Optimized Workflow for Proteomics and Phosphoproteomics with Limited Tissue Samples

Abstract

Proteomics and phosphoproteomics play crucial roles in elucidating the dynamics of post-transcriptional processes. While experimental methods and workflows have been established in this field, a persistent challenge arises when dealing with small samples containing a limited amount of protein. This limitation can significantly impact the recovery of peptides and phosphopeptides. In response to this challenge, we have developed a comprehensive experimental workflow tailored specifically for small-scale samples, with a special emphasis on neuronal tissues like the trigeminal ganglion.

Our proposed workflow consists of seven steps aimed at optimizing the preparation of limited tissue samples for both proteomic and phosphoproteomic analyses. One noteworthy innovation in our approach involves the utilization of a dual enrichment strategy for phosphopeptides. Initially, we employ Fe-NTA Magnetic beads, renowned for their specificity and effectiveness in capturing phosphopeptides. Subsequently, we complement this approach with the TiO2-based method, which offers a broader spectrum of phosphopeptide recovery. This innovative workflow not only overcomes the challenges posed by limited sample sizes but also establishes a new benchmark for precision and efficiency in proteomic investigations.

INTRODUCTION:

The study of the proteome involves a comprehensive assessment of protein function and structure to gain a nuanced understanding of protein expression in specific tissues or cells (Breitkopf and Asara 2012, Al-Amrani, Al-Jabri et al. 2021). Phosphorylation of proteins, a critical mechanism regulating cell signaling processes such as gene expression and membrane transport, plays a pivotal role in both physiological and pathological events. However, the detection and identification of phosphorylated peptides on a proteome-wide scale present significant challenges due to the low abundance of phosphorylated proteins and potential losses during sample preparation (Dunn, Reid et al. 2010).

The trigeminal ganglion, a critical component of primary sensory ganglia, is responsible for transmitting pain and itch sensations. Its intricate structure comprises trigeminal ganglion cell bodies surrounded by a single layer of satellite glial cells, while Schwann cells wrap both distal and central afferent neuronal processes. The ganglion also accommodates fibroblasts, collagen fibers, small blood vessels, and various immune cells (Messlinger and Russo 2019). Additionally, each mouse trigeminal ganglion is approximately 0.1g in size. As a result, studying the proteome and phosphoproteome of tiny neuronal tissues such as the trigeminal ganglion, presents challenges due to the limitations imposed by tissue size and cell type composition.

To address these challenges, our customized workflow employs a high concentration of 5% SDS as a lysis buffer for protein extraction from the trigeminal ganglion. Additionally, a three-step process, incorporating Fe-MTA magnetic beads enrichment and TiO2 enrichment, is implemented to enhance the yield of phosphopeptides. This protocol provides a comprehensive workflow outline facilitating high-quality phosphoproteomic analysis with limited neuronal tissue samples. Detailed procedures cover sample lysis, protein digestion, clearance, enrichment, and LC-MS/MS analysis.

In the document presented, we detail a comprehensive suite of protocols for protein analysis. Basic Protocol 1 outlines the procedures for total protein extraction and digestion, providing a foundation for subsequent analyses. Basic Protocol 2 focuses on the labeling of peptide. samples and their clean-up, essential for accurate detection and quantification. Basic Protocol 3 introduces the use of Fe-NTA magnetic beads for phosphopeptide enrichment, a critical step for phosphoprotein analysis. Protocol 4 extends this enrichment process by incorporating a TiO2 methodology following the magnetic bead procedure, enhancing specificity and yield. Protocol 5 further refines phosphopeptide enrichment through a secondary Fe-NTA technique, ensuring comprehensive phosphoprotein characterization. Finally, Basic Protocols 6 and 7 describe the fractionation of peptides, followed by LC-MS/MS analysis and database search, culminating in a robust approach for protein identification and quantification. NOTE: Mice to be used for experiments should be purchased from approved animal vendors that provide healthy and pathogen-free mice suitable for research. The whole procedure to be conducted on animals must be approved by the appropriate Institutional Animal Care and Use Committee (IACUC) and conform to governmental regulations.

BASIC PROTOCOL 1

Basic protocol title:

Protein extraction and digestion

Introductory paragraph:

Protein extraction is a pivotal initial step in proteomics, particularly when dealing with limited tissue samples or total protein. Achieving efficient protein extraction from neuronal tissues, such as the trigeminal ganglion, requires careful selection of the lysis buffer to optimize results1. This particular part of the protocol focuses on the use of the 5% SDS lysis buffer (Hu, Doyle et al. 2022, Wang, Veth et al. 2023) for conducting protein extraction.

Protein digestion is a critical tool for identifying, characterizing, and measuring proteins in proteomic research (Switzar, Giera et al. 2013). Predominantly, proteomics experiments rely on digestion of the protein into peptides prior to mass spectrometric (MS) analysis. Basic Protocol 1 describes the use of dithiothreitol (DTT) and subsequent iodoacetamide (IAA) for the reduction and alkylation of proteins, respectively, which leads to their denaturation. This refined protocol has been adapted from the original S-trap digestion method.

Materials:

Lysis buffer: 5% SDS (see Reagents and Solutions)

Pierce^™ BCA Protein Assay Kits (Thermo Fisher Scientific, cat. no. 23225)

S-Trap^™ micro columns (≤ 100 μg) (Protifi, cat. no. C02-MICRO-10)

Trimethylammonium bicarbonate (TEAB) 50mM (see Reagents and Solutions)

0.5M Dithiothreitol (DTT) (see Reagents and Solutions)

0.55M Iodoacetamide (IAA) (see Reagents and Solutions)

Trypsin Gold, Mass Spectrometry Grade (Promega, cat. no. V5280)

12% Phosphoric acid solution (see Reagents and Solutions)

0.2% Formic acid in water (see Reagents and Solutions)

1.5 ml Protein LoBind^® tubes (Eppendorf, cat. no. 022431081)

KIMBLE^® KONTES^® DUALL^® Tissue Grinder, 1ml (DWK life sciences, cat. no. 885460–0020)

Incubator (Benchmark Scientific, cat. no. RS7166) or equivalent equipment

Centrifuge (Eppendorf, cat. no. 5425 R) or equivalent refrigerated centrifuge

SpeedVac (CentriVap-84 Cold Trap, LabConco, cat. no. 7460020

Refrigerated CentriVap Benchtop Vacuum Concentrator with glass lid, LabConco, cat. no. 7310021) or equivalent equipment

Vortex Machine (Scientific Industries, inc., cat. no. SKU:SI-0236)

Protein extraction

Protocol steps with step annotations:

• 1
  Dissect trigeminal ganglion tissue (Katzenell, Cabrera et al. 2017) from mice and promptly freeze it using a dry ice. Store the tissue in −80°C freezer.

