Compartment-Resolved Proteomics with Deep Extracellular Matrix Coverage

Materials and reagents

Reagents

1. Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) (Roche, catalog number: 10812846001)

2. 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS) (Sigma-Aldrich, catalog number: C3023)

3. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E4884)

4. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888)

5. Halt protease inhibitor cocktail (Thermo Fisher, catalog number: 78429)

6. Guanidine hydrochloride (Gnd-HCl) (Sigma-Aldrich, catalog number: G4505)

7. Hydroxylamine-HCl (NH₂OH–HCl) (Sigma-Aldrich, catalog number: 159417)

8. Ammonium bicarbonate (ABC) (Sigma-Aldrich, catalog number: A6141)

9. Double-distilled water (ddH₂O)

10. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D9779)

11. Iodoacetamide (IAM) (Sigma-Aldrich, catalog number: I1149)

12. Potassium carbonate (K₂CO₃) (Sigma-Aldrich, catalog number: 209619)

13. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S5881)

14. Urea (Sigma-Aldrich, catalog number: U5128)

15. Buffer A: 0.1% formic acid (FA) in H₂O, Optima (Thermo Fisher, catalog number: LS118)

16. Buffer B: 0.1% FA in 80% ACN, Optima (Thermo Fisher, catalog number: LS122500)

17. Sequencing-grade modified trypsin (Promega, catalog number: V5111)

18. Lysyl Endopeptidase^®, mass spectrometry grade (Lys-C) (FUJIFILM Wako Pure Chemical Corporation, catalog number: 125-05061)

19. Optional (if necessary based on protocol notes):

    1. Gel-loading pipette tips (Corning, catalog number: CLS4853)

    2. Optima^TM acetone (Thermo Fisher, catalog number: A929)

    3. Pierce^TM 660 nm protein quantification assay (Thermo Fisher, catalog number: 22660)

       
       

1. High salt buffer (see Recipes)

2. Gnd-HCl solution (see Recipes)

3. DTT solution (see Recipes)

4. IAM solution (see Recipes)

5. Hydroxylamine (HA) buffer preparation (pH 9.0) (see Recipes)

6. Urea solution (see Recipes)

7. Digest master mix (per sample) (see Recipes)

   
   

1. High salt buffer

   Reagent	Final concentration	Amount
   Tris-HCl (1M, pH 7.4)	50 mM	5 mL
   CHAPS	0.25%	25 mg
   EDTA (500 mM)	25 mM	5 mL
   NaCl	3 M	17.53 g
   Halt protease inhibitor (100×)	1×	1 mL
   Total	n/a	100 mL

   Combine buffer components and bring to 100 mL with ddH₂O. Buffer without protease inhibitors can be prepared in advance and stored at 4 °C for up to one month. Protease inhibitors must be added fresh before use.

   
   

2. Gnd-HCl solution

   Reagent	Final concentration	Amount
   Gnd-HCl	6 M	57.32 g
   ABC (1 M, pH 8.0)	100 mM	10 mL
   Total	n/a	100 mL

   Dissolve Gnd-HCl in ddH₂O before adding 10 mL of 1 M ABC. Adjust pH to 9.0 using NaOH. Add ddH₂O to 100 mL. Prepare fresh before use.

   
   

3. DTT solution

   Reagent	Final concentration	Amount
   DTT	100 mM	15.4 mg
   ABC (1 M)	100 mM	1 mL
   Total	n/a	10 mL

   Combine components and add ddH₂O to 10 mL. Prepare fresh before use.

   
   

4. IAM solution

   Reagent	Final concentration	Amount
   IAM	500 mM	92.5 mg
   ABC (1 M)	100 mM	1 mL
   Total	n/a	10 mL

   Combine components and add ddH₂O to 10 mL. Prepare fresh before use and store in the dark.

