High Calorie Diet (Western Diet) in Atp7b-/- (Wilson Disease model) mice compared to C57BL/6J mice

TMT proteomics

Protein extracts were reduced with 10µL of 50mM ditheothreitol,
alkylated with 10µL of 100mM iodoacetamide in the dark for 30 minutes and
TCA/Acetone precipitated (100 µg). Protein pellets were resolubilized in 100µL
of 100mM triethyl ammonium bicarbonate (TEAB) and were digested overnight at
37°C by adding 10 µg of Trypsin/LysC mixture (V5071, Promega) in 100 mM
TEAB. Individual samples (100 µg) were
labeled with a unique isobaric mass tag reagent (TMT 16-plex, Thermo Scientific)
according to manufacturer instructions. Both pairing and labeling order of TMT
reagent and peptide sample were randomized.
Briefly, TMT-16 plex reagents (0.5 µg vials) were allowed to come to room
temperature before adding 20 µL of anhydrous acetonitrile, then vortexed and
centrifuged. The entire TMT reagent vial
was added to the 100 µg peptide sample and reacted at room temperature for 1
hour. 5% hydroxylamine (8 µL) was then
added to quench the reaction. The TMT
labeled samples were combined and vacuum centrifuged to dryness removing the
entire liquid. The TMT labeled peptides
were resuspended in 2mL of 10mM TEAB and separated into 84 fractions at
250µL/min using a 0 - 90% acetonitrile gradient in 10 mM TEAB on a 150mm x
2.1mm ID Waters XBridge 5µm C18 using an Agilent 1200 capillary HPLC in normal
flow mode and Agilent 1260 micro-fraction collector. The 84 fractions were concatenated into 24
fractions by combining all odd rows of each column 1 through 12 into 12
fractions and all even rows of each column into another 12 fractions.

Peptide fractions analyzed by liquid
chromatography interfaced with tandem mass spectrometry (LC/MSMS) using an
Easy-LC 1200 HPLC system (www.thermofisher.com) interfaced with an Orbitrap
Fusion Lumos Tribrid Mass Spectrometer (www.thermofisher.com). Fractions were
resuspended in 20ul loading buffer (2% acetonitrile in 0.1% formic acid) and
analyzed by reverse phase liquid chromatography coupled to tandem mass
spectrometry. Peptides (20% each
fraction) were loaded onto a C18 trap (S-10µM, 120Å, 75 µm x 2 cm, YMC, Japan) and subsequently
separated on an in-house packed PicoFrit column (75um x 200mm, 15u, +/-1um tip, New Objective) with C18
phase (ReproSil-Pur C18-AQ, 3µm, 120Å, www.dr-maisch.com) using 2-90%
acetonitrile gradient at 300 nl/min over 120 min. Eluting peptides were sprayed
at 2.0 kV directly into the Lumos.

Survey scans (full MS) were acquired from 350-1800 m/z with data
dependent monitoring with a 3sec cycle time. Each precursor individually
isolated in a 0.7 Da window and fragmented using HCD activation collision
energy 38 and 15s dynamic exclusion, first mass being 120 m/z. Precursor and
the fragment ions were analyzed at resolutions 120,000 and 30,000, respectively,
with automatic gain control (AGC) target values at 4e5 with 50ms maximum
injection time (IT) and 1e5 with 120ms maximum IT, respectively.

Isotopically resolved masses in precursor (MS) and fragmentation
(MS/MS) spectra were processor in Proteome Discoverer (PD) software (v2.4,
Thermo Scientific). All data were
searched using Mascot (2.6.2; www.matrixscience.com) against the 2017_Refseq 83_mouse database.
The following criteria were set for the database search: All species; trypsin
as the enzyme, allowing one missed cleavage; cysteine carbamidomethylation and
N-terminal TMTpro label as fixed modifications; TMT label on lysine, methionine
oxidation, asparagine and glutamine deamidation as variable modifications. Mass
tolerance was 3ppm and 0.01Da for the precursors and peptides, respectively. Identifications
from mascot searches were filtered at 1% False Discovery Rate (FDR) confidence
threshold, based on a concatenated decoy database search, using the Proteome
Discoverer.