  If the tissue is dense, such as bone, the use of liquid nitrogen is preferred.

• 2Pre-cool a grinder and pestle on ice.
• 3
  Take the tissue out from the freezer. Transfer the tissue into a 1-ml homogenizer. Add 100 μl of 5% SDS lysis buffer and thoroughly homogenize sample at room temperature.

  This step should not be done on ice, as SDS may precipitate at low temperatures, potentially affecting protein extraction from the tissue.

  Be cautious and ensure that the grinder and pestle match, and that the top of the pestle can reach the bottom of the grinder. Otherwise, the tissue may get stuck at the bottom, resulting in inhomogeneous samples and poor protein recovery.

• 4Transfer the homogenized tissue and buffer into a 1.5-ml microcentrifuge tube with low protein binding properties, then boil the mixture for 2 minutes.
• 5Centrifuge samples at 14,000 × g for 10 minutes and collect the supernatants. Store the samples at 4°C.
• 6Determine the protein concentration using a BCA protein assay kit. After testing the concentration, retain the sample for digestion as outlined in Basic Protocol 2.

Protein digestion

Protocol steps with step annotations:

• 7
  Take 100 μg protein aliquot from the total protein extract supernatant from Basic Protocol 1 for each sample.

  Ideally, the volume should be less than 30 μl, which will be convenient for the subsequent steps. If the volume of 100 μg protein exceeds 30 μl, the amounts of DTT and IAA added in the following steps will need to be adjusted accordingly.

• 8
  Add DTT to achieve a final concentration of 2 mM and incubate at 56°C for 30 min.

  This step is for reducing disulfide bonds.

  Do not heat to >60°C if there is urea in buffer, it will result in carbamylation of lysine and protein N-termini.

  For example, If the current protein aliquot is 30 μl, add 1.2 μl 50 mM DTT (diluted 10-fold from stock solution) to the protein sample.

• 9
  Add iodoacetamide (IAA) to a final concentration of 5 mM and incubate at room temperature for 45 min. Keep the reaction in the dark.

  This step is to alkylate free cysteines.

  For example, if the current sample volume is now 31.2 μl, add 3 μl of 55 mM IAA (diluted 10-fold from stock solution) to the sample.

• 10
  Add 12% aqueous phosphoric acid to the sample at a 1:10 volume/volume (v/v) ratio, achieving a final concentration of 1.2% phosphoric acid. After adding, vortex the sample for thorough mixing.

  For example, if the sample volume is now 34.2 μl, add 3.4 μl 12% phosphoric acid.

  This step is essential since the protein trap binds at pH ≤ 1.

• 11
  Add 165 μl binding/wash buffer to every 27.5 μl acidified protein solution. Mix well by vortexing.

  If enough protein is present, solution should turn opaque, indicating colloidal formation.

• 12Transfer to top of S-Trap column; if total volume is > 200 μl at this point, repeat this step and step 7 with ~200 μl each time.
• 13
  Centrifuge the micro column at 4,000 × g for 1 to 2 min at 4000 × g, 4°C, until all buffer has passed through the S-Trap column.

  Protein will be captured and held in the spin column’s protein-binding matrix.

• 14
  Wash captured protein by adding 150 μl S-Trap buffer and repeating centrifugation three times.

  During centrifugation, rotate the S-Trap micro units 180° after each centrifugation cycle, especially when using a fixed-angle rotor. Marking the outside edge of the unit can help to recognize 180° rotation for each cycle.

• 15Move S-Trap micro column to a clean 2-ml sample tube for protease digestion.
• 16
  Add 20 μl of 50mM TEAB containing trypsin at 1:10 –1:25 wt:wt (compare with current sample volume) to the top of the micro column.

  For effective digestions, do not apply <0.75 μg trypsin.

  Make certain that no air bubbles are present between the protease digestion solution and the protein trap.

• 17Cap the S-Trap column loosely to limit evaporation loss.
• 18
  Incubate at 47 °C for 1hr for trypsin.

  Preferably use a water bath or non-moving thermomixer.

  Avoid shaking.

• 19
  Elute peptides with 40 μl of 50 mM TEAB, and then centrifuge 1 min at 4000 × g, 4°C.

  Do not centrifuge the digestion before applying TEAB.

  Apply TEAB directly into the trap containing the digestion buffer that was incubate.

• 20Add 40 μl 0.2% aqueous formic acid to the column and then centrifuge 1 min at 4,000 × g, 4°C.
• 21Add 40 μl 50% acetonitrile containing 0.2% formic acid and then centrifuge (4,000 × g, 1min).
• 22Pool eluted peptides and dry down in SpeedVac. Eluted peptides are then resuspended for TMT labeling (Basic Protocol 3).

Basic Protocol 2: TMT labeling and peptides Clean-up

Introductory paragraph:

After protein digestion, when dealing with multiple samples, tandem mass tags (TMT) can be employed for labeling. TMT reagents utilize the principle of isotopes. Different isotopic labels are conjugated to specific amino acid residues in peptides, allowing for the labeling of peptides from different sources. The TMT labeling reagents enable simultaneous identification and quantification of proteins in diverse samples using tandem mass spectrometry. The protocol is adapted from TMTpro^™ Label Reagent set user guide.

Peptide cleanup is essential for the removal of secondary metabolite contamination produced during sample preparation (Waas, Pereckas et al. 2019). These compounds can negatively impact the efficiency of subsequent phosphopeptide enrichment processes. To ensure an optimal protein yield and to prepare high-quality protein samples for mass spectrometric analysis, Basic Protocol 2 second section outlines the procedure for peptide clean-up using the Thermo Fisher Scientific prep kit.

