comprehensive systems biology analysis of a 7-month cigarette smoke inhalation study in c57bl/6 mice

Smoking of combustible cigarettes has a significant impact on human health.
Use of systematic toxicology methods in models of chronic blocked lung disease (C57BL/6 mice)
, We evaluated the health effects of aerosol derived from prototype modified risk tobacco products on mice (pMRTP)
Compared with traditional cigarettes.
We studied physiological and histological endpoints parallel to transcription, lipid, and proteomics in mice exposed to reference cigarettes (3R4F)
Smoke or pMRTP aerosol for up to 7 months.
We also include a stop Group and a switch-to-pMRTP group (
3 r4f after 2 months of exposure)
Except for control (fresh air-exposed)
Group, to understand the potential risk reduction of switching to pMRTP compared to continuous 3R4F exposure and stop.
This manuscript describes the design, setup, and implementation of the study, as well as the generation, processing, and quality control analysis of toxicology and \"omics\" data sets accessible in public repositories for further analysis.
Chronic pulmonary disease (COPD)
It is the main cause of chronic morbidity and mortality worldwide, usually caused by smoking.
At least in part, rodent models of cigarette smoke can be used to study biological processes related to the development of chronic lung obstruction (CS)exposure.
There is a moderate degree of celase inhibitor Serpina-in mice-
1, this is more obvious in women, and it develops to swelling of the air and Pinghua of the cup-shaped cells after smoke exposure.
They smoke easily-
Induced Airway remodeling is therefore a useful model for studying the occurrence and development of asthma, characterized by reduced lung function, Abnormal airway inflammatory response, and small airway remodeling, and the early stages and aspects of human chronic lung obstruction are common in the destruction of the pulmonary tissue.
To investigate the effect of tobacco aerosol inhalation on the development of swollen/chronic lung obstruction, we evaluated mice exposed to smoke/aerosol from two tobacco products (regular reference cigarettes (3R4F)
And prototype improved risk tobacco products (pMRTP), in a 7-
Study on monthly inhalation
Female mice were randomly divided into the following five groups :(1)
Sham exposed to filter air conditioning (2)
3R4F, exposed to the mainstream smoke of 3R4F cigarettes (
Total particulate matter of 750 mg (TPM)/mu2009target dose), (3)
PMRTP exposed to MRTP prototype aerosol (
Test the nicotine level in the atmosphere to match the nicotine level in the I . 3R4F Groupe. , 34u2009mg/m), (4)
Stop, be exposed to 3R4F for 2 months, then be exposed to a filtered air conditioner for 5 months, and (5)
Switch to pMRTP and be exposed to 3R4F for 2 months and then to pMRTP aerosol for 5 months.
Animals are exposed to 4 Thanh/day, 5 days/week for up to 7 months.
Regularly analyze the test environment in the animal breathing area to ensure the quality and repeatability of exposure throughout the study period.
Biological Monitoring of animals ()
Demonstrate that the aerosol is effectively absorbed and absorbed
Life observations confirmed that animals were well tolerated for both 3R4F and pMRTP exposure concentrations.
The lung pathology and functional changes in a specific animal group were assessed by standard tissue pathology and morphometric analysis, as well as lung puffs volume: 55 yml;
Frequency of puffs: 30 m/s, poor ventilation, 30-
Rotary smoking equipment (15 ports used)
Equipped with programmable double syringe pump (PDSP)
And active side flow exhaust (type PMRL-G, SM2000).
Mainstream cigarette (CS)
Dilute to a target TPM concentration of about 34 mg TPM/m with filtered air conditioning fresh air.
4 μg nicotine/l aerosol.
The PMRTP aerosol concentration matches the nicotine level used for 3R4F exposure, I . E. e. , 34.
4 μg nicotine/l aerosol.
The smoking conditions of PMRTP are: 12 puffs per branch;
Puffs: 55 ml, puffs frequency: 30 m/s, using a modified SM2000 machine equipped with PDSP.
PMRTP aerosol or 3R4F CS is delivered through a glass pipe from the smoking machine to the exposure room.
Total flow rate through the whole-
The body exposure room is adjusted to ≥ 80 liters/minute (
Achieve target nicotine concentration by adjusting diluted Air).
For the sham group, mice were exposed to filtered air-conditioned fresh air and the flow rate was similar to that in the aerosolexposed groups. Whole-
The body exposure chamber is used to provide controlled and repeatable exposure conditions.
The study inhalation phase began in March 12, 2012 and ended in October 28, 2012.
It was 16 at first.
Daily adaptation period: the target 3R4F TPM concentration was 200 µm on study day 1-4, 400 µm on study day 5, 8 and 9, and 10-12 days of normal study time.
Study from the 15 th (
Not exposed on weekends)
The target concentration is 50 µm.
PMRTP uses a similar adaptation period
In exposed mice, the concentration of pMRTP aerosol was adjusted to match the nicotine level of 3R4F aerosol.
Since then, mice have been exposed to fresh filtered air or to CS or pMRTP aerosol with a target concentration of 34.
4 μg of nicotine/l per day, 5 days per week.
Animals were exposed to filtered fresh air 30 min after completing the first hour of exposure and 60 min after completing the second and third hours of exposure (Exposure block)
Prevent acute carbon monoxide poisoning (for 3R4F)
And control of carbon and oxygen hemoglobin (COHb)
No more than 50% levels.
Mice with predetermined anatomy were exposed to the same smoking regimen for at least 2 consecutive days prior to autopsy (
Including weekends in case of anatomy on Monday or Tuesday).
The position of the cage in the exposure chamber rotates twice a week in each column of the chamber from top to bottom to minimize the impact of the position.
Weigh regularly before distribution.
At the point in time assigned to the group, weight measurement was performed and used for the distribution procedure, which ensured a similar weight distribution between the groups and an even distribution of weight within the exposure group.
Thereafter, weight was taken twice a week during the whole study of calibration and verification of balance.
At the time point of the autopsy, the mice were weighed to determine the dose of anesthesia used (
Weight before anesthesia).
If weighed twice on the same day, the average weight is recorded.
The data generated by the weight measurement can be found in the \"weight ISA File (Metadata)
\"Original data file of weight \"[: Figshare ].
Got blood from vintage
Direct Orbital sinus-
Coated blood vessels.
Analysis requires at least 50 μ l of whole blood.
Samples were analyzed within 2 hours of collection using the Sysmex XT 2000 I clinical blood analyzer.
The following parameters are measured by the instrument: red blood cells (RBC)and platelets (PLT)
DC detection technology (
Impedance method
With hydraulic focus, hemoglobin (HGB)
According to the hemoglobin method and red blood cell pressure of sodium 12 alcohol (HCT)
Cumulative pulse height measurement method is adopted.
And white blood cells (
Differential and relative Count)
, And the number of net-woven red blood cells was estimated using the flow cell method of semiconductor laser.
From these parameters, the average cell volume (MCV)
Calculate red blood cells and HCT, mean cell hemoglobin (MCH)
Calculate the concentration of red blood cells and HGB and mean cell hemoglobin (MCHC)
Calculated for HGB and HCT.
All samples were repeatedly analyzed and the results were accepted only if the second measurement was not the same as 10% of the first measurement;
An additional set of measurements were also taken when the acceptance criteria were not met.
The data generated by the Hematology measurement can be in the \"Hematology ISA file (Metadata)
And the documents \'raw data of Hematology [: Figshare ].
Blood COHb is determined by measuring the absorbance of several wavelengths of mixed hemoglobin in blood dissolved by luminosity.
This is done in 3, 4 and 6 months (see ).
Plasma levels of nicotine and cotinine were determined using established LC-MS/MS methods (
ABF, Munich, Germany)
From the blood sample taken on the 7 th
Monthly anatomy (see ).
Urine samples were collected overnight after exposure and stored at-80 °c and then used for high
High performance liquid chromatography (LC)
Analysis of nicotine metabolism3′-
Tinning, Tinning, nicotine-N′-
1-3 after diethyl-3 oxide and nicotine per group2-
Thiobabbit uric acid derivative as described.
The value of the latest time point measured (6 months)are given in .
According to the method described earlier, on the day after the last exposure, a full autopsy was performed without an empty stomach.
Animals are randomly placed into exposed groups, including sub-groups, when assigned
Group defining the time point of autopsy (
Month 1, 2, 3, 4, 5, 7)
And endpoint type, and limit to maintain a similar weight mean between groups.
Thereafter, the groups remained unchanged throughout the course of the study.
Because the number of mice to be dissect at one point in time is large, the anatomical process lasts for several days.
Before each anatomy, the animals were randomly assigned when preparing the anatomy schedule.
The anatomy coordinator ensures that there is no bias in the spread of animals in the exposure group during each anatomy day and anatomy.
On the day of anatomy, the animals were dissect according to the anatomical schedule.
The person who performed the anatomy did not know the original treatment group to which the animal belonged.
Different anatomical protocols are used according to the endpoint in the focus ().
Mice were injected with e-barbital by abdominal cavity (100u2009mg/kg).
Blood collection through vintage
The Orbital sinus of the mouse after reaching the depth plane of anesthesia.
Two equal samples of 100 µμ l whole blood were collected in RNAprotect tubes (
Qiagen, HILDEN, Germany)
Used for nucleic acid extraction.
Blood is inverted at least 10 times and upright overnight at room temperature to ensure effective cell cracking before storage at-80 °c
The rest of the blood is to use. . .
Coated tube, inverted 10 times, placed for at least 30 minutes, then stored at-80 °c
Completely draw blood by cutting off the aorta, and then dissect the mouse to collect the lungs.
Exposure of respiratory organs by making an initial mid-
Exposed abdominal incision in the chest area. The rib-
By cutting the place close to the breastbone, exposing the lungs, trachea and throat, the cage was opened.
Carefully cut any connected tissue so that the lungs, throat and trachea are removed from the chest as a unit.
After taking it out, a small incision was made in the middle
The trachea point between the throat and the bifurcation.
The trachea sleeve is then inserted into the incision and tied with a ligation method.
The casing is connected to a fixed bottle through a surgical pipe.
The lungs are instilled and the back is down. Fixative (EGAFS; glycerin (5%), acetic acid (5%)Formaldehyde (4%), NaCl (0. 4%))
Inject under the water pressure equivalent to the 15 cm water column.
After perfusion, the trachea was ligated and the casing was removed.
Then, in EGAFS, the lungs were instilled in 48 km/h in a \"inflated\" manner and then transferred to ethanol (70%)
Until histological treatment.
\"Omics\" organization collection 0.