Consensus Step workflow- Analysis settings
Processing node 1: PSM Grouper
1. Peptide Group Modifications:
- Site Probability Threshold: 75

Processing node 2: Peptide Validator
1. General Validation Settings:
- Validation Mode: Automatic (Control peptide level error rate if possible)
- Target FDR (Strict) for PSMs: 0.01
- Target FDR (Relaxed) for PSMs: 0.05
- Target FDR (Strict) for Peptides: 0.01
- Target FDR (Relaxed) for Peptides: 0.05

2. Specific Validation Settings:
- Validation Based on: q-Value
- Target/Decoy Selection for PSM Level FDR Calculation Based on Score: Automatic
- Reset Confidences for Nodes without Decoy Search (Fixed score thresholds): False

Processing node 3: Peptide and Protein Filter
1. Peptide Filters:
- Peptide Confidence At Least: High
- Keep Lower Confident PSMs: False
- Minimum Peptide Length: 6
- Remove Peptides Without Protein Reference: False

2. Protein Filters:
- Minimum Number of Peptide Sequences: 1
- Count Only Rank 1 Peptides: False
- Count Peptides Only for Top Scored Protein: False

Processing node 4: Protein Scorer
No parameters

Processing node 5: Protein Grouping
1. Protein Grouping:
- Apply strict parsimony principle: True

Processing node 6: Peptide in Protein Annotation
1. Flanking Residues:
- Annotate Flanking Residues of the Peptide: True
- Number Flanking Residues in Connection Tables: 1

2. Modifications in Peptide:
- Protein Modifications Reported: Only for Master Proteins

3. Modifications in Protein:
- Modification Sites Reported: All And Specific
- Minimum PSM Confidence: High
- Report Only PTMs: True

4. Positions in Protein:
- Protein Positions for Peptides: Only for Master Proteins

Processing node 7: Protein FDR Validator
1. Confidence Thresholds:
- Target FDR (Strict): 0.01
- Target FDR (Relaxed): 0.05

Processing node 8: Protein Annotation
1. Annotation Aspects:
- 1. Aspect: Biological Process
- 2. Aspect: Cellular Component
- 3. Aspect: Molecular Function
- 4. Aspect: None
- 5. Aspect: None
- 6. Aspect: None

Processing node 9: Protein Marker

5. Annotate Species:
- As Species Map: False
- As Species Names: False

6. Mark Additional Entities:
- Annotation Groups: False
- Pathway Groups: False
- Modification Sites: True
- Peptide Isoform Groups: True

Processing node 10: Reporter Ions Quantifier
1. General Quantification Settings:
- Peptides to Use: Unique
- Consider Protein Groups for Peptide Uniqueness: True
- Use Shared Quan Results: True
- Reject Quan Results with Missing Channels: False

2. Reporter Quantification:
- Reporter Abundance Based On: S/N
- Apply Quan Value Corrections: True
- Co-Isolation Threshold: 30
- Average Reporter S/N Threshold: 4
- SPS Mass Matches [%] Threshold: 55

3. Normalization and Scaling:
- Normalization Mode: Total Peptide Amount
- Scaling Mode: On All Average

4. Exclude Peptides from Protein Quantification:
- For Normalization: Use All Peptides
- For Protein Roll-Up: Use All Peptides
- For Pairwise Ratios: Exclude Modified

5. Quan Rollup and Hypothesis Testing:
- Protein Ratio Calculation: Protein Abundance Based
- Maximum Allowed Fold Change: 100
- Imputation Mode: None
- Hypothesis Test: ANOVA (Individual Proteins)

6. Quan Ratio Distributions:
- 1st Fold Change Threshold: 2
- 2nd Fold Change Threshold: 4
- 3rd Fold Change Threshold: 6
- 4th Fold Change Threshold: 8
- 5th Fold Change Threshold: 10

Processing node 11: Result Statistics
No parameters

Processing node 12: Display Settings
1. General:
- Filter Set:
### Filter Set MasterProteinFilter.filterset contains the following filters:
### Row Filter for TargetProtein:
### Master is equal to Master
###

'magellan filter set' 1 'MasterProteinFilter.filterset' FiltersetProperties 1 'LastFileName' 'C:\Users\frank.berg\Desktop\MasterProteinFilter.filterset' Filter 'TargetProtein' 1 NARY_AND 1 = FilterConditionProperties 1 'NamedComparableFilterCondition/DisplayPropertyHint' 'Master' property 'Thermo.PD.EntityDataFramework.MasterProteinAssessment, Thermo.Magellan.EntityDataFramework' 'IsMasterProtein' constant 'Thermo.PD.EntityDataFramework.MasterProteinAssessment, Thermo.Magellan.EntityDataFramework' 'IsMasterProtein'

Processing node 13: Data Distributions
1. ID Distributions (Bottom-up):
- Peptides to Use: Only unique peptides based on protein groups