Muscle Biopsy Sample Preparation and Proteomics Analysis Based on UHPLC-MS/MS

Materials and reagents

Biological materials

1. Human vastus lateralis muscle biopsy sample

1. Povidone-iodine (Sigma-Aldrich, catalog number: PVP-I)

2. 2% lidocaine with epinephrine (Hospira, catalog number: 0409-4279-16)

3. Glucose solution (Sigma-Aldrich, catalog number: G8769)

4. Physiological saline (Sigma-Aldrich, catalog number: S8776)

5. Liquid nitrogen (Air Products, catalog number: NI)

6. Isopentane (Sigma-Aldrich, catalog number: 277258)

7. Tris-HCl (Sigma-Aldrich, catalog number: T5941)

8. Triton X-100 (Sigma-Aldrich, catalog number: T8787)

9. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P2714)

10. BCA Protein Assay Kit (Thermo Fisher, catalog number: 23227)

11. Trichloroacetic acid (TCA) (Sigma-Aldrich, catalog number: T6399)

12. Acetone, HPLC grade (Sigma-Aldrich, catalog number: 34850)

13. Triethylammonium bicarbonate (TEAB) (Sigma-Aldrich, catalog number: T7408)

14. Trypsin (Promega, catalog number: V5111)

15. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632)

16. Iodoacetamide (IAA) (Sigma-Aldrich, catalog number: I1149)

17. Formic acid (FA), HPLC grade (Sigma-Aldrich, catalog number: 56302)

18. Acetonitrile (ACN), HPLC grade (Sigma-Aldrich, catalog number: 34967)

19. Water, HPLC grade (Thermo Fisher, catalog number: W7-4)

20. EASY-nLC 1200 solvent A (0.1% formic acid in water) (Thermo Fisher, catalog number: LS1224)

21. EASY-nLC 1200 solvent B (0.1% formic acid in acetonitrile) (Thermo Fisher, catalog number: LS1225)

22. FAIMS Pro compensation voltage solution (Thermo Fisher, catalog number: FMSP 0003)

1. PBS (see Recipes)

2. Triton lysis buffer (see Recipes)

3. LC-MS/MS mobile phase A (see Recipes)

4. LC-MS/MS mobile phase B (see Recipes)

1. PBS

   Reagent	Final concentration	Quantity or Volume
   NaCl	137 mM	8.01 g
   KCl	2.7 mM	0.2 g
   NaHPO	10 mM	1.42 g
   KHPO	1.8 mM	0.24 g
   Distilled H2O	n/a	Up to 1 L




2. Triton lysis buffer

   Reagent	Final concentration	Quantity or Volume
   Tris-HCl	50 mM	60.57 mg
   NaCl	150 mM	87.66 mg
   EDTA	1 mM	0.292 mg
   Triton X-100	1%	10 μL
   Protease inhibitor	1%	10 μL
   Distilled H2O	n/a	Up to 1 mL




3. LC-MS/MS mobile phase A

   Reagent	Final concentration	Quantity or Volume
   Formic acid	0.1%	1 mL (for 1 L)
   Acetonitrile	2%	20 mL (for 1 L)
   Water	--	Up to 1 L




4. LC-MS/MS mobile phase B

   Reagent	Final concentration	Quantity or Volume
   Formic acid	0.1%	1 mL (for 1 L)
   Acetonitrile	90%	900 mL (for 1 L)
   Water	--	Up to 1 L

Laboratory supplies

1. Disposable semi-automatic biopsy needle (Bard, catalog number: 1520-5010)

2. Pre-cooled tweezers (VWR, catalog number: 82027-530)

3. Aluminum foil (Reynolds, catalog number: 614)

4. Liquid nitrogen storage container (Thermo Fisher, catalog number: 11887163)

5. Cryogenic vials (Nalgene, catalog number: 5000-1020)

6. Mortar and pestle set (Bel-Art, catalog number: H37260-0000)

7. Microcentrifuge tubes (Eppendorf, catalog number: 0030120086)

8. 1.5 mL microcentrifuge tubes (Eppendorf, catalog number: 022363204)

9. 50 mL conical tubes (Falcon, catalog number: 352070)

10. 1,000 μL pipette tips (Rainin, catalog number: RT-L1000F)

11. 200 μL pipette tips (Rainin, catalog number: RT-L200F)

12. 10 μL pipette tips (Rainin, catalog number: RT-L10F)

13. Sterile gloves (Medline, catalog number: MDS195285)

14. Sterile surgical drapes (3M, catalog number: 1010)

15. Sterile gauze pads (Dynarex, catalog number: 3344)

16. Surgical mask (Medline, catalog number: NON27378)

17. Medical adhesive tape (3M, catalog number: 1530-1)

18. Medical bandages (Johnson & Johnson, catalog number: 2015)

19. 96-well plates (Thermo Fisher, catalog number: 260836)

Equipment

1. Ultrasound machine (GE Healthcare, model: LOGIQ E9)

2. -80 °C freezer (Thermo Fisher, model: ULT2186)

3. Centrifuge (Eppendorf, model: 5424)

4. Ultrasonic homogenizer (Qsonica, model: Q55)

5. Vortex mixer (VWR, model: 945302)

6. UHPLC system (Thermo Fisher, model: EASY-nLC 1200)

7. nanoACQUITY HPLC HHS T3 column (Waters, catalog number: 186003538)

8. Mass spectrometer (Thermo Fisher, model: Orbitrap Exploris 480)

9. FAIMS Pro Interface (Thermo Fisher, catalog number: FAIMS01-10000)

10. Water bath (Grant Instruments, model: JB Academy)

11. pH meter (Mettler Toledo, model: SevenCompact)

12. Magnetic stirrer (IKA, model: RCT basic)

13. Analytical balance (Sartorius, model: Entris II)

14. Refrigerated microcentrifuge (Eppendorf, model: 5418 R)

15. Electronic pipettes (Eppendorf, model: Xplorer plus)

Software and datasets

1. UniProt (https://www.uniprot.org/) (Access date, 2021/08)

2. Proteome Discoverer (v2.4.1.15, Thermo Scientific)

Procedure

1. Muscle biopsy

   1. Preparation

      1. Ensure all equipment and reagents are prepared and sterile.

      2. Set up a sterile area and prepare a disposable semi-automatic biopsy needle.

   2. Biopsy procedure

      1. Position the subject comfortably and identify the biopsy site (typically the vastus lateralis muscle).

      2. Disinfect the target puncture site three times using povidone-iodine, expanding the circular disinfection area around the puncture point. Inject 3–5 mL of 2% lidocaine containing 50 μL of epinephrine for local anesthesia of the skin and subcutaneous tissue. Ensure the needle does not penetrate the deep fascia to avoid anesthetic contamination of muscle tissue. Provide the subject with sufficient glucose to prevent hypoglycemia and stabilize their emotions.

      3. The physician holds the disposable semi-automatic biopsy needle and slowly inserts the needle through the skin and subcutaneous tissue, using musculoskeletal ultrasound to guide the needle and avoid important nerves and blood vessels. Upon reaching the deep fascia, increase the force and rotate the needle to penetrate the fascia. Use ultrasound to confirm the needle's position in the vastus lateralis muscle. The assistant gently compresses the muscle around the puncture site, while the physician triggers the needle mechanism to cut the muscle (Figure 1A).

         
         

         
         Figure 1. Muscle biopsy. A. Biopsy procedure. B. Muscle sample preservation.

         
         

      4. Given the small diameter and cutting window of the biopsy needle, multiple cuts (5–7 times) may be necessary to obtain sufficient muscle tissue (~50 mg).

   3. Sample preservation

      1. Remove the muscle tissue from the biopsy needle using pre-cooled tweezers, rinse multiple times with saline, wrap in foil, quickly freeze in liquid nitrogen–cooled isopentane, and temporarily store in a liquid nitrogen container. Finally, store all muscle samples in a -80 °C freezer for proteomic analysis (Figure 1B).

   4. Post-procedure care

      1. Remove the biopsy needle and apply a medical dressing to the puncture site.

      2. Instruct the subject to sit quietly for 15–20 min, applying continuous pressure to the puncture site to prevent bleeding. Advise the subject not to shower for 24 h and to keep the wound clean and dry.

      3. Continuously monitor and record the subject's condition and wound recovery until fully healed.




2. Preparation of muscle samples for proteomic analysis

   1. Homogenization

      1. Retrieve muscle samples from the -80 °C freezer and place them on ice.

      2. Weigh the tissue sample into a liquid nitrogen–cooled mortar and grind it into a fine powder.

   2. Lysis

      1. Add four times the volume of lysis buffer (see Recipes) to the powdered muscle samples. Perform ultrasonic lysis to homogenize the muscle tissue.

      2. Centrifuge at 12,000× g for 10 min at 4 °C, discard the pellet, and transfer the supernatant to a new tube.

      3. Determine protein concentration using a BCA assay kit.

   3. Protein digestion

      1. Digest equal amounts of muscle sample protein from each group (100 μg to 1 mg). Adjust the volume with lysis buffer.

      2. Slowly add TCA to a final concentration of 20%, vortex to mix, and precipitate at 4 °C for 2 h.

      3. Centrifuge at 4,500× g for 5 min at 4 °C, discard the supernatant, and wash the pellet 2–3 times with pre-cooled acetone.

      4. Air-dry the pellet (15–30 min), add TEAB to a final concentration of 200mM, and sonicate to disperse the pellet. Sonication time is 10–30 s per cycle; power is 30%–50% output; 3–5 cycles with 30 s to 1 min cooling on ice between cycles.

      5. Add DTT to a final concentration of 5 mM and reduce at 56 °C for 30 min.

      6. Add IAA to a final concentration of 11 mM and incubate in the dark at room temperature for 15 min.

      7. Add trypsin at a 1:50 ratio (enzyme:protein, m/m) and digest overnight at 37 °C.




3. UHPLC-MS/MS analysis

   1. Calibration process of UHPLC-MS/MS

      1. Calibration of the instrument: To ensure accuracy, the UHPLC-MS/MS system is typically calibrated by injecting standard solutions with known concentrations. The calibration process aims to validate the performance of chromatographic separation and mass spectrometric detection and ensure a good linear response across the concentration range of the target analyte.

      2. Optimization of mass spectrometry parameters: Calibration of the mass spectrometry component involves optimizing ion source conditions (e.g., spray voltage, ion source temperature) and settings of the mass analyzer to ensure accurate detection of compounds across different molecular weights, with stable peak shapes and responses.

      3. Mass calibration: Using standard mass calibration substances (such as known peptides or small molecules), the mass accuracy of the spectrometer is checked regularly, and necessary corrections are made to ensure that the mass deviation is within the permissible range.

   2. Preparation of calibration solutions

      1. Selection of standards: The standards used in the experiment should be high-purity target compounds, usually with a purity of over 98%. These standards could be purified forms of the target analyte, isotope-labeled compounds, or peptides with known sequences.

      2. Preparation of stock solutions: First, weigh the standard and dissolve in an appropriate solvent (e.g., 0.1% formic acid solution or acetonitrile) to prepare a high-concentration stock solution. The stock solution should have an accurate concentration, typically 1 mg/mL or higher.

      3. Dilution of standard solutions: From the stock solution, prepare a series of diluted solutions of different concentrations to cover the expected concentration range of the samples. The concentration of the standard solutions usually ranges from 1 ng/μL to 500 ng/μL, depending on the experimental requirements.