Materials:

Tandem Mass Tag (TMT) pro reagent (Thermo Fisher Scientific, cat. no. A52045)

50 mM TEAB (see Reagents and Solutions)

5% hydroxylamine (see Reagents and Solutions)

20% formic acid solution (see Reagents and Solutions)

EasyPep^™ Mini MS Sample Prep Kit (Thermo Fisher Scientific, cat. no. A4006)

SpeedVac (CentriVap-84 Cold Trap, LabConco, cat. no. 7460020; Refrigerated CentriVap Benchtop Vacuum Concentrator with glass lid, LabConco, cat. no. 7310021) or equivalent equipment

pH paper (Thermo Fisher Scientific, cat. no. 14–853-150R)

Protocol steps with step annotations:

• 1Just before using the kit, let the TMTpro^™ Label Reagents reach room temperature inside the foil pouch.
• 2Add 100 μl 50 mM TEAB to resuspend peptides for each sample. Mix and vortex thoroughly.
• 3
  Add 40 μl of TMT^™ reagent, which is dissolved in 100% acetonitrile, to each buffered peptide sample. Allow this mixture to incubate at room temperature for 30–60 mins.

  When using TMTpro^™ label reagent, apply 0.1–1 mg of the label reagent for every 10–100 ug of protein digest.

• 4To stop and acidify labeling reaction, add 50 μl of 5% hydroxylamine and 20% formic acid solution to each labeling reaction.
• 5
  Take 1 μl drop from each tube to verify pH < 4 using pH paper.

  If pH >4, then add more drops of 20% formic acid to the sample.

• 6
  After labeling, combine the samples into one tube and then proceed with the clean-up procedure.

  Since each sample has been labeled, combining the samples will save time in the subsequent procedures.

Peptide Cleanup

Protocol steps with step annotations:

• 7Take off the white cap located at the bottom of the Peptide Clean-up column. Gently loosen the green top cap and put it into a 2 ml microcentrifuge tube.
• 8Centrifuge the column at 3,000 × g for 2 minutes to remove any remaining liquid from the column, and then dispose of the liquid that flows through.
• 9Transfer the protein digest sample, which has a total volume of approximately 300 μl, into the dry Peptide Clean-up column.
• 10
  Centrifuge the column at 1,500 × g for 2 minutes and dispose the flow-through.

  You can continue using the same 2 ml microcentrifuge tube until step 10.

• 11Add 300 μl of Wash Solution A into the column.
• 12Centrifuge once more at 1,500 × g for 2 minutes and discard the flowthrough.
• 13Add 300 μl of Wash Solution B into the column.
• 14Centrifuge at 1,500 × g for 2 minutes and discard the flowthrough.
• 15Repeat steps 7 and 8 one more time.
• 16Transfer the Peptide Clean-up column to a fresh 2 ml microcentrifuge tube.
• 17Add 300 μl of Elution Solution to the column.
• 18Centrifuge at 1,500 × g for 2 minutes to collect the purified peptide sample.
• 19
  Use a vacuum centrifuge to dry the peptide sample. After drying, take 10% of the protein sample for fractionation (basic protocol 6) and analysis of background proteome. The remaining sample then proceed to phosphopeptide enrichment using Fe-NTA magnetic beads.

  Set vacuum centrifuge temperature around 30°C to avoid overheating during drying.

  Depending on the centrifuge, this step could take approximately 1–2 hours.

  The dried peptide may be invisible. If nothing is apparent in the tube, proceed to the next step.

  The dried peptide can be stored frozen at −80°C overnight.

Basic Protocol 3: IMAC Fe-NTA magnetic beads phosphopeptide enrichment

Introductory paragraph

Mass spectrometry is pivotal for identifying protein phosphorylation sites and quantifying changes in phosphorylation levels. Nonetheless, analyzing protein phosphorylation via MS presents challenges, such as low stoichiometry, high hydrophilicity, suboptimal ionization, and incomplete fragmentation of phosphopeptides. Due to the comparatively low occurrence of phosphorylation modifications in complex protein mixtures, it becomes essential to enrich phosphopeptides to enhance the effectiveness of MS analysis.

PTMScan^® Phospho-Enrichment IMAC Fe-NTA Magnetic Beads utilize immobilized metal affinity chromatography to capture phosphorylated peptides. The negatively charged phosphate groups bind to the positively charged metal ions on the beads. Combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), this method facilitates the isolation, identification, and quantification of numerous phosphorylated cellular peptides. It achieves a high level of specificity and sensitivity, offering a comprehensive snapshot of phosphorylation in cellular and tissue samples (Liu, Rossio et al. 2022). Basic Protocol 3 second section describes the detailed steps for using magnetic beads to enrich phosphopeptides.

Materials:

PTMScan^®Phospho-Enrichment IMAC Fe-NTA Magnetic Beads (Cell Signaling Technology, cat. no. 20432)

Acetonitrile (ACN, Thermo Fisher Scientific, cat.no. 51101)

Ammonium hydroxide 28% (Sigma, cat. no. 338818)

20% Trifluoroacetic acid (TFA, see Reagents and Solutions)

Loading Buffer (see Reagents and Solutions)

Wash Buffer (see Reagents and Solutions)

Elution Buffer (see Reagents and Solutions)

Parafilm (USA Scientific, cat. no. 3023–4526)

1.5 ml Protein LoBind^® tubes (Eppendorf, cat. no. 022431081)

Magnetic stand (Cell Signaling Technology, cat. no. 7017)

Speedvac (CentriVap-84 Cold Trap, LabConco, cat. no. 7460020

Refrigerated CentriVap Benchtop Vacuum Concentrator with glass lid, LabConco, cat. no. 7310021) or equivalent equipment

Rotator (Barnstead/Thermolyne Labquake Shaker, Model 400110, cat. no. 26806) or equivalent instrument

Protocol steps with step annotations:

1. Add 0.95 mL Loading Buffer, which contains 0.1% TFA and 85% ACN to the digested peptide solution. Mix thoroughly by vortexing.

2. Centrifuge at 10,000 × g for 5 min at 4°C in a microcentrifuge to clear solution.

   There may be a significant insoluble pellet, but this should not be a concern as the majority of the peptides will remain in solution.

3. Rotating the PTMScan^® IMAC Fe-NTA Magnetic Beads end-over-end on a rotator for 10 minutes to resuspend the beads.

   This can be done at room temperature.

4. Cut off 2 mm from the tip of a 20 μl pipette tip using a razor blade or scissors. Use this tip to take 20 μL of bead slurry and transfer to a 1.5 mL microcentrifuge tube.

5. Add 1 ml Wash Buffer to wash the IMAC magnetic beads. Allow the beads to settle on a magnetic stand. Carefully remove the wash solution, taking care not to dislodge any beads. Wash 3 times.

6. Transfer the cleared peptide solution into the microcentrifuge tube containing IMAC beads.

   Do not dislodge any pelleted material, pipet sample directly on top of the beads at the bottom of the tube to ensure immediate mixing.

7. Tighten the cap on the tube. Seal the top of the microcentrifuge tube with parafilm to prevent any potential leakage. Incubate on the rotator for 30 minutes at room temperature.

8. After incubation, allow the magnetic beads to settle on the magnetic stand, then discard the supernatant carefully. Do not remove any beads. The depleted peptide solution may be frozen at −80° C and saved for later step 17 SMOAC enrichment (labeled W1).

9. Add 1 mL of Wash Buffer to wash beads, gently rotate the tube until the beads are completely resuspended.

10. Allow the beads to settle on the magnetic stand, then transfer the supernatant into a separate tube, labeled W2.

11. Wash two more times, transferring each subsequent supernatant into distinct tubes labeled W3 and W4, respectively.

12. Add 50 μL of elution buffer, which contains 50% acetonitrile and 2.5% ammonia, to elute the phosphopeptides from the beads. Tap the bottom of the tube to resuspend the beads and then allow the beads to settle.

13. Rinse a fresh microcentrifuge tube with 0.5 mL acetonitrile to remove any potential contaminants, vortex it, and discard the rinse.

14. Transfer the supernatant containing the eluted phosphopeptides to the rinsed tube.

15. Add 40 μL of 20% TFA to acidify the eluate.

16. Repeat the elution step (Step 12–14) once more and combine the resulting eluates.

17. Dry the eluted phosphopeptides and the combined washed supernatant (W1-W4) in a SpeedVac at 25°C (about 4–5 hours due to large volume).

18. Freeze the eluted phosphopeptides at −80°C. The W1-W4 tubes will then be used for Basic Protocol 4.

Basic Protocol 4: TiO2 enrichment

Introductory paragraph

The Thermo Fischer Scientific^™ High-Select^™ TiO2 Phosphopeptide Enrichment Kit offers an effective method for isolating phosphorylated peptides from complex protein digests and fractions, facilitating their analysis through mass spectrometry. TiO2 exhibits a strong affinity for phosphopeptides, as well as other acidic peptides, and is water insoluble. Phosphopeptides can be effectively captured by TiO2 beads in an acidic loading buffer and subsequently released using an alkaline elution buffer. This carefully optimized procedure, along with the specific buffers, enhances phosphopeptide yield, enabling direct MS analysis without requiring extra graphite or C18 purification steps. This method is adapted from the Thermo Fisher Scientific High-Select^™ SMOAC protocol.

Materials:

High-Select^™ TiO2 Phosphopeptide Enrichment Kit (Thermo Fisher Scientific, cat. no. A32993)

Water, LC-MS Grade (Thermo Fisher Scientific, cat. no. 51140))

0.1% Formic acid, LC-MS Grade (Thermo Fisher Scientific, cat. no. 85170)

Centrifuge (Eppendorf, cat. no. 5425 R) or equivalent refrigerated centrifuge

SpeedVac (CentriVap-84 Cold Trap, LabConco, cat. no. 7460020; Refrigerated CentriVap Benchtop Vacuum Concentrator with glass lid, LabConco, cat. no. 7310021) or equivalent equipment

2 ml Protein LoBind^® tubes (Eppendorf cat. no. 022431102)

pH paper (Thermo Fisher Scientific, cat. no. 14–853-150R) or equivalent pH meter

Protocol steps with step annotations:

1. Take all the solutions from the refrigerator to equilibrate them at room temperature.

2. To prepare the column, place a centrifuge column adaptor in a 2 ml Protein LoBind^® microcentrifuge tube and insert a TiO₂ Spin Tip into the adaptor.

3. Add 20 μl of wash buffer and centrifuge at 3,000 × g for 2 minutes.

4. Add 20 μl of Binding/Equilibration Buffer and centrifuge at 3,000 × g for 2 minutes.

5. Discard the flowthrough. Keep the microcentrifuge tube for step 10.

6. Transfer the prepared TiO₂ Spin Tip and adaptor into a new 2 ml microcentrifuge tube.

7. Add 150 μl of Binding/Equilibration Buffer to completely suspend lyophilized peptide from basic protocol 3 step 18 (W1-W4).

   Vortex the tube to make sure the sample is entirely dissolved. Lyophilized peptide must be entirely dissolved for optimal results.

8. Add 150 μL of suspended peptide sample to the spin tip. Centrifuge at 1,000 × g for 5 minutes.

9. Reapply sample in the microcentrifuge tube to the spin tip. Centrifuge at 1,000 × g for 5 minutes.

   If needed, keep the flowthrough for analysis. This fraction is called TiO2-FT

10. Transfer the TiO2 Spin Tip and adaptor into the collection tube prepared from step 5.

11. Add 20 μL of Binding/Equilibration buffer to wash column. Centrifuge at 3,000 × g for 2 minutes.

12. Add 20 μL of Wash buffer to wash the column. Centrifuge at 3,000 × g for 2 minutes. Save the wash fraction 1.

13. Transfer the TiO2 Spin Tip and adaptor into a fresh 2.0 ml microcentrifuge tube.

14. Repeat steps 11 and 12.

    Reordering the wash column steps leads to a notably increased occurrence of non-specific peptide binding.

15. Add 20 μL of LC-MS grade water. Centrifuge at 3,000 × g for 2 minutes. Save the wash fraction 2.

16. Eliminate surplus liquid by gently blotting the bottom of the spin tip using a clean paper towel, such as a Kimwipe.

17. Place the spin tip and adaptor in a new 2.0 ml microcentrifuge tube.

18. Add 50 μl of Phosphopeptide Elution Buffer. Centrifuge at 1,000 × g for 5 minutes.

19. Repeat step once.

    Use same 2ml microcentrifuge tube to combine the flowthrough.

20. Promptly dry the eluate in a speed Vac concentrator to eliminate Phosphopeptide Elution Buffer.

    It is important to note that eluates cannot be stored in phosphopeptide Elution Buffer due to its high pH, which may lead to the loss of phosphopeptides.

21. Suspend the eluate with 50 μl 0.1% formic acid for direct MS analysis.

    If the starting peptide sample amount is less than1 mg, suspend dried elute using 25 μl of 0.1% formic acid.

22. Combine TiO2-FT from step 9, with the saved wash fraction 1 & 2 from step 12 & 15.

23. Dry the combined sample by using Speed Vac. The dried sample can be stored at −80C until the next procedure.

    Do not over dry. It is typical to observe some translucent jelly-like material at the end of dry step.

    The drying procedure can take around 2–4 hours.

Basic Protocol 5: Fe-NTA phosphopeptide Enrichment

Introductory paragraph

The Thermo Fisher Scientific^™ High-Select^™ Fe-NTA Phosphopeptide Enrichment Kit offers a swift and effective means of isolating phosphorylated peptides, with a specificity exceeding 90%. The streamlined process allows for the enrichment of phosphopeptides from protein digests or peptide fractions, priming them for mass spectrometry analysis. Each spin column, provided within the kit, is equipped with a specialized resin designed for phosphopeptide capture, boasting exceptional binding and recovery capabilities, accommodating up to 150 μg of phosphopeptides per column. This method is derived from the High-Select Fe-NTA Phosphopeptide Enrichment SMOAC protocol.

Materials:

High-Select Fe-NTA Phosphopeptide Enrichment Kit (Thermo Fisher Scientific, cat. no. A32992)

Water, LC-MS Grade (Thermo Fisher Scientific, cat. no. 51140)

0.1% Formic acid, LC-MS Grade (Thermo Fisher Scientific, cat. no. 85170)

2 ml Protein LoBind^® tubes (Eppendorf, cat. no. 022431102)

Protocol steps with step annotations:

1. To equilibrating the column, remove the spin column bottom closure and loosen the screw cap.

2. Put column in a 2 ml microcentrifuge collection tube. Centrifuge at 1,000 × g for 30 seconds to eliminate storage buffer.

3. Take off the screw cap and set aside for use in later Phosphopeptide Binding (step 8).

4. Add 200 μl of Binding/Wash Buffer. Centrifuge at 1,000 × g for 30 seconds and discard the flowthrough.

5. Repeat step 4 one more time.

6. Use a white Luer plug to cap the bottom of the column. Transfer the column with the plug into the empty microcentrifuge tube.

7. Add 200 μl of Binding/Wash Buffer Completely suspend lyophilized peptide sample. Use vortex mixer with tube stand if necessary.

   To achieve the best outcomes, ensure the lyophilized peptide sample is fully dissolved in the Binding/Wash Buffer.

   If desired, use pH paper to confirm that the pH of the resuspended sample is below 3.If the pH is higher than 3, add 1 or several drops of formic acid to keep the pH low.

8. Add 200 μL of the peptide solution to the prepared spin column and secure the screw cap.

9. Mix the resin with the sample by gently tapping the bottom plug while holding the screw cap for 10 seconds, ensuring the resin becomes fully suspended.

   Avoid using vortex or turning the column upside down to prevent resin from splashing against the column walls, as this could lead to an increase in nonspecific peptide attachment.

10. Incubate for 30 minutes at room temperature.

    Gently mix the resin every 10 minutes as described in step 9.

11. Gently remove both the bottom plug and top screw cap. Do not compress the bottom plug which could cause liquid to flow back into the column.

12. Put the column into the microcentrifuge tube. Centrifuge at 1,000 × g for 30 seconds. Discard the flowthrough.

13. Add 200 μL of Binding/Wash Buffer to the column to wash column. Centrifuge at 1,000 × g for 30 seconds.

14. Repeat wash step 13 two more times for a total of 3 washes. Discard the flowthrough.

15. Add 200 μL of LC-MS grade water for an additional wash. Centrifuge at 1,000 × g for 30 seconds. Discard the flowthrough.

16. To elute column, first place column in a new microcentrifuge tube. Add 100 μL of Elution Buffer to the column. Centrifuge at 1,000 × g for 30 seconds. Keep the flowthrough. Repeat this step once.

    It is normal for the resin to change to a brown color during this process.

17. Immediately dry the collected eluate using a speed vacuum concentrator to eliminate the Elution Buffer.

    High pH Elution Buffer will lead to loss of phosphates on phosphopeptides.

18. Add 70 μL of 0.1% formic acid to suspend the dried eluate and then direct LC-MS analysis.

    If starting peptide sample amounts is less than 1mg, suspend dried elute in 40 μL of 0.1% formic acid.

Basic Protocol 6: High pH peptide fractionation

Introductory paragraph

The Thermo Fisher Scientific^™ Pierce^™ High pH Reversed-Phase Peptide Fractionation Kit is specifically designed to improve protein identification in complex samples via liquid chromatography-mass spectrometry (LC-MS) analysis, utilizing a high-pH reversed-phase chromatography approach. This method efficiently separates peptides based on their hydrophobic properties, offering a strong alternative to low-pH reversed-phase LC-MS gradients and demonstrating its distinct advantage over strong cation exchange (SCX) fractionation by eliminating the need for an additional desalting process before LC-MS analysis. The kit’s protocol is a refined version of the original method used in the Pierce High pH Reversed-Phase Peptide Fractionation Kit.

Materials

Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher Scientific, cat. no. 84868)

0.1% Trifluoroacetic acid (TFA) (see Reagents and Solutions)

Acetonitrile (ACN), LC-MS Grade (Thermo Fisher Scientific, cat. no. 51101)

Water, LC-MS Grade (Thermo Fisher Scientific, cat. no. 51140)

2 ml Protein LoBind^® tubes (Eppendorf, cat. no. 022431102) or Thermo Fisher Scientific^™ Pierce^™ Low Protein Binding Microcentrifuge Tubes, 2.0mL (cat. no. 88379 or 88380)

Microcentrifuge with adjustable rotor speed up to 7,000 × g.

SpeedVac (CentriVap-84 Cold Trap, LabConco, cat. no. 7460020; Refrigerated CentriVap Benchtop Vacuum Concentrator with glass lid, LabConco, cat. no. 7310021) or equivalent equipment

Protocol steps with step annotations:

1. Take off and discard the protective white tip from the bottom of the column. Then, place the column into a 2.0 ml tube. Centrifuge at 5,000 × g for 2 minutes. Discard the flowthrough.

