Proteomics Documentation

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Sample preparation for this project was conducted utilizing the S-Trap^TM spin micro column digestion method by PROTIFI.

 

The full protocol with reagents and solutions can be found here.

 

Samples were additionally desalted with Pierce^TM C18 Spin Tips according to manufacture specification.

 

            The full protocol can be found here.

Sample preparation for this project was conducted utilizing the S-Trap^TM spin mini column digestion method by PROTIFI.

 

The full protocol with reagents and solutions can be found here.

 

Samples were additionally desalted with Pierce^TM C18 Spin Tips according to manufacture specification.

 

            The full protocol can be found here.

The FASP Protocol is detailed below.

Solutions and Reagents

UA: 8 M urea in 100 mM ABC pH 8.0.

IAA solution: 500 mM iodoacetamide in UA buffer

DTT solution: 100 mM DTT in UA buffer

50 mM ABC in water.

0.1% FA in water.

0.02% Protease Max

Trypsin in 25mM ABC

 

Protocol

1. Quantifying protein concentration within samples, aliquot desired amount of each sample (0.2-400 μg, up to 200 μL) into a 1.75 mL tube. Add enough UA buffer to make 250 μL.
1. Note: Tissue samples are first frozen and milled in liquid nitrogen with a mortar and pestle. Samples are then lyophilized prior to protein quantification.
2. Add 100 mM DTT to each sample to a final concentration of 10 mM (28 μL for 250 μL) Incubate at 37°C for 30 minutes.
3. Add 2.2 molar excess (over DTT) of 500 mM IAA to each sample (12.3 μL for 250 μL). Incubate 15 minutes in the dark at room temperature.
4. While samples are reducing and alkylating, add 100 µL 0.1% FA to the filter unit and centrifuge at 14,000 x g for 15 minutes.
5. Add 100 µL UA to the filter unit and centrifuge at 14,000 x g for 15 minutes.
6. Load reduced and alkylated samples onto equilibrated FASP filter. Spin at 14,000 x g for 20 minutes.
7. Discard the flow-through from the collection tube.
8. Add 200 µL of UA to the filter unit and centrifuge at 14,000 x g for 20 minutes. Repeat 1X.
9. Discard flow-through from collection tube. 
10. Add 100 µL of 50 mM ABC to the filter unit and centrifuge at 14,000 x g for 15 minutes. Repeat 2X.
11. Transfer the filter units to new collection tubes.
12. Prepare digest master mix:
13. Add 50-100 µL of digest master mix and mix for 1 minute.
14. Incubate the units in a wet chamber at 37°C for 4 -18 hours.
15. Centrifuge the filter units at 14,000 x g for 15 minutes.
16. Add 75 µL of 0.2% FA and centrifuge the filter units at 14,000 x g for 15 minutes. Repeat 1X.

17. Perform C18 clean-up.
18. Transfer sample from FASP collection tube to auto sampler vial.
19. Speed vac until approximately 2-5 μL of sample remains.
20. Acidify with 0.1% FA to final concentration of by bringing up to desired volume.

Samples were additionally desalted with Pierce^TM C18 Spin Tips according to manufacture specification.

 

          The full protocol can be found here.

 

 

Sample preparation for this project was conducted utilizing a modified FASP protocol with three fractions including the cellular, soluble ECM, and the insoluble ECM factions.

 

The 3-fraction ECM protocol for extraction is detailed below.

 

Buffers

HS Buffer: 50mM Tris-HCl (pH 7.4), 0.25% CHAPS, 25mM EDTA, 3M NaCl

sECM Buffer: 6M Gnd-HCl, 100 mM ABC pH 9.0

NH₂OH Buffer (1 M NH₂OH-HCl, 4.5 M guanidine-HCl, 0.2 M K₂CO₃, pH adjusted to 9.0 with NaOH)

iECM Buffer: 6M guanidine hydrochloride, 100mM ABC pH 9.0, 1M K₂CO₃, 50% NaOH (w/v)

 

Protocol

1. Pre-weigh the whole organ if available.
2. Pulverize or mill in liquid N₂ and lyophilize samples.
3. Weigh ~5mg of lyophilized tissue per sample supplemented with 100mgs of 1mm glass beads.