   3. Establishment of calibration standards

      1. Calibration curve: Inject different concentrations of the standard solutions into the UHPLC-MS/MS system, record the mass spectrometric response (such as peak area or peak height) for each concentration, and plot a calibration curve. The calibration curve should exhibit good linearity across the measured concentration range to ensure the accuracy of quantitative analysis.

      2. Use of internal standards: To compensate for variability during sample preparation and detection, internal standards (such as isotope-labeled peptides or compounds) may be used. Internal standards can be added to both the sample and the standard solution to correct for experimental errors.

   4. Quality control and validation

      1. Regular calibration: The calibration process should be conducted regularly, especially if the instrument undergoes maintenance or there are changes in experimental conditions. Regular validation of the instrument’s performance ensures consistency and reliability in calibration results.

      2. Use of quality control samples: Quality control (QC) samples can be used to assess the instrument’s performance and ensure that the mass spectrometric response falls within the expected range.

   5. Validation of calibration results

      1. Linear validation: Ensure that the calibration curve maintains a good linear relationship across the entire analytical range (usually requiring an R² greater than 0.99).

      2. Limit of detection (LOD) and limit of quantification (LOQ): Based on the calibration curve, determine the instrument’s LOD and LOQ to ensure that accurate detection results can still be obtained at low concentrations.

   6. Sample loading and separation

      1. Inject 1 μg of peptide dissolved in 0.1% formic acid into an EASY-nLC1200 system using a nanoACQUITY HPLC HHS T3 column (75 μm × 150 mm, 1.8 μm). The mobile phase A is 0.1% formic acid with 2% acetonitrile; mobile phase B is 0.1% formic acid with 90% acetonitrile.

      2. Set the gradient: 0–68 min, 6%–23% B; 68–82 min, 23%–32% B; 82–86 min, 32%–80% B; 86–90 min, 80% B. Maintain the flow rate at 450 nL/min.

   7. Ionization and mass spectrometry

      1. The separated peptides are ionized using an NSI source and analyzed by an Orbitrap Exploris 480 mass spectrometer. Set the capillary temperature to 260 °C and ionization voltage to 2.3 kV. The FAIMS compensation voltage (CV) is set to -45V, -65V.

   8. Mass spectrometry analysis

      1. Both the parent ions and their fragments are detected using the high-resolution Orbitrap. The primary scan range is 400–1,200 m/z with a resolution of 60,000; the secondary scan range starts at 110 m/z with a resolution of 15,000. TurboTMT is off.

   9. Data acquisition

      1. Use data-dependent acquisition (DDA) to select the top 25 precursor ions for fragmentation using HCD with a collision energy of 27% and secondary analysis.

      2. To optimize the use of the mass spectrometer, set the automatic gain control (AGC) to 100%, the signal threshold to 5E4 ions/s, maximum injection time to Auto, and dynamic exclusion to 20 s.

Data analysis

1. Mass spectrometry data analysis

   1. Quality control

      1. Peptide length distribution: Most effective peptides should be between 7 and 20 amino acids in length, with the ideal length being 8–15 amino acids (Figure 2A).

         
         

         
         Figure 2. Quality control. A. Peptide length distribution. The colors represent the amount of charge carried by the peptide. B. Peptide count distribution. C. Protein coverage distribution. The colors represent protein coverage. D. Protein molecular weight distribution.

      
      

      2. Peptide count distribution: At least 2–3 unique peptides should be detected for each protein to ensure reliability and reproducibility (Figure 2B).

      3. Protein coverage distribution: Generally, the protein coverage should exceed 20%–30%, with high-abundance proteins ideally achieving 50% or more (Figure 2C).

      4. Protein molecular weight distribution. A wide range of molecular weights should be covered, typically from 5 to 250 kDa, ensuring the detection of proteins of various sizes within this range (Figure 2D).

