GB/T 42699.2-2023Textiles - Qualitative and quantitative proteomic analysis of some animal hair fibres - Part 2: Peptide detection using MALDI-TOF-MS (English PDF)

6.4 Sodium dihydrogen phosphate solution (

0.2 mol/L). -- Sodium dihydrogen phosphate 24 g Dissolve in water to a volume of 1 L.

6.5 Disodium hydrogen phosphate solution (

0.2 mol/L). -- Disodium hydrogen phosphate

28.4 g Dissolve in water to a volume of 1 L.

6.6 Phosphate buffer solution (pH=7.8,

0.2 mol/L). --

0.2 mol/L sodium dihydrogen phosphate solution (6.4) --

0.2 mol/L disodium hydrogen phosphate solution (6.5) 100 mL Add

0.2 mol/L sodium dihydrogen phosphate solution (6.4) to

0.2 mol/L disodium hydrogen phosphate solution (6.5) and adjust the pH of the solution to 7.8.

6.7 Sodium dodecyl sulfonate (SDS), with a purity of >=99.5%.

6.8 Dithiothreitol (DTT), with a purity of >=97%.

6.9 SDS buffer solution. --

0.2 mol/L phosphate buffer solution (pH=7.8) (6.6) 50 mL

7.7 Pipette tip. The tip of the pipette is equipped with chromatography media for concentrating and purifying samples.

7.8 MALDI-TOF mass spectrometer. It is used for the detection of the relative molecular mass of peptides.

7.9 Centrifuge tube.

1.5 mL; it is capable of withstanding speeds of 6500 g and above. 7.10 96-well microplate. A plate with 96 wells made of inert material, suitable for micro-volume applications such as storage, and sample or reagent transfer.

7.11 pH meter. with an accuracy of 0.01.

7.12 Centrifuge. with a speed of 6500 g and above.

8.1 Sample preparation

8.1.1 Cut a representative and sufficient sample into small pieces with scissors and place (12.5±2.5) mg of the specimen into a

1.5 mL centrifuge tube (7.9) containing

0.25 mL of SDS buffer solution (6.9) and 6 grinding balls.

8.1.2 The fibre specimen is ground for 30 minutes in a ball mill (7.1) at a power of

8.2.1 Add

0.25 mL of the extraction solution (6.10) to a centrifuge tube (7.9) using a micropipette (7.6) and mix using a vortex mixer (7.3).

8.2.2 Place the centrifuge tube containing the specimen in a metal bath (7.2) at 95 °C for 15 min.

8.2.3 Centrifuge at 6500 g for 1 min at room temperature. Add 10 µL of DTT solution (6.12) and then react in a metal bath at 95 °C for 15 min.

8.2.4 At room temperature, add 50 µL of IAA solution (6.11) and react for 15 min to alkylate the protein and prevent the formation of disulfide bonds.

8.2.5 Add 20 µL of DTT solution (6.12) to terminate the reaction.

8.2.6 Centrifuge at 6500 g for 5 minutes at room temperature, and transfer the supernatant to another reaction tube to obtain the protein extract for later use.

8.3 Partial purification of extracted proteins using SDS-PAGE

8.3.1 The protein extract (8.2.6) is purified by SDS-PAGE electrophoresis using a small-sized polyacrylamide gel (6.13) and a small protein electrophoresis apparatus (7.4) to remove proteins with excessively large or small molecular weights.

8.3.2 Dilute the protein extract (8.2.6) 3 times with the sample loading buffer (6.18), and add 5 µL~10 µL of the diluted sample solution into the wells of a small-sized polyacrylamide gel (6.13).

8.3.3 After electrophoresis for 15 minutes, remove the gel. Stain with Coomassie Brilliant Blue (6.19).

8.3.4 The protein bands are concentrated in three regions. upper, middle and lower (see Figure A.1). Use a gel cutter or blade (7.5) to cut and recover the middle band with a diameter of about 1 mm (about 45,000 Daltons) (see the example in Appendix A).

8.3.5 Place the gel in a 96-well microplate (7.10), wash with 100 µL of washing buffer (6.23) for 10 min, and repeat the washing twice for 1 min each time to remove Coomassie Brilliant Blue. Add 100 µL of acetonitrile (6.22) to wash the gel, remove the acetonitrile after 1 min, and air-dry the gel for (10±5) min.

8.4 Trypsin digestion of proteins

8.4.1 After the gel dries, add 35 µL of trypsin solution (6.27) and incubate in a water bath at 50 °C for 1 h.

8.4.2 Purify the tryptic digest using a pipette tip with chromatography medium (7.7), elute with 1.5 µL of matrix (6.30), and transfer to a target plate for MALDITOF-MS.

8.5 MALDI-TOF-MS analysis of peptides

8.5.1 Analyze peptides using the reflectance or linear mