In the 1980s, with the emergence of electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI)—two soft ionization techniques suitable for protein research—mass spectrometry became a highly important tool for the identification of biological macromolecules. Currently, mass spectrometry is widely used in various fields, including the identification of molecular markers, bacterial identification, protein expression in tissues and whole organisms, and protein post-translational modifications.

Tandem mass spectrometry (MS/MS) can directly determine the amino acid sequence of proteins with high reliability. Peptides generated by trypsin digestion of proteins are introduced into the mass spectrometer. The first-stage mass spectrometry measures the molecular weights of the peptides. The peptides are then fragmented to generate a series of ions, including the N-terminal ion series (b-series) and the C-terminal fragment ion series (y-series). The mass spectrometer then detects the masses of these fragment ions in the second-stage mass spectrometry.

Experimental Steps

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Experimental Protocol for In-Gel Protein Digestion

1. Cut the target protein spot into small pieces of approximately 1–2 mm in size. (Do not grind or crush, as fine particles may block the capillary LC column.)

2. Transfer the gel pieces into a 1.5 mL microcentrifuge tube.

3. Add 20 μL of wash buffer and gently wash the gel pieces at room temperature overnight.

4. Carefully remove the wash buffer using a pipette.

5. Add 200 μL of wash buffer and wash the gel pieces at room temperature for 2–3 hours.

6. Carefully remove the wash buffer using a pipette.

7. Add 200 μL of acetonitrile and dehydrate at room temperature for approximately 5 minutes.

8. Carefully remove the acetonitrile using a pipette.

9. Dry under vacuum centrifugation at room temperature for 2–3 minutes.

10. Add 30 μL of 10 mmol/L DTT solution and reduce the proteins at room temperature for 0.5 hours.

11. Carefully remove the DTT solution using a pipette.

12. Add 30 μL of 10 mmol/L iodoacetamide and alkylate the proteins at room temperature for 0.5 hours.

13. Carefully remove the iodoacetamide solution using a pipette.

14. Add 200 μL of acetonitrile and dehydrate at room temperature for approximately 5 minutes.

15. Carefully remove the acetonitrile using a pipette.

16. Add 200 μL of 100 mmol/L ammonium bicarbonate solution and incubate at room temperature for 10 minutes.

17. Carefully remove the ammonium bicarbonate solution using a pipette.

18. Add 200 μL of acetonitrile and dehydrate at room temperature for approximately 5 minutes.

19. Carefully remove the acetonitrile using a pipette.

20. Dry under vacuum centrifugation at room temperature for 2–3 minutes.

21. Prepare trypsin reagent by adding 1000 μL of ice-cold 50 mmol/L ammonium bicarbonate and 20 μg of trypsin. The final trypsin concentration is 20 ng/μL.

22. Keep on ice until use.

23. Add 30 μL of the trypsin solution to the sample.

24. Allow the sample to rehydrate on ice for 10 minutes, with occasional vortex mixing.

25. Centrifuge for 30 seconds.

26. Remove excess trypsin solution.

27. Add 5 μL of 50 mmol/L ammonium bicarbonate, vortex to mix.

28. Centrifuge for 30 seconds.

29. Incubate at 37°C for overnight digestion.

30. Add 5 μL of 50 mmol/L ammonium bicarbonate, incubate for 10 minutes with occasional gentle vortex mixing.

31. Centrifuge for 30 seconds.

32. Collect the supernatant.

33. Add 30 μL of extraction buffer, incubate for 10 minutes with occasional gentle vortex mixing.

34. Centrifuge for 30 seconds.

35. Collect the supernatant and combine with the extract from the previous step.

36. Add 30 μL of extraction buffer to the tube containing the gel pieces, incubate for 10 minutes with occasional gentle vortex mixing.

37. Centrifuge for 30 seconds.

38. Collect the supernatant and combine with the extract from the previous step.

39. Evaporate the combined extract in a vacuum centrifuge at room temperature until the volume is less than 20 μL.

Featured Products

J&K Scientific offers reagents, pipettes, and consumables for protein mass spectrometry identification.

Wash Buffer：

• 949300 Methanol, 99.5%, for analysis CAS:67-56-1
• 991956 Acetic acid, 99.5%, for analysis CAS:64-19-7

Ammonium Bicarbonate Solution：

• 430860 Ammonium bicarbonate, for synthesis, 21 - 22% NH₃ CAS:1066-33-7

DTT Solution:

• 963376 DL-Dithiothreitol, 98%, for bio CAS:3483-12-3

Iodoacetamide Solution:

• 519692 Iodoacetamide, 98%, reagent grade CAS:144-48-9

1% TFA Solution：

• 252705 Trifluoroacetic acid, 99% CAS:76-05-1

LC-MS/MS Reagents：

• 923394 Formic acid, 88%, for analysis CAS：64-18-6
• 925301 Acetonitrile, 99.9%, HPLC/PREP CAS：75-05-8

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