1. Add 200μl/mg of HS buffer supplemented with 10ul/mL Halt Protease Arrest. Bead beat samples for 3 minutes at 4°C. Vortex samples at 4°C for 20 minutes. Centrifuge samples at 18,000 x g (4°C) for 20 minutes and remove supernatant. Repeat 2X. Combine supernatant and store fraction 1 at -80°C.
2. Add 200μl/mg sECM Buffer. Bead beat samples for 2 min at 4°C. Vortex at room temp for 19 hours (O/N). Centrifuge at 21,000 x g for 15 minutes and remove supernatant.
3. Add 200ul/mg NH₂OH Buffer. Bead beat power 8, 2 minutes. Vortex for 4 hours at 42°C with mild agitation. Centrifuge at 21,000 x g for 15 minutes and remove supernatant.

7. All fractions can be frozen at -80°C until FASP.
8. Digest enough for replicates and protein concentration should be scaled to the experiment. Average loads are 30 μg.

The FASP Protocol is detailed below.

Solutions and Reagents

UA: 8 M urea in 100 mM ABC pH 8.0.

IAA solution: 500 mM iodoacetamide in UA buffer

DTT solution: 100 mM DTT  in UA buffer

50 mM ABC in water.

0.1% FA in water.

0.02% Protease Max

Trypsin in 25mM ABC

 

Protocol

1. After quantifying samples, aliquot desired amount of each sample (0.2-400 μg, up to 200 μL) into a 1.75 mL tube. Add enough UA buffer to make 250 μL.
2. Add 100 mM DTT to each sample to a final concentration of 10 mM (28 μL for 250 μL) Incubate at 37°C for 30 minutes.
3. Add 2.2 molar excess (over DTT) of 500 mM IAA to each sample (12.3 μL for 250 μL). Incubate 15 minutes in the dark at room temperature.
4. While samples are reducing and alkylating, add 100 µL 0.1% FA to the filter unit and centrifuge at 14,000 x g for 15 minutes.
5. Add 100 µL UA to the filter unit and centrifuge at 14,000 x g for 15 minutes.
6. Load reduced and alkylated samples onto equilibrated FASP filter. Spin at 14,000 x g for 20 minutes.
7. Discard the flow-through from the collection tube.
8. Add 200 µL of UA to the filter unit and centrifuge at 14,000 x g for 20 minutes. Repeat 1X.
9. Discard flow-through from collection tube. 
10. Add 100 µL of 50 mM ABC to the filter unit and centrifuge at 14,000 x g for 15 minutes. Repeat 2X.
11. Transfer the filter units to new collection tubes.
12. Prepare digest master mix:
13. Add 50-100 µL of digest master mix and mix for 1 minute.
14. Incubate the units in a wet chamber at 37°C for 4 -18 hours.
15. Centrifuge the filter units at 14,000 x g for 15 minutes.
16. Add 75 µL of 0.2% FA and centrifuge the filter units at 14,000 x g for 15 minutes. Repeat 1X.
17. Perform C18 clean-up.
18. Transfer sample from FASP collection tube to auto sampler vial.
19. Speed vac until approximately 2-5 μL of sample remains.
20. Acidify with 0.1% FA to final concentration of by bringing up to desired volume.

 

 

Samples were additionally desalted with Pierce^TM C18 Spin Tips according to manufacture specification.

 

            The full protocol can be found here.

Data collection was carried out on a Q Exactive HF Mass Spectrometer (ThermoFisher Scientific) coupled with an Easy nLC 1000 (ThermoFisher Scientific) liquid chromatography–mass spectrometry (LC–MS) interface.

• Samples were loaded on a C18 column (100 µM inner diameter x 20 cm) packed in-house with 2.7 µm Cortecs C18 resin.

   

• LC mobile phase solvents consisted of 0.1% formic acid in water (buffer A) and 0.1% formic acid in 80% acetonitrile (buffer B, Optima LC/MS, Fisher Scientific).

   

• Flow rate was set at 400 nL/min with the following a linear separation gradient:

  Sample Type	Separation Gradient
  Biofluids, Tissues, and Cell cultures	4% buffer B for 3 min, followed by a linear gradient 4 to 32% buffer B from 3 to 102 min, 32 to 55% buffer B from 102 to 107, 55 to 95% buffer B from 107 to 108. Flow at 95% buffer B was maintained from 108 for 117 min to remove the remaining peptides.

   

• The data-dependent acquisition was performed, and the top 15 most abundant precursors were considered for MS/MS analysis.

   

• The mass spectrometer was operated in the positive ion mode.

Data collection was carried out on a Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) coupled to an EASY-nLC 1200 (Thermo Fisher Scientific) through a nanoelectrospray liquid chromatography–mass spectrometry (LC–MS) interface.

• Samples were loaded onto a 20 μL loop using the autosampler. The analytical column was then switched on-line at 400 nL/min over an in-house-made 100 μm i.d. × 150 mm fused silica capillary packed with 2.7 μm CORTECS C18 resin (Waters; Milford, MA).

   

• LC mobile phase solvents consisted of 0.1% formic acid in water (buffer A) and 0.1% formic acid in 80% acetonitrile (buffer B, Optima LC/MS, Fisher Scientific, Pittsburgh, PA).

   

• After 22 μL of sample loading at a maximum column pressure of 700 bar, each sample was separated on a 120 min gradient at a constant flow rate of 400 nL/min.

   

• Separation gradient was established according to the following sample types.

  Sample Type	Separation Gradient
  Biofluids and Tissues	6% buffer B from 0 to 3 min, followed by a linear gradient from 6 to 42% buffer B from 3 to 105 min. Gradient elution was followed by a linear increase to 55% buffer B from 105 to 110 min and further to 95% buffer B from 110 to 111 min. Flow at 95% buffer B was maintained from 111 to 120 min to remove the remaining peptides.
  Cell (Cell culture and ECM extraction)	6% buffer B from 0 to 3 min, followed by a linear gradient from 6 to 42% buffer B from 3 to 105 min. Gradient elution was followed by a linear increase to 55% buffer B from 105 to 110 min and further to 95% buffer B from 110 to 111 min. Flow at 95% buffer B was maintained from 111 to 120 min to remove the remaining peptides.
  sECM (ECM extraction only)	6% buffer B from 0 to 3 min, followed by a linear gradient from 6 to 36% buffer B were utilized from 3 to 105 min. Gradient elution was followed by a linear increase to 55% buffer B from 105 to 110 min and further to 95% buffer B from 110 to 111 min. Flow at 95% buffer B was maintained from 111 to 120 min to remove the remaining peptides.
  iECM (ECM extraction only)	6% buffer B from 0 to 3 min, followed by a linear gradient from 6 to 24% buffer B were utilized from 3 to 105 min. Gradient elution was followed by a linear increase to 55% buffer B from 105 to 110 min and further to 95% buffer B from 110 to 111 min. Flow at 95% buffer B was maintained from 111 to 120 min to remove the remaining peptides.

   

• Instrument control and data acquisition were performed using Xcalibur (version 4.5) software.

   

• The following parameters were utilized for data-dependent acquisition in positive ion mode: mass range m/z 375–1600, higher energy collisional dissociation (HCD) MS/MS (30% collision energy) using the standard automatic gain. control (AGC) target and a 35 ms maximum injection time with an isolation width of 1.6 m/z.

   

• MS1 and MS2 detection at resolutions of 120,000 and 50,000, respectively. Dynamic exclusion was 45 s. Singel Singly charged ions were excluded from HCD selection.

Data collection was carried out on a TIMS Q-TOF mass spectrometer (Bruker Daltonics) coupled with an Evosep One liquid chromatography-mass spectrometry (LC-MS) interface.

• Digested peptides were loaded on to Evotips following the manufacture protocol and analyzed directly using an Evosep One liquid chromatography system (Evosep Biosystems, Denmark). Peptides were separated on a 75 µm i.d. × 15 cm separation column packed with ReproSil 1.9 µm C18 beads, 120A resin (Evosep Biosystems, Denmark) and over a predetermined 44-minute gradient according to manufacture settings.

 

• LC mobile phase solvents consisted of 0.1% formic acid in water (buffer A) and 0.1% formic acid in 80% acetonitrile (buffer B, Optima LC/MS, Fisher Scientific).

 

• The system was coupled to the timsTOF Pro mass spectrometer (Bruker Daltonics, Bremen, Germany) via the nano-electrospray ion source (Captive Spray, Bruker Daltonics).

   

• Instrument control and data acquisition were performed using Compass Hystar (version 6.0) with the timsTOF Pro (Bruker Daltonics, Bremen, Germany) operating in parallel accumulation-serial fragmentation (PASEF) mode.

   

• The following parameters were utilized for data-dependent acquisition in positive ion mode: mass range 100-1700m/z, ion mobility scan from 0.7 to 1.40 Vs/cm², ramp accumulation times were 100ms; capillary voltage was 1400 V, dry gas 3.0 L/min and dry temp 180°C.

   

• The PASEF settings were as follows: 10 MS/MS scans (total cycle time, 1.1 s); charge range 0 – 5; active exclusion for 0.40min; scheduling target intensity 12500 cts/s; intensity threshold 1000 cts/s; collision-induced dissociation (CID) energy 20 eV to 59 eV.

Data collection was carried out on a TIMS Q-TOF SCP mass spectrometer (Bruker Daltonics) coupled with a nanoElute liquid chromatography-mass spectrometry (LC-MS) interface.

• Samples were loaded on a IonOpticks C18 column (75 µM inner diameter x 25 cm) with a flow rate of 300 nL/min. 

   

• LC mobile phase solvents consisted of 0.1% formic acid in water (buffer A) and 0.1% formic acid in 80% acetonitrile (buffer B, Optima LC/MS, Fisher Scientific).

   

• The system was coupled to the timsTOF SCP mass spectrometer (Bruker Daltonics, Bremen, Germany) via the nano-electrospray ion source (Captive Spray, Bruker Daltonics).

   

• Separation gradient was established according to the following sample types.

Sample Type	Separation Gradient
Biofluids, Tissues, Cell cultures	a linear gradient from 4 to 25% buffer B from 0 to 60 minutes, followed by a linear gradient from 25 to 35% buffer B from 60 to 79.5 minutes. Gradient elution was followed by a linear increase to 95% buffer B from 79.5 to 82.0 minutes. Flow at 95% buffer B was maintained from 82.0 to 90 min to remove the remaining peptides.

 

• Instrument control and data acquisition were performed using Compass Hystar (version 6.0) with the timsTOF SCP operating in parallel accumulation-serial fragmentation (PASEF) mode.

   

• The following parameters were utilized for data-dependent acquisition in positive ion mode: mass range 100-1700 m/z, ion mobility scan from 0.7 to 1.30 Vs/cm²; ramp accumulation times were 166 ms; capillary voltage was 4500 V, dry gas 8.0 L/min and dry temp 200°C.

   

• The PASEF settings were as follows: 5 MS/MS scans (total cycle time, 1.03 s); charge range 0–5; active exclusion for 0.2 min; scheduling target intensity 20,000 cts/s; intensity threshold 500 cts/s; collision-induced dissociation (CID) energy 10 eV to XX eV.

Data for this project was analyzed with the MSFragger utilizing the following parameters.

 

• Precursor/fragment tolerance was set as the following.

  Instrument	Precursor Tolerance (ppm)	Fragment Tolerance (ppm)
  QE HF	±10	15
  Fusion	±10	15
  timsTOF	±15	25
  SCP	±15	25

   

• Data was searched against the appropriate UniProt/SwissProt database. Database was adapted with 50% decoys and MSFragger common contaminants. Common databases including the following:

  Database	Scientific Name	Proteome ID
  Swiss-Port	Homo sapiens	UP000005640
  Swiss-Port	Mus musculus	UP000000589

   

• All searches were carried out with trypsin enzymatic cleavage.

   

• Variable modifications were set as the following: methionine oxidation (15.9949), N-terminal modification (42.016), Gln->pyro-Glu (N-term Q). Fixed modification was set as cysteine (57.021465).

   

• Match between runs was utilized within MS1 quantification.

   

• Searches were performed with all replicates from each sample condition grouped to obtain protein coverage across all analyzed replicates.

   

• Results were filtered to 1% FDR at the peptide and protein level.

Data for this project was analyzed with the PEAKS X Pro version 10.6 (Bioinformatics Solutions, Inc.) utilizing the following parameters.

 

• Precursor/fragment tolerance was set as the following.

  Instrument	Precursor Tolerance (ppm)	Fragment Tolerance (ppm)
  QE HF	±10	15
  Fusion	±10	15
  timsTOF	±15	25
  SCP	±15	25

   

• Data was searched against the appropriate UniProt/SwissProt database. Common databases including the following:

  Database	Scientific Name	Proteome ID
  Swiss-Port	Homo sapiens	UP000005640
  Swiss-Port	Mus musculus	UP000000589

   

• All searches were carried out without cleavage limitations.

   

• Variable modifications were set as the following: carbamidomethyl (C), oxidation (M), hydroxyproline oxidation (P), Gln->pyro-Glu (N-term Q), and acetyl (Peptide N-term).

   

• Searches were performed with all replicates from each sample condition grouped to obtain protein coverage across all analyzed replicates.

   

• Results were filtered to 1% FDR at the peptide and protein level.