   Do not exceed recommended centrifugation speeds.

2. Take off the top screw cap and add 300 μl of ACN into the column. Secure the cap, place the spin column back into a 2.0 ml microcentrifuge tube and centrifuge at 5,000 × g for 2 minutes. Discard flowthrough.

3. Repeat wash step 2 one more time.

4. Add 0.1% TFA to the spin column to wash. Centrifuge at 5,000 × g for 2 minutes. Discard flowthrough. The column is now ready for use.

5. Prepare elution solutions as outlined in Table 1.

   Allocating 300 μL for each solution per sample. Adjust the volume of elution solutions based on the number of samples to be fractionated if processing more than three samples.

6. Dissolve the peptide sample, ranging from 10–100 μg, in 300 μl of 0.1% TFA.

   Ensure the peptide is fully dissolved and does not contain organic solvents like ACN or DMSO.

7. Transfer the spin column to a fresh 2 ml tube. Add 300 μl of the sample solution onto the column, reapply the cap on top. Centrifuge at 3,000 × g for 1 minutes. Keep eluate flowthrough fraction as “flow-through”.

8. Place the column into a new 2 ml tube. Add 300 μL of water onto the column. Centrifuge at 3,000 × g for 1 minute. Retain eluate as “wash” fraction.

   For TMT-labeled samples, perform an extra wash using 300 μl of 5% ACN with 0.1% triethylamine (TEA) to remove any unbound TMT reagent.

9. Put the column into a new 2 ml tube. Add 300 μl of the elution solution (5% ACN, 0.1% TEA). Centrifuge at 3,000 × g for 2 minutes to collect the fraction.

10. Repeat step 9 for each elution step, using new 2 ml tubes and the respective elution solutions detailed in Table 1 for each fraction.

11. Use a SpeedVac or similar vacuum concentrator to evaporate all the liquid from each sample tube until dry.

Table 1.

Preparation of elution solutions for Thermo Fisher Scientific TMT-labeled peptides.

Fraction No.	Acetonitrile (%)	Acetonitrile (μL)	Triethylamine (0.1%) (μL)
Wash	5.0	50	950
1	10.0	100	900
2	12.5	125	875
3	15.0	150	850
4	17.5	175	825
5	20.0	200	800
6	22.5	225	775
7	25.0	250	750
8	30	300	700
9	37.5	375	625
10	50	500	500

Basic protocol 7: LC-MS/MS analysis and database search

Introductory paragraph

In a typical LC-MS/MS analysis, peptides are separated based on hydrophobicity before entering mass spectrometry. Mass spectrometer operates in a “data dependent” mode where eluted peptides are isolated and fragmented inside the mass spectrometer. Accurate mass of the intact peptide and its fragments are recorded in the mass spec raw data. The raw data is then processed by a search engine find the best match between each mass spectrum with a peptide from the designated protein database. After applying FDR (false discovery rate) control, peptides with high confidence are inferred to proteins. A parsimony rule is applied so that the smallest list of proteins is reported to represent all peptides identified. Peptide quantification is based on abundance of TMT reporter ions.

Materials

Water, LC-MS Grade (Thermo Fisher Scientific, cat. no. 51140),

Acetonitrile (ACN), LC-MS Grade (Thermo Fisher Scientific, cat. no. 51101)

0.1% Formic acid, LC-MS Grade (Thermo Fisher Scientific, cat. no. 85170)

High resolution mass spectrometer (orbitrap Fusion Lumos from Thermo Fisher Scientific)

Nanoscale HPLC system (Dionex Ultimate3000nano RSLC) with autosampler or equivalent system

Trap column Acclaim PepMap 100 trap column (75 μm × 2 cm)

Capillary analytical column: 75 μm × 250 mm Acclaim PepMap 100 column (3 μm, 100 Å)

Data processing software: Proteome Discoverer 2.5

Protocol steps with step annotations:

1. Resuspend dry samples obtained from basic protocol 6 in an appropriate volume of 0.1% formic acid before LC-MS analysis.

2. Load 1 μg of the tryptic digest from last step onto an Acclaim PepMap 100 trap column (75 μm × 2 cm) and desalt it at a flow rate of 4 μl/min for 5 minutes for each run.

3. Elute peptides onto a 75 μm × 250 mm Acclaim PepMap 100 column (3 μm, 100 Å).

4. Perform chromatographic separation using a two-part solvent system, with solvent A being 0.1% formic acid and solvent B combining 0.1% formic acid with 80% acetonitrile, at a flow rate of 300 nl/min.

5. Implement a gradient from 1% of solvent B to 42% over 150 minutes.

6. Follow this by washing the column with 80% solvent B for 5 minutes and re-equilibrating it at 1% solvent B for 10 minutes before introducing the next sample.

7. Detect precursor masses in the Orbitrap with a resolution of 120,000 (m/z 200).

8. Detect HCD fragment masses in the Orbitrap at a resolution of 50,000 (m/z 200), employing data-dependent MS/MS with a cycle time of 2 seconds and a dynamic exclusion period of 20 seconds.

9. The proteomics and phosphoproteomics data are subsequently analyzed using Proteome Discoverer (V2.5) against human protein database (Uniprot) with the following search parameters: full trypsin cleavage with maximum 1 missed cleavage. Precursor tolerance 10 ppm, fragment tolerance 0.02 Da. Static modification: carbamidomethylation of Cysteine, TMTPro labeling of lysine and peptide N-terminus. Dynamic modification: oxidation of methionine, phosphorylation of serine, threonine, and tyrosine, and protein N terminal acetylation.

10. A representative example of the results, similar to those depicted in Figure 1D, can be visualized as a volcano plot.

Fig. 1.

A. Different lysis buffers yield varying amounts of proteins from trigeminal ganglion tissue. Fig. 1. B. Ten samples are abundantly and evenly labeled with TMTpro labeling reagents. Fig. 1. C. A Venn diagram depicting how each step of the 3-step phosphopeptide enrichment process captures different quantities and groups of proteins. Fig. 1. D. A volcano plot presenting the quantitative analysis of the global phosphopeptides dataset from the trigeminal ganglion, as identified by mass spectrometry. In the treated group, the analysis revealed 61 upregulated and 33 downregulated peptides within the total proteome dataset. Peptides with fold changes >1.5 (p<0.01, t-test) were identified as being significantly expressed in the treatment group. The x-axis represents the log2 fold change, and the y-axis represents the −log10 FDR-adjusted p-value.