   
   

5. Hydroxylamine (HA) buffer preparation (pH 9.0)

   Reagent	Final concentration	Amount
   Gnd-HCl solution (Recipe 2)	70% v/v	70 mL
   NH2OH–HCl	1 M	7 g
   NaOH (50% w/v)	4% w/v	8 mL
   K2CO3 (1 M)	0.2 M	20 mL
   Total	n/a	100 mL

   Prepare Gnd-HCl solution according to Recipe 2. Dissolve NH₂OH–HCl in 50 mL of Gnd-HCl solution. Add NaOH, mix, then add K₂CO_3. Add Gnd-HCl solution to 90 mL before adjusting pH to 9.0 using HCl. Add Gnd-HCl solution to 100 mL. Prepare fresh before use.

   
   

6. Urea solution

   Reagent	Final concentration	Amount
   Urea	8 M	24.02 g
   ABC (1 M)	100 mM	5 mL
   Total	n/a	50 mL

   Combine components and add ddH₂O to 50 mL. Prepare fresh before use.

   
   

7. Digest master mix (per sample)

   Reagent	Final Concentration	Amount
   ABC (1 M)	25 mM	2.5 μL
   Lys-C	1:400 enzyme:protein ratio	0.075 μg (for 30 μg protein)
   Total	n/a	100 μL

   
   

Laboratory supplies

1. MS-compatible pipette tips (any brand)

2. Safe-Lock 1.75 mL tubes (Eppendorf, catalog number: 003012361)

3. 2 mL snap cap tubes (any brand)

4. Glass beads, 1 mm diameter (Next Advance, catalog number: GB10)

5. 10 kDa molecular weight cutoff (MWCO) filters (Sartorius, catalog number: VN01H02)

6. Pierce^TM quantitative colorimetric peptide assay (Thermo Fisher, catalog number: 23275)

7. Flat-bottom, clear 96-well plate (Grenier, catalog number: 655101)

8. Empore sealing tape for 96-well plate (Millipore Sigma, catalog number: 66881-U)

9. Pierce^TM C18 spin tips (Thermo Fisher, catalog number: 84850)

10. Verex 300 μL vials with pre-slit cap (Phenomenex, catalog number: AR0-39S0-13-C/AR0-8972-13-C)

Equipment

1. Ceramic mortar and pestle (any brand)

2. Measuring pipettes, p1000, p200, p10 (any brand)

3. FreeZone 4.5L benchtop freeze dryer (Labconco, catalog number: 7750020)

4. Clear complete fast-freeze flask, any size (Labconco)

5. Scroll vacuum pump (Labconco, catalog number: 7587000)

6. Bullet blender (Next Advance, model: BBX24)

7. Centrifuge (Eppendorf, model: 5430R)

8. Vortex mixer with 1.5 mL tube attachment (Labnet, model: VX-200)

9. Orbital shaker, Orbit M60 (Labnet, catalog number: LS-2020-M60)

10. Spark^® microplate reader (TECAN, catalog number: 30086376)

11. CentriVap vacuum concentrator (Labconco, catalog number: 7810016)

12. Fusion Lumos Tribrid mass spectrometer (Thermo Fisher)

13. EASY-nLC 1200 System (Thermo Fisher, catalog number: LC140)

14. Analytical column made in-house: 100 μm i.d. × 150 mm fused silica capillary packed with 2.7 μm CORTECS C18 resin (Waters). Commercial alternative: EASY-Spray C18 column, 75 μm i.d. × 150 mm fused silica capillary packed with 3 μm resin (Thermo Fisher, catalog number: ES900)

Software and datasets

1. XCalibur Version 4.5 (Thermo Fisher)

2. Mascot Server Version 2.5 (Matrix Science)

3. Proteome Discoverer Version 2.5 (Thermo Fisher)

4. MSFragger (Nesvizhskii Lab) [13]

5. UniProtKB (https://www.uniprot.org/help/uniprotkb)

6. CRAPome Contaminant Database [14]

7. MatrisomeDB [15]

Procedure