5-3 Thanh after the last exposure (Omics1)
Including acute exposure effects or within 16-24 hours of last exposure (Omics2)
Determine only the longer-
Lasting exposure.
As mentioned above, mice were anesthesia with E. obarbital.
No complete blood draw was performed using 27-1 to promote lung perfusion through the right brain Chamber
G needle with icecold 0.
Add a 100 salt solution under a static hydraulic pressure of 9% cm to clear the lungs in the remaining blood cells.
Do outlet cutting at the right atrium and descending aorta to drain.
Complete perfusion of the liver and lungs occurs within 2 min, and the paleness of the organs indicates this.
After perfusion, the heart is collected first, then the left and right lobe are collected.
Subsequently, the entire liver and the extracted nasal epithelial cells were dissect simultaneously by two isolates.
Collect all tissues within 15 minutes from the start of perfusion to avoid tissue degradation and maintain high quality RNA.
The organ was broken immediately.
Freeze in liquid nitrogen before storage at-80 °c
Efforts to minimize RNA degradation, such as the use of RNaseZAP (
Carlsbad life technology, California, USA)
Strictly observe the cleaning of tools and surfaces.
18-24 hours after the last exposure, animals from a specialized group were measured using unrestricted full body volume photography (
FlexiVent system SCIREQ, Montreal, Canada)
As mentioned earlier.
Anesthesia of mice by abdominal cavity (i. p. )
Injection of ketanone and 0 of 50 mg/kg.
33 mg/kg medetom set.
Subsequently, the trachea of the mouse was cut, and the trachea was cut and connected to the computer.
Small animal ventilator control.
I caused paralysis before the measurement. p.
Taking Panku ammonium bromide (0. 8u2009mg)
In order to avoid the interference of spontaneous breathing after default ventilation (
Baseline, no maneuver). Regular quasi-
The frequency of sine ventilation was 150 times/min, and the tidal volume was 10 kg/kg.
Flexible disturbance maneuvers, including maximum lung capacity (
The so-called tlc scireq), quasi-Static pressure-
Single chamber volume loop (snapshot)
Constant phase model (primewave-3)
, And negative pressure forced to expire.
Lung F4/80-data generatedAPC)
And DNA content (PI). The sub-
CD4 T-cell division of lymphocytes in LPScells, CD8+ T-cells, and B-
Analysis of cells (Ly6g-PE, CD4-PerCP, CD8-PE-Cy7, B220-APC-Cy7, CD45-FITC, CD69-APC).
Only the measurement of> 500 events in the lymphocytes gate is considered to be an effective calculation of the proportion of lymphocytes.
To check non
Specific staining, other samples were stained with appropriate Isotype control antibodies.
The measurement results were corrected by automatic fluorescence/non-fluorescence
Specific fluorescence.
The data generated from the assessment of face s in the LPs can be found in the \"face s analysis report in The LPs\" and the \"cell count and enzyme activity in the free lung cells in the LPs\" files [: Figshare ].
Metal enzyme (MMP)
Based on the cracking of Chromium fluoride, the activity in asthma culture medium was determined using commercially available analytical kits
Dissolution of gelatin in LPS labeled gelatin (
Enzyme-cut gel enzyme/enzyme assay kit;
Molecular Probes, Eugene, United States of America).
After collection, save the samples of LPs at-70 °c until analysis, no freezing-thaw cycles.
Samples are analyzed within 3 months of collection ().
The data generated by the measure of the protein hydrolysis activity of LPs can be found in the [file] \"The number of free lung cells in the LPs and the enzyme-promoting activity\": Figshare ]. Cell-
A large number of free sample preparation lesions were used to carry out lung and lung irrigation fluid (
Rule-based medicine in Austin, Texas)
Reuse bead arrays (RodentMAP v 2. 0)().
Six samples were shipped on dry ice, each corresponding to one of the six exposure periods (
Exposure for 1, 2, 3, 4, 5, 7 months)
, 6 batches of measurements were made in a total of about 7 months.
Therefore, the list of analytes measured changes over time (
When the vendor changes the content of the RodentMAP)
, Three specific analytes were measured only on samples of the first four batches of goods, five specific analytes were measured on samples of the last two batches of goods, and 55 others on all samples
A total of 63 analytes were identified that were commonly considered as biomarkers of inflammatory response.
The data generated by the MAP evaluation of the LPs can be found in the \"analysis of the LPs\" file [: Figshare ].
The left and right lobe of 10 mice per exposure group and time point was paraffin-embedded.
The left lung was used for morphological measurement and tissue pathology assessment, and the right lung was stored as a reserve tissue. Step-
Serial segment (4u2009μm thick)
Made of paraffin.
Embedded in the left lobe every 15 μm, stained with Sumueosin (Sigma), Periodic acid-Schiff (PAS)-alcian blue (Merck), and resorcin-fuchsin (Ellipsiz).
Digitize the tissue slices using the Aperio scanner (
Aperio technology).
Five lung sections were selected for tissue pathology assessment based on the following strategies: This method provides central and peripheral aspects of lung essence for tissue pathology investigations.
Evaluation of high-foot Cup cells (main bronchus)and bronchus-
Related lymph tissue (BALT)
Because the main trachea performed best at this level, surgery was performed on lung section 3. XXL-PAS-
Positive macrophages are a type of lung macrophages confined to the lung cavity and also counted from Section 3.