      5. Filter data at the spectrum, peptide, and protein levels with an FDR of 1%.

   2. Data processing

      1. Use Proteome Discoverer (v2.4.1.15) to analyze the secondary mass spectrometry data. Set the database to Homo_sapiens_9606_SP_20210721.fasta (20,387 sequences) with a decoy database to calculate the false discovery rate (FDR) and a contaminant database to eliminate common contaminants. Set enzyme to Trypsin (Full) with up to two missed cleavages, minimum peptide length of six residues, and a maximum of three modifications per peptide. Set the precursor mass tolerance to 10 ppm and fragment mass tolerance to 0.02 Da.

      2. The mass tolerance for precursor ions is set to 10 ppm, and to 0.02 Da for fragment ions; the maximum allowed retention time deviation is 10 min.

      3. Search engines choose Sequest HT. Set Carbamidomethyl (C) as a fixed modification; Oxidation (M), Acetyl (N-terminus), Met-loss (M), Deamidated (N, Q), and Met-loss + acetyl (M) as variable modifications. Set FDR for protein, peptide, and PSM identification to 1%.

      4. In the Minora detection process, set the minimum trace length for signal identification to 5, Max ΔRT of Isotope Pattern Multiplets [min] to 0.02, and the confidence of the Peptide-Spectrum Match to HIGH.

      5. Use UNIQUE and RAZOR peptides for quantification based on intensity values. Protein abundance was calculated as summed abundance.




2. Quantitative analysis

   1. Normalization

      Normalize raw mass spectrometry data both row-wise and column-wise. Divide each protein's quantification value by its average value across all samples (row-wise). Then, divide each protein in a sample by the median value of that sample (column-wise) to obtain normalized data for bioinformatics analysis.

Validation of protocol

This protocol or parts of it has been used and validated in the following research article(s):

1. Du et al. [10]. Proteomic Profiling of Muscular Adaptations to Short-Term Concentric Versus Eccentric Exercise Training in Humans. Mol Cell Proteomics (Supplemental Table S2)]

General notes and troubleshooting

General notes

Problem 1: How to recalibrate the gradient and how to optimize the column temperature.

1. Recalibrate the gradient

   1. Check the gradient program: Begin by reviewing the existing gradient program to determine whether it is appropriate for separating the target compounds. If the gradient time or solvent ratio is incorrect, it may lead to retention time shifts or poor separation of the target analytes.

   2. Optimize start and end conditions: Adjust the starting and ending solvent ratios based on the polarity and chemical nature of the sample. For polar compounds, use a higher proportion of the aqueous phase at the start. For hydrophobic compounds, start with more of the organic phase.

   3. Extend or shorten gradient time: If peaks are not well-separated, consider extending the gradient time to allow more time for different compounds to separate. If the peaks are too broad, resulting in lower resolution, shorten the gradient time.

   4. Validate with standards: Use known standard samples to check if the new gradient can effectively separate the target compounds and ensure stable retention times. After adjusting the gradient, record the retention time for each compound and ensure it remains consistent.

2. Optimize column temperature

   1. Determine the optimal temperature range: Typically, column temperature is optimized between 25 °C and 60 °C. Higher temperatures can speed up the analysis process and reduce retention times but may lead to decreased resolution. Lower temperatures, on the other hand, increase resolution but result in longer analysis times.

   2. Monitor peak shape and resolution: Gradually adjust the temperature (e.g., by increasing or decreasing it by 5 °C at a time) and observe changes in peak shape and resolution. Generally, as temperature increases, sample mobility improves, and peaks become narrower. However, excessive temperature increases may lead to peak fronting or peak loss.

   3. Balance analysis time and resolution: Select a temperature that maintains good separation while also not excessively prolonging the analysis time, ensuring sufficient resolution.

   4. Consider sample stability: For heat-sensitive samples, avoid using excessively high column temperatures to prevent sample degradation in the column.

Problem 2: How to increase the sample volume within the linear range of the instrument.