REAGENTS AND SOLUTIONS:

100 mM triethyl ammonium bicarbonate (TEAB)

• 500 μl 1M TEAB (Thermo Fisher Scientific, cat. no. 90114) store protected from direct sunlight in a dry, cool, and well-ventilated area.

• 4.5 ml Water LC-MS Grade (Thermo Fisher Scientific, cat. no. 51140)

50mM TEAB

• 1 ml 100mM TEAB (refer to the materials mentioned above for preparing 100mM TEAB)

• 1 ml μl Water, LC-MS Grade (Thermo Fisher Scientific, cat. no. 51140)

Dithiothreitol (DTT) 500 mM in 50 mM TEAB

• 7.2 mg DTT (Millipore Sigma, cat. no. D0632) storage at 2–8 °C

• 100 μl 50 mM TEAB (refer to the materials mentioned above for preparing 50mM TEAB)

Iodoacetamide (IAA) 0.55 M

• 10.2 μg IAA (Thermo Fisher Scientific, cat. no. A39271)

• 100μl 50mM TEAB (refer to the materials mentioned above for preparing 50mM TEAB)

5% hydroxylamine

• 50 μl hydrosylamine (Thermo Fisher Scientific, cat. no. 90115)

• 450 μl 100 mM TEAB (refer to the materials mentioned above for preparing 100mM TEAB)

5% SDS

• 5 ml 10% SDS solution, Molecular Biology Grade (Promega, cat. no. V6551). Store at room temperature.

• 5 ml Water LC-MS Grade (Thermo Fisher Scientific, cat. no. 51140)

10ml 0.1% trifluoroacetic acid (TFA)

• 10 μl Pierce^™Trifluoroacetic Acid (TFA) Sequencing grade (Thermo Fisher Scientific, cat. no. 28904) Store in original container protected from direct sunlight in a dry, cool and well-ventilated area.

• 10 ml Water, LC-MS Grade (Thermo Fisher Scientific, cat. no. 51140)

20% trifluoroacetic acid (TFA)

• 10 ml Pierce^™ Trifluoroacetic Acid (TFA) Sequencing grade (Thermo Fisher Scientific, cat. no. 28904)

• 40 ml Water, LC-MS Grade (Thermo Fisher Scientific, cat. no. 51140)

0.2% Formic Acid 1 ml

• 2 μl Formic Acid, LC-MS Grade (Thermo Fisher Scientific, cat. no. 85178) Use only under a chemical fume hood. Keep containers tightly closed in a dry, cool and well-ventilated place. Keep away from heat, sparks and flame. Containers should be vented periodically in order to overcome pressure buildup.

• 998 μl Water, LC-MS Grade (Thermo Fisher Scientific, cat. no. 51140)

• 20% Formic Acid 1ml

• 200 μl Formic Acid, LC-MS Grade (Thermo Fisher Scientific, cat. no. 85178) Store between 2–25 °C, in a dry, cool, well-ventilated area.

• 800 μl Water, LC-MS Grade (Thermo Fisher Scientific, cat. no. 51140)

10ml 12% phosphoric acid solution

• 1.4 ml of phosphoric acid (85%) (Sigma Aldrich, cat. no. 345245) Storage with corrosive hazardous materials, do not storage in metal containers. Tightly closed.

• 8.6 ml water LC-MS Grade (Thermo Fisher Scientific, cat. no. 51140)

Loading Buffer (0.1% TFA, 85% acetonitrile) 5 ml

• 4.25 mL Pierce^™ Acetonitrile (ACN), LC-MS Grade (Thermo Fisher Scientific, cat. no. 51101)

• 0.725 mL Water, LC-MS Grade (Thermo Fisher Scientific, cat. no. 51140)

• 25 μL 20% TFA

Wash Buffer (0.1% TFA, 80% acetonitrile) 50 ml

• 40 mL ACN (Thermo Fisher Scientific, cat. no. 51101)

• 9.75 mL Water, LC-MS Grade (Thermo Fisher Scientific, cat. no. 51140)

• 250 μL 20% TFA

Elution Buffer: (50% acetonitrile, 2.5% ammonia) 5ml

• 0.45 mL 28% Ammonia (Sigma, cat. no. 338818) Store between 2–8°C

• 2.05 mL Water, LC-MS Grade (Thermo Fisher Scientific, cat. no. 51140)

• 2.5 mL ACN.

COMMENTARY:

In a global phosphoproteomic analysis, the use of a large number of cells, tissues, or other biological material is recommended due to the inherently low levels of phosphorylated proteins in most biological systems and the potential loss occurring across multiple steps of sample preparation. Limited sample size poses a challenge in achieving optimal protein and peptide recoveries. To address this, we present a detailed method for enriching phosphoproteins, allowing for the use of limited sample size tissues while maintaining high-quality phosphoproteomic data. This method is suitable for small tissue samples while ensuring high-quality phosphoproteomic data. Our approach involves a 3-step sequential enrichment process: initially using Fe-NTA magnetic beads from Cell Signaling Technology, followed by TiO2 enrichment, and concluding with secondary Fe-NTA phosphopeptide enrichment.

Critical parameters:

Lysis buffer

In the lysis stage, selecting the appropriate lysis buffer type is critical. This buffer aids in breaking down cells or tissues for protein extraction. Both the type and volume of the buffer might require adjustments based on the initial sample and the target protein concentration. Factors to consider include the cell or tissue’s type and size, along with the need for effective protein extraction. In our study, we evaluated three different lysis buffers for trigeminal ganglion tissue: T-per (Thermo Fisher Scientific, cat. no. 78510) with protease/phosphatase inhibitors, EasyPep^™ Mini MS Sample Prep Kit lysis buffer (Thermo Fisher Scientific, cat. no. A40006), and a 5% SDS solution. Following the application of these lysis buffers, we proceeded with the Basic Protocol 1 for protein digestion and then assessed the protein yield. Our findings indicate that the 5% SDS lysis buffer outperforms the others, recovering a significantly higher number of proteins, as illustrated in Fig.1. Further details on these findings are provided in the ‘Understanding Results’ section.