Cell size varies from 18-28 μm.
All other pathological endpoints were evaluated for each lung slice (
5 independent data sets).
Air swelling and fibrodegeneration were evaluated by semi-tissue pathology
Quantitative methods (
Histovia GmbH auverat, Germany)
Use severity score according to defined grading system: 0 = no difference with normal form;
1 = the difference between normal form and normal form is the smallest;
2 = some areas are slightly different compared with normal morphology, and some areas are slightly changed;
3 = slightly changed compared with normal morphology (
Local severity and spread distribution);
4 = some areas are slightly different compared with normal morphology, and some areas have undergone serious changes;
5 = the overall serious change compared with the normal form (
Local severity and spread distribution).
Please note that, to the extent possible, a complete severity scale was used, and reference slides for strong cigarettes were used
The smoke induction effect score was 5 points.
This means that scores should be given relative weight in this particular study, and that the results found may be more severe in other experimental settings.
Morphological assessment of pulmonary edema was performed on a central slice (section 4)
Scan the lungs of each mouse on a digital image using version 4 of the Visiopharm integral system video software. 2. 9. 0 (
Holland, Denmark).
Morphological measurement of mean string length (Lm)
And damage Index (DI)
Performed using a lung slide of about 30%, which was randomly selected using an automatic image capture program.
For measurements of airway attachments, 100% of the slides were evaluated.
The mean string length is determined using three randomly oriented lines per image, protected at 100 u2009 μm.
The damage index is determined using the grid structure of 4 × 5 measurement points per image.
Determine the number of fine trachea attachments on the four count frames of each image, equivalent to 30% of the entire slide.
Notes and data generated from the assessment of lung tissue pathology and tissue morphology measurement can be found in the \"tissue morphology measurement ISA File (Metadata)
\'And \'raw data for the measurement of pathology and morphology of lung tissues \'[: Figshare ].
Cut the frozen left lung into segments (20u2009μm thick)
With a low temperature thermostat (Leica CM3050 S)
The month and month collected the sharp force of the sterilized test tube (Including 1% 2-Alcohol)lysis buffer (Qiagen)
For RNA isolation.
In order to perform proteomics analysis, the frozen right lung is cut into slices (40u2009μm thick)
Use a low temperature thermostat, transfer to air traffic control and store at-80 °c until further processing is required.
All samples were randomly frozen in a batch.
Parallel Analysis of transcript, protein and lipid levels of tissues obtained from \"omics\" animals ().
Separation of total RNA from tissues (
Lung, nasal epithelial and liver)
Use the miRNeasy mini kit (Qiagen)
Animal blood protection kit using RNeasy (Qiagen)
, RNAse dissolved in the appropriate volume-
Free water quantified using Nanodrop 1000 spectrometer (
American, horse, Waltham, hot science)
Check the quality using the Agilent 2100 biological analyzer (
Agilent Technology).
Total RNA of blood samples (80u2009ng)was reverse-
Ovation RNA amplification system V2 (
NuGEN, San Carlos, California, United States of America).
100 ng of total RNA is reversed for tissue samples
Transcription to cRNA using Affymetrix®HT 3\'ivt express kit (
Affymetrix, Santa Clara, California, United States of America).
The cDNA/cRNA was then purified using magnetic beads to remove unbound nucleotide triphosphate, salt, enzyme, and inorganic phosphate.
The purified cDNA/cRNA was quantified and labeled using nucleotide-bound enzyme connections to biotin.
Fragmented, labeled cDNA (50u2009μl)/cRNA (33,3u2009μl)
Add the main mixture of 170/216, 7 μ l hybrid cocktail (
Affymetrix, Santa Clara, California)
And then 2 min (cDNA)or 5u2009min (cRNA)
At 99 ° C and 5 ° C, centrifuge for 1 min at 45 ° C and then for 1 min at Vmax.
Then a total of 200 u2009 μ l of cDNA/cRNA cocktails were bred on MG430 2. 0 GeneChips (Affymetrix).
These arrays were incubated at a speed of 60 r in the GeneChip hybrid oven 645 at 45 °c for 1 yy ± 3 h. p. m.
After hybridization, the array is cleaned and dyed on the fluid station FS450 using the Affymetrix GeneChip command console software (
AGCC software version 3. 2)
Fs450_0004 agreement for the Nugen agreement (blood samples)
Fs450_0001 of the IVT express protocol (tissue samples)
, Then scan 3000 7G using the GeneChip scanner.
The original image of the scanner is saved as a DAT file.
Use the AGCC Viewer software application to check if each image has artifacts, an overall strength distribution, a chessboard in the corner, a central cross that ensures sufficient grid alignment, and readability of array names.
The AGCC software automatically grids the DAT file Image and extracts the probe cell strength into the CEL file.
These documents were further processed (MAS5. 0)
Using Affymetrix expression console software (Build version 1. 3. 1. 187)
First quality check of data.
The data generated by the transcription group analysis has been submitted to ArrayExpress [: ArrayExpress].
As mentioned earlier, plasma was obtained and stored at-80 °c before analysis.
Lung use of \"Omics2\" mice 27-
G needle with icecold 0.
9% remove contaminated blood cells from normal saline.
Superior, middle and late after Heart Dissection
Collect the caval leaves of the right lung and capture
Freeze in liquid nitrogen immediately before storage at-80 °c
Again, a piece of liver is frozen and stored in the same way.