In LC–MS methods, increasing the sample amount must ensure it remains within the linear range of the instrument to avoid signal saturation and ensure quantitative accuracy. The following steps and considerations should be followed when increasing the sample amount:

1. Confirm the instrument's linear range: The linear range of the instrument refers to the range in which the analyte concentration has a linear relationship with the detection signal (e.g., peak area). In this range, the signal increases linearly with the sample concentration. Create a calibration curve by injecting standards at different concentrations to determine the linear range. This curve should cover the expected sample concentrations and typically uses at least five different concentration points.

2. Gradually increase sample amount: If the sample amount needs to be increased, it is recommended to gradually increase the concentration or injection volume while monitoring the signal response. Stop increasing the sample amount before the signal enters the nonlinear range. The sample amount can be increased by injecting a larger volume into the liquid chromatography system, for example, increasing from 10 to 20 μL. However, ensure that the injection volume does not exceed the sample loop capacity.

3. Evaluate signal saturation: While increasing the sample amount, monitor the signal intensity of the mass spectrometer. If the signal intensity starts to show nonlinear growth or approaches the saturation point, reduce the sample concentration or injection volume accordingly. Increasing the sample amount may lead to increased co-eluting substances, causing ion suppression and affecting the detection of analytes. Therefore, closely monitor for ion suppression and adjust sample preparation methods if necessary.

4. Appropriate sample dilution: For high-concentration samples, perform dilution to ensure that the sample concentration falls within the instrument's linear range. The dilution factor should be chosen based on the linear range of the calibration curve. Internal standards can help correct signal deviations that may result from increased sample amounts, ensuring the accuracy of the quantification results.

5. Optimize mass spectrometer parameters: When increasing the sample amount, adjust the ion source parameters of the mass spectrometer (e.g., spray voltage, gas flow, temperature) to prevent ion source overload. By optimizing the scan range or using selected reaction monitoring (SRM) mode, focus on the target analytes and minimize interference from other substances.

6. Maintain good peak shape: Increasing the sample amount may cause peak broadening, affecting the separation. Ensure that the column selection is appropriate, and that flow rate, gradient, and other parameters are optimized to maintain good peak shape. Excessive sample amounts may overload the column, leading to peak broadening or tailing. Monitor the peak shape when increasing the sample amount to ensure the column is not overloaded.

7. Repeat experiments and validation: After each increase in sample amount, repeat the experiment to ensure consistency and reproducibility, avoiding random deviations. After increasing the sample amount, validate the calibration curve again to confirm that the linear range and detection limits remain unaffected.

Troubleshooting

Problem 1: Poor chromatographic separation.

Possible cause: Incorrect solvent composition or gradient program.

Solution: Double-check the solvent preparation and gradient program. Ensure that solvent bottles are correctly connected and that the mobile phase composition matches the method. If the problem persists, consider recalibrating the gradient and optimizing the column temperature.

Problem 2: Low signal intensity in MS.

Possible cause: Insufficient sample loading.

Solution: Increase the sample injection volume within the linear range of the instrument. Ensure that samples are properly dissolved in the appropriate solvent. Check for clogs or blockages in the sample introduction system and clean or replace them as necessary.

Problem 3: Poor ionization efficiency.

Possible cause: Contaminated ion source or electrospray emitter.

Solution: Clean the ion source and electrospray emitter according to the manufacturer's instructions. Use appropriate solvents for cleaning to avoid damaging sensitive components. Regularly perform maintenance procedures recommended by the instrument manual to ensure optimal ionization conditions.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (82071413, 82271438). This protocol was adapted and modified from Du et al. [10].

Competing interests

The authors declare no competing interests.

Ethical considerations

This protocol was conducted according to the Declaration of Helsinki's ethical principles. The local ethics committee of Sports Science Experimental Ethics Committee at Beijing Sport University approved the study (Permission number: 2020148H).

References

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