-glucose (UDPG) | 30μL |
| 1M Tris-HCl(PH8.5) | 15μL |
| 100 mM Ginsenoside substrate | 0.15μL |
| Crude enzyme extract | 251.85μL |
1. Taking 3mL of fermentation liquor into a crushing tube, centrifuging (13000 rpm/min,
3min), and discarding an upper
layer of culture medium;
2. Add a proper amount of small glass bead (diameter: 0.5mm) and 1mL of an extracting
agent (chloroform: methanol =
1:1) into that precipitate, and shaking and crushing for 5min;
3. Carry out ultrasonic crushing for 30 minute, and keeping that temperature not higher
than 50 deg C;
4. Centrifuging (13000 rpm, 3min), taking the supernatant and placing in a refrigerator
at -20℃ for later use.
Chromatographic separation was performed on a VP-ODS C18 column (150 mm × 4.6 mm,5 μm)
using a gradient elution system.
Separation conditions:
Acetonitrile (solvent A) and deionized water (solvent B).
The column temperature was 35 ℃, the injection volume was 20 μL, the flow rate
was 1 mL/min, and the wavelength of UV detector was 203 nm.
Elution conditions:
0 ~ 25 min: 19% B, 81% A;25 ~ 50 min: 29% B, 71% A.
A GL Sciences Inertsil ODS 3 column (2.1 × 150 mm, 5 μm) was used for gradient elution.
Separation conditions:
Acetonitrile (solvent A), 0.1% phosphoric acid (solvent B).
The column temperature was 35 ℃, the injection volume was 5 μL, the flow rate was 0.3
mL/min, and the wavelength of UV
detector was 203 nm.
Elution conditions:
0 ~ 3min 80%B；5min 70%B；10min 60%B；16min 50%B；20min 15%B；22 ~ 28min 0%B；30 ~ 35min 80%B.
1. LC
A GL Sciences Inertsil ODS 3 column (2.1 × 150 mm, 5 μm) was used for gradient elution.
Separation conditions:
Acetonitrile (mobile phase A), 0.1% formic acid (mobile phase B).
The column temperature was 35 ℃, the injection volume was 3 μL, the flow rate was 0. 3
mL/min, and the wavelength of UV
detector was 203 nm.
Elution conditions:
0 ~ 3min 80%B；5min 70%B；10min 60%B；16min 50%B；20min 15%B；22 ~ 28min 0%B；30 ~ 35min 80%B.
2. MS
(1) Samples were collected by Waters mass spectrometer with electrospray ionization
(ESI) in positive and negative ion
modes. Capillary voltages in positive and negative modes were 2.6 kV and -2.2 kV,
respectively; mass spectra were
scanned from 200 to 1100 m/Z.
(2) Other conditions are as follows:
Cone voltage, 40 V; desolvation temperature, 400 ° C; cone gas flow, 50 L/H; source
temperature, 150 °C; Desolventizing
gas flow, 800L/h. The high energy channel voltage is 20 ~ 40 V, and the low energy
channel voltage is 6 V.
(3) Mass spectrometry data were collected using the MSEcentroid mode. Sodium formate was
used to correct the mass of the
instrument, and leucine-enkephalin (100ng/ml, m/Z 556.2771 [M+H]+, m/z 554.2615 [M-H]-)
was used to correct the
instrument in real time, and the flow rate was set at 20 μl/min. In order to evaluate
the stability of the device during
sample testing, 4 tests were performed randomly on the QC samples from the entire sample
list.