Protein concentration test

Determining protein concentration is a critical step in preparing samples for various downstream applications. Although this protocol does not delve deeply into the specific method used for determining protein concentration, it acknowledges the variability in available methods. Researchers often use techniques such as the BCA assay to quantify protein content.

Protein clean-up

Protein clean-up is crucial for removing contaminants and substances that might interfere with subsequent analyses. The timing of this clean-up, either before or after TMT labeling, is flexible. However, performing clean-up after TMT labeling can improve efficiency by allowing the combination of labeled samples into a single tube. This step streamlines the process, simplifying downstream processing and analysis for a more efficient workflow.

Phosphopeptide enrichment

When considering the sequence of phosphopeptide enrichment methods, it is advantageous to employ the CST Fe-NTA magnetic Beads method before utilizing the TiO2 and SMOAC methods. This is primarily because the final step in the SMOAC method involves repeating the Fe-NTA enrichment, which, when combined with the initial Fe-NTA step, yields a more abundant result. This observation is corroborated by the Venn diagram presented in Fig1. C, which demonstrates that each enrichment step captures distinct phosphopeptides. We will delve into a more comprehensive discussion of these findings during the “Understanding Results” session.

Troubleshooting

Please see Table 2 for troubleshooting guide.

Table 2.

Troubleshooting Guide for proteomic and phosphoproteomics analysis for limited tissue

Problem	Possible Cause	Solution
Low protein yield	Insufficient lysis of the tissue	To enhance tissue lysis, consider increasing the volume of the lysis buffer or incorporating additional mechanical disruption methods. These steps can help prevent the formation of an insufficiently lysed tissue pellet after centrifugation.
Reduced effectiveness in Tryptic digestion of proteins	The concentration of protease might be insufficient. Additionally, the presence of bubbles on top of the trap could hinder the exposure of the sample to the digestive protease.	Adjust the enzyme-to-protein ratio or extend the digestion time, such as by allowing for an overnight digest in an incubator at 37 °C. To ensure there are no bubbles, conduct a visual inspection. If bubbles are present, gently flick the container until they rise to the surface.
Poor peptide recovery	The column dried out during digestion due to being uncapped	Rehydrate the protein-trapping matrix to solubilize the peptides. Add digestion buffer without the enzyme and allow it to sit for 30 minutes, followed by centrifugation. Subsequently, repeat the washing and elution steps
Low binding efficiency of Fe-NTA magnet beads	Wrong elution buffer used	Double-check all buffer layouts. Continue with the process, as the subsequent TiO2 step may still capture phosphopeptides.
Spin tip clogged	Particles present in the sample caused by the incomplete dissolution of the protein digest sample in Binding/Equilibration Buffer	To ensure complete dissolution of the digested peptide sample, use a vortex mixer or centrifuge the sample prior to applying it to the spin tip.

UNDERSTANDING RESULTS:

This protocol outlines a workflow for preparing limited protein tissue samples, applicable to other neuronal tissues. We conducted tests with various lysis buffers, as showed in Fig 1.A, and observed that different lysis buffers yielded varying protein quantities. When we employed T-per and added proteases, it resulted in a total recovery of 1995 proteins. By using a miniprep kit from Thermo Fisher Scientific, we successfully identified 1283 proteins. The utilization of a 5% SDS lysis buffer allowed us to identify a total of 3662 proteins. The Venn diagram illustrates the overlap and unique proteins identified with different lysis buffers. Consequently, for neuronal tissues such as trigeminal ganglion or dorsal root ganglion, we recommend using a 5% SDS lysis buffer.

Following protein digestion and clean-up, TMT labeling is executed to consolidate the protein samples for subsequent workflow steps. This process is vital for ensuring efficient downstream analysis. Post-labeling, we routinely assess the effectiveness of TMT labeling. Typically, we analyze 1 μg of the labeled sample to confirm that each sample has been sufficiently and uniformly labeled, as illustrated in Figure 1B. Ensuring even labeling of samples is crucial, as it significantly impacts the accuracy and reliability of subsequent experimental stages, including quantitative analysis and the detection of subtle changes in protein expression across different samples.

In our protocol, we outline a three-step enrichment process aimed at improving phosphopeptide identification. We successfully identified a total of 4,454 phosphopeptides. Figure 1C details the number of phosphopeptides identified via each enrichment step. The initial Fe-NTA method led to the identification of 1,433 phosphopeptides, followed by the TiO2 step, which uncovered an additional 199 phosphopeptides. The third step, employing Thermo Fisher Scientific Fe-NTA, revealed 62 more phosphopeptides. There is a final step of fractionation of the combined sample from the above three-step enrichments, resulting in a total of 4,454 phosphopeptides. Each method uniquely captures different proteins, thereby enhancing the overall yield in phosphoproteomics. Furthermore, although not illustrated in the figure, we identified a total of 2,923 phosphoproteins, corresponding to 13,460 peptide groups.

The volcano plot in Figure 1D displays the global phosphoproteome data from trigeminal ganglion. It plots log2 fold changes on the x-axis against p-values on the y-axis. Using high-sensitivity LC-MS/MS for proteomics and phosphoproteomics, we identified peptides and phosphopeptides with differential expression in the trigeminal ganglia. A peptide is deemed differentially expressed if it exhibits a fold change greater than 1.5 and a p-value below 0.05. The plot, generated by Proteome Discoverer 2.5, shows the overall peptide profile, highlighting peptides with significant increases or decreases. In the treated group, analysis revealed 61 upregulated and 33 downregulated peptides within the total proteome dataset.

Time Considerations:

Basic Protocol 1 first section takes approximately 1–2 hours, including the BCA assay process.

Basic Protocol 1 second section requires around 4–5 hours, which includes the drying process. Drying times may vary depending on the equipment used.

Basic Protocols 2 together take about 1–2 hours.

Basic Protocol 3 lasts 5–6 hours and incorporates the drying process.

Basic Protocol 4 is completed in approximately 2–3 hours, including the drying process.

Basic protocol 5 takes around 4–5 hours including drying process.

Basic Protocol 6 takes around 6 hours.

DATA AVAILABILITY STATEMENT

Data sharing is not applicable to this article as no new data were created or analyzed in this study.