The tissue sample is crushed with a CP02 CryoPrep dry crushing system (Covaris).
Slowly thaw plasma samples on ice.
All samples are homogenized in ice
Cold 70% methanol-HO 0. 1% butyl-hydroxy-toluene (BHT)
The concentration is 100 ml.
The homogenized samples were stored at-80 °c before lipid extraction and analysis.
Zoora biological science Oy (Espoo, Finland).
96-robot assistance
Good sample preparation and extraction using Hamilton MSI system (
Hamilton Robotics Co. , Ltd. , Bona Duz, Switzerland).
An improved Folch scheme for methane, methanol and acetic acid for liquids-
Liquid extraction for the extraction of a spectrum of broad lipid types.
This extraction method is effective and robust in a wide range of lipid concentrations.
This method is used for the extraction of Gan oil, glycerin phosphate, steroid Ester and nerve sheath, except for the neurosheath and the thorn gosine-1-
Phosphate extracted with 1. 1u2009ml of ice-
Cold methanol containing 0. 1% BHT.
The Hamilton robot system was used to extract the glutin using methods described by Fong and colleagues, with a slight modification.
The process of Eicosanoids extraction is considered.
Before extraction, the sample is added with an internal standard of known quantities (IS, see ).
As described below, this set of ISs is used to quantify endogenous lipids in samples and controls.
After the lipid extraction, the sample is dried under mild nitrogen.
Samples of Shotgun gun lipid OMICS, sphinglipid OMICS, and glutin OMICS were found in methane: Methanol (1:2, v/v)
And nerve sheath ammonia-like/sheath ammonia-1-
The phosphate and epoxy resin were reorganized from methanol.
The final extract was stored at-20 °c through qualitative spectrum analysis.
A shotgun lipidmics was carried out on QTRAP 5500 (Sciex).
As mentioned earlier, the quantification of molecular gan grease, glycerin, and steroid Ester was evaluated by a bird\'s gun method lipid OMICS.
The sample is loaded to 96-well plates (twin.
Tec PCR plate 96 for Eppendorf AG, Hamburg, Germany)
Sealed with aluminum foil (
Eppendorf AG) hot sealing foil.
An equal sample of inhalation and infusion of 10 μ l.
As mentioned earlier, pioneer ion and neutral loss scans were performed in positive and negative ion modes.
Ionization by electric spray (ESI)
The applied voltage is usually 1. 3u2009kV and −1.
3 kv in positive and negative ion modes, respectively.
The gas pressure is usually set to 0.
There are 75 psi in both polarity modes.
The following MS settings are used in positive ion mode: Curtain gas;
Collision gas;
6. interface heater;
Tap potential;
30. Inlet potential 10. outlet potential of collision unit; 20.
The following settings are used in negative ion mode: Curtain gas;
Collision gas;
6. interface heater;
Tap potential;
-100, Inlet potential-10 and outlet potential of collision unit; −20.
Q1 and Q3 four bars run in unit resolution mode.
As mentioned earlier, the molecular nerve sheath, glucose nerve sheath, emulsion nerve sheath, and spherical nerve sheath were analyzed.
In short, single species were isolated using Acquity BEH 18, 2.
Columns with a grain size of 1 × 50mm. 7u2009μm (
United States, MA, Milford, Waters)
Evaluation on a UHPLC system consisting of ctc htc pal automatic sampler (
CTC analysis company in Zwingen, Switzerland)
And Rhine fast plate pump (
Flux instruments of Swiss Reinach).
Use a 25 min gradient of 10mm ammonium acetate in water containing 0. 1% acid (mobile phase A)
Sodium acetate with 10mm: 2-propanol (4:3, v/v)containing 0. 1% acid (mobile phase B)was used.
The temperature of the column oven is set to 60 °c and the flow rate is 500 μl/min.
QTRAP 5500 mass spectrometer (
Sciex in Concord, Canada)
It is equipped with an electric spray ion source for mass spectrometry determination.
The instrument is monitored in multiple reactions (MRM)
The pattern in positive ion mode, as mentioned earlier.
78 rm conversions were monitored using a stay of 20 MS.
Q1 and Q3 four bars run in unit resolution mode.
The collision energy is set to 40 ev and glucose-45 ev
And globotriaoceramides lactosylceramides and 66 asiev.
Nitrogen is used as a collision gas.
Set the ESI voltage to 5000 v and set the ion source temperature at 400C.
Stephens and Stephens1-
Phosphate was analyzed on a similar system.
Separate individual species using AQUASIL 18,2.
1 × 50mm column with a grain size of 5 μm (
Hot Fisher Science in San Jose, USA).
Use the 19 min gradient of 5mm ammonium acetate in ultra-inpure water (UPW):methanol (1:1)with 0. 1% acid (mobile phase A)
5 mM ammonium acetate was 0 in methanol. 1% acid (mobile phase B)
And ammonium acetate of 10 mM, 0 in acetone. 1% acid (mobile phase C)was used.
The temperature of the column oven is set to 60 °c and the flow rate is 50 μ l/min.
Perfect for Stephanie and Stephanie1-
Phosphate of a single species was monitored in multiple Response Monitoring (MRM)
Mode in positive ion mode.
22 rm conversions were monitored using a stay time of 25 MS.
Q1 and Q3 four bars run in unit resolution mode.
Collision energy 22 eV is determined to be the nerve sheath of sphingosines and 21 u2009 eV-1-phosphates.
Nitrogen is used as a collision gas.
Set the ESI voltage to 4500 v and set the temperature of the light source to °c.
As mentioned earlier, the analysis was carried out in addition to ammonium acetate and 0 of 10 mM.
Use 1% of ammonium A in all solvents instead of ammonium.
The analysis was evaluated on 6500 QTRAP (
Sciex in Concord, Canada)
Similar UHPLC Systems are equipped as described above.
Separate individual species using Acquity BEH 18, 2.
Columns with a grain size of 1 × 50mm. 7u2009μm (
United States, MA, Milford, Waters).
The 32 min gradient using 10mm ammonium acetate in methanol was 0. 1% acid (mobile phase A)
, Ammonium acetate of 10 mM, 0 in acetone. 1% acid (mobile phase B)
And add 10 mM ammonium acetate to the HPLC water, with 0. 1% acid (mobile phase C)was used.
The temperature of the column oven is set to 45 °c and the flow rate is 500 μl/min.
The analysis was evaluated on QTRAP 6500 (
Sciex in Concord, Canada)
A similar UHPLC system is equipped.
Single species were monitored in multiple Response Monitoring (MRM)
Mode in negative ion mode.
103 rm conversions were monitored using 30 MS of stay time.
Q1 and Q3 four bars run in unit resolution mode.
The collision energy of GM1s is set to 80 ev, the collision energy of GM3s is set to 70 ev, the collision energy of GM3s is set to 60 ev, and the collision energy of GDs is set to 50
Nitrogen is used as a collision gas.
The ESI voltage is set to-4500 v and the power supply temperature is set to 40000 °c.
The Eicosanoids were analyzed as described earlier.
Instrument settings similar to sphingolip OMICS were used.
Jupiter separated a single species using the phenomenon of 250 × 2.
0mm columns with a grain size of 5 μm (
Phenomenex, Torrance, California, United States of America).
18 min gradient using water: acetone: acid (63:37:0. 02)(mobile phase A)
And acetone: acetone (50:50)(mobile phase B)was used.
The temperature of the column oven is set to 60 °c and the flow rate is 300 μ l/min.
The analysis was evaluated on QTRAP 5500 (
Sciex in Concord, Canada).
Single species were monitored in multiple Response Monitoring (MRM)
Mode in negative ion mode.
A stay time of 15 MS was used to monitor 103 rm conversions that were run twice.
Q1 and Q3 four bars run in unit resolution mode.
The collision energy is set according to The Deems et. al. .
Nitrogen is used as a collision gas.
The ESI voltage is set to-4500 v and the power supply temperature is set to 525 °c.
Process Mass spectrometry data files using LipidView v1. 0.
99 and MultiQuant 2.
0 software generates a list of lipid names and peak regions.
As mentioned earlier, the Shotgun lipidmics data is processed in the LipidView.
In short, endogenous species were identified based on their characteristic fragment ions, neutral loss, and parent ions.
Such as m/z 184.
1. it is the characteristic head-based ion of Ester-based bile salt (PC)
And sphingomyins (SM)
Used to identify peaks observed in PIS 184 mass spectrometry along with maternal mass.
1 in positive ion mode.
In a similar way, the monitored lipid-based ions were used to identify molecular species in the negative ion mode.
For example, identifying PC 16: 0-18: 1 requires a corresponding signal from 16: 0 (PIS of m/z 255. 2)and the 18:1 (PIS of m/z 281. 2)scans.
Rm data is processed in MultiQuant.
Selected lipid characteristic ions and their parental mass were used together with retention time to identify endogenous species.
Information related acquisition (IDA)
An experiment used to confirm identity.
Use rm as a survey scan to trigger IDA and then use enhanced product ions (EPI)
Scanning of the two strongest ions
Quantification of identified lipids by standardizing against their respective internal standards and matrix types, E. G. g.
, The volume of the plasma and presented accordingly (e. g. , μ m for plasma).
Total lipid concentration calculated by summation
Increase the concentration of species belonging to the same category.
By dividing all observed lipid concentrations by the total class concentration in the sample, the percentage of molar distribution is produced.
The data filtering of the final data set is based on the frequency of individual lipid molecules observed throughout the collected data.
Molecules observed from less than 75% of the samples, as well as those lacking lipid-specific internal standards, were excluded.
Molecules whose concentration is four or four times lower than the median value of the group are considered outliers and excluded.
Before merging the final lipid group data set, the filtration procedure was performed on the polar pattern and lipid group evaluation, respectively.
SAS 9 is used for all calculations and data processing. 3 (SAS Institute).
Samples at 2 and 3 month time points were analyzed from 7 month time points, respectively.
Changes in the instrument control samples between batches were observed in neuroamine (
Cer, LacCer, Glc/GalCer and Gb3 lipids)
Therefore, the correction factor is applied to the endogenous lipid concentration of these platforms.
Due to the use of the correction factor, the concentration of neuroamine and icanka substances at the 7-month point in time is relative, not absolute.
Group comparisons within a specific time point are not affected by the use of correction factors.
The change in endogenous lipid concentration is due to the internal standard (IS)issues (eicosanoids)
Changes in total lipid extraction (
Ceramic varieties).
It was observed from the control sample that the concentration of eicosanoid IS was different between the two batches.
The change IS most likely due to evaporation of the solvent in the IS vial, even if the vial IS not opened and stored as recommended by the manufacturer.
Calculate the correction factor according to the instrument control sample (ICs)
Included in all extracts used to monitor the quality of lipid omics analysis.
The pooled human plasma is extracted in ICs, not in the sample, and the concentration of ISs is the same as in the sample, and for ICs and samples, the extraction is the same.
Run 10 copy ICs in each sample set.
Corrected the endogenous lipid concentration of eicosanoids and neuroceramic lipid species on the sample at 7 months by correction factor (k)
From the equation below.
Peak area ratio (
Endogenous area/IS area)
First calculated from 10 repeated IC runs, then the correction factor is determined based on the equation using the average of these 10 ratios.
Obviously, the correction factor is determined only for the lipid detected in the IC.
For neuroamine species, calculations are performed based on lipid extraction of tissue type, for example, comparing ICs in lung sample batches.
To calculate the correction concentration, it is the quantity (pmol)
Multiply by k in a sample.
In order to identify high-quality data, various control measures were evaluated.
Data that meet all application acceptance criteria are accepted.
In the control of all analytical instruments (IC)
Quality Control (QC)
Application of blank and calibration line.
ICs is based on the extraction of fresh human plasma (i. e.
, Collection extraction)
And analyze in a similar way to the sample.
ICs is used to monitor the performance and variation of mass spectrometry analysis.
Dependent on analysis and molecules (i. e. , abundance)
Different thresholds are applied, but typical in the range of 20-50%.
The sample was re-
Run if the threshold is exceeded.
QCs provide services in the same way as ICs, and in addition to the sample matrix being the same as the sample to be analyzed, they are individually extracted to include monitoring of extraction efficiency.
Typically a slightly higher change threshold is applied to QCs.
Blanks are used to monitor background noise and are excluded from the sample if the signal of lipid molecules in the blanks exceeds 25%.
Calibration line for monitoring linear response of mass spectrometer.
This analysis is accepted based on the linearity of the calibration line.
Linear regression must exceed 0.
95 based on at least four of the six non-Chinesezero standards.
Note the lipid according to the shorthand notes described and the lipid map nomenclature.
In short, PC, diammonium phosphate (PE)
, Nitroyl Ser (PS)
4) polylyyl myo (PI)
Phosphate (PA)
, Glycerin (PG)
, And diester of glycerin (DAG)
Lists two fat bases separated by Hyphen, E. G. g.
, PC 16: 0-18: 1, use slash, e when the position is unknowng.
Computer 16: 0/16: 0, when know.
Lysoc and lysoPE are abbreviated as LPC and LPE, respectively, and the abbreviation of cholesterol ester is CE. Ether-
Displayed as pc o (alkyl), PC P (alkenyl), PE O (alkyl), and PE P (alkenyl).
Ether-fat
Connecting lipid and N-
SM, neuroamine (Cer)
The wetland after the sheath fat and the giangin.
Sheath fat is: nerve sheath alcohol (SPH), sphingosine-1-phosphate (S1P), sphinganine-1-phosphate (SA1P)
Sugar amine (GlcCer)
Milk neural amine (LacCer)
And globotriaosylceramide (Gb3).
GM1, GM3, GQ1, and gt2 are nodes.
Fatty acids and twenty-carbon six-acid are 22-carbon six-acid (DHA)
Four sour peanuts (AA)
Twenty-Carbon five-officer acid (EPA), 12-
Hydroxyeicosatraenoic acid (12-HETE), 11-
Hydroxyeicosatraenoic acid (11-HETE), 15-
Hydroxyeicosatraenoic acid (15-HETE), 8-
Hydroxyeicosatraenoic acid (8-HETE), 5-
Hydroxyeicosatraenoic acid (5-HETE)
, E 2 (PGE2)
P1 D2 (PGD2), 13-
Hydroxoctadecadienoic acid (13-HODE), 9-
Hydroxoctadecadienoic acid (9-HODE), 6-
1 Alpha of ketone front ring (6-keto-PGF1alpha)
F2 alpha, P1 (PGF2alpha)
, Tromboxane B2 (TXB2)
, Tromboxane B3 (TXB3), 12-
8 hydrogen toluene acid (12-HEPE), 14,15-
2 Oxygen sugar acid (14_15-DHET), 11,12-
2 Oxygen sugar acid (11_12-DHET), 8,9-
2 Oxygen sugar acid (8_9-DHET), 5,6-
2 Oxygen sugar acid (5_6-DHET)
Eighteen carbonated (13-HOTrE), 12-oxo-
Twenty-four-acid12-oxoETE), 5-oxo-
Twenty-four-acid5-oxoETE), 5-
Hydroxyicosapentaenoic acid (5-HEPE), 15-
Hydroxyicosapentaenoic acid (15-HEPE).
The data from the analysis of lipid OMICS has been submitted to the metabolic OMICS [: Metabolism].
Sample of right lung tissue of 6 mice (i. e.
, Six biological repeats)
Each exposure condition and time point were analyzed.
Lung tissues exposed for 1, 3, 5 and 7 months and 3, 5 and 7 months with 3R4F and pMRTP can be used for quantitative proteomics analysis.
The lower right lung is frozen.
Cut into 40 μm thick slices (see above).
Slice evenly in tissue cracking buffer (
BioRad, Hercules, California, United States of America)
Use tissue II beads-
Crushing System of Mill (Qiagen)
Divided into two tubes.
Two for the first tube. dimensional (2D)-
Page gel swimming instrument (PAGE)
Analysis for relative and absolute quantitative isopressure labels and second (iTRAQ)analysis.
The protein extracted in the cracking buffer is acetone-
Precipitate and re-hang in the ReadyPrep 2D starter kit, re-dissolve/sample buffer (8u2009M urea, 2% 3-[(3-Cholamidopropyl)dimethylamino]-1-
C Shu Ester, 50mm sulfur Sulu, 0. 2% Bio-
PH gradient buffer pH 3-10, brom phenol blue; BioRad).
Determination of protein concentration using modified Bradford protein (BioRad).
Isoelectric focusing on a fixed pH gradient strip of 11 cm (
IPG Readystrip 3-10 NL, BioRad company)
On the E-focusing system such as Ettan IPGphor 3, there is a nonlinear gradient of pI 3-10 (
GE Healthcare (UK).
Resuspended protein applied to night between the zone rehydration then focus on 2 u2009 h 150 u2009 V when 1 u2009 h 500 u2009 V when in 1,000 u2009 V 1 u2009 h, then 4 u2009 h 8,000 u2009 V to 25,000-30,000.
After focusing on the electricity, use balance buffer 1 (
7 M urea, 75 mM Tris-HCl (pH 8. 8), 29.
3% glycerin, 2% SDS, 1% dithiol, brom phenol Blue)
During 20 minutes, then base with balance buffer 2, which is the same as buffer 1, but replaces 1% of dithiol with 2. 5% imitation iodine.
These proteins are applied to a standard XT Bis-
Tris gel, 12%, IPG + 1 well, 11 cm IPG strip, 13. 3×8. 7u2009cm (W×L)(BioRad). Second-
Dimension Separation is performed in sequence using standard Dodeca units (BioRad).
Visually separate spots using SYPRO Ruby staining (
Invitrogen, Bud, CA, USA).
Scan the dyed 2D gel on the image scanner (
Typhoon scanner Typhoon 9500, GE Healthcare), and spot-
2D-intensity calibration, spot detection, background extraction and matching
Execute the page using the SameSpots software (Nonlinear).
The spots of interest are cut out of the gel, cleaned, and in-
Gel digested with pancreatic enzyme Gold of 20 ng, mass spectrometry level (
Promega, Madison, United States of America)
Using the ETTAN biogas digester robot (GE Healthcare).
Peptide mass fingerprint data and mass spectrometry spectra obtained from matrix
Analytical Ionization Time by auxiliary laserof-
Flight Mass Spectrometry (
Super limit MALDI-TOF/TOF-MS;
USA, MA, Bianca, Brook).
They got in touch with the mascot search program and the Swissprot database.
Data generated by proteomics 2D
Page analysis can be found in the \"proteomics 2D gel ISA File (Metadata)
, \"2D gel images for proteomics\" and \"2D-PAGE data—
Standardized matrix document [: Figshare ].
The protein extracted in the cracking buffer was precipitated with acetone and then suspended at 0.
5 u2009 m triethyl ammonium bicarbonate, 1 m urea, 0. 1% SDS (Sigma-
Aldridge, St. Louis, Missouri).
As mentioned above, the protein concentration was determined using an improved Bradford protein assay (BioRad).
ITRAQ 8-processed a total of 50 μg of protein
Plex labeling according to manufacturer\'s instructions (
American, MA, Framingham, AB Sciex). Trypsin (Promega)
Added to the sample at 1: 10w/w)
Then digested overnight at 37 °c.
Then mark the sample with a reporter
Ion labeling in different treatment groups.
A general reference mixture containing 50 μg of all protein extracts at each point in time was also prepared and used with iTRAQ reporter-ion tag (
ITRAQ channel 114).
All labeled samples belonging to a set of iTRAQ are gathered together and dried in SpeedVac.
On September, the sample was diluted using 1 cc 18 reverse phasePak columns (Waters)
According to the manufacturer\'s instructions. For SDS-
Removal and reduction of PEG residue from the optimum cutting temperature embedded media, 0.
Remove column using 5 ml bed volume detergent (
Pierce, Rockford, United States of America).
Dry the sample using SpeedVac and re-hang in nanoLC buffer (
5% acetone, 0. 2% acid (both Sigma-Aldrich)).
Simple to use-
NanoLC 1000 instrument online connection with Q Exactive qualityanalyzer (
Thermal Science).
The peptide is graded on 50 cuccm C18RP RSLCspray column (
2 μm granularity;
Thermal Science)
At a flow rate of 200nl nl/min, a 200 min gradient was obtained from nanoLC buffer (
5% acetone, 0. 2% formic acid)
With 40% acetone, 0. 2% formic acid.
Each sample is injected twice with two different analytical methods: one is fast and the other is a sensitive method as described above.
Search for two mass spectrometry runs together according to the mouse reference protein set (
Uniprot version Aug_2013 ,)
Vers using the protein group Finder1. 4. 0. 288 software (
Thermal Science). Mascot (v. 2.
Ma, Boston, MA)
Use SequestHT as a search tool to merge the list of proteins obtained.
The filter node of the protein group discovery software is used to estimate the peptide-Level adjustment-values (q-values)
And the peptide is filtered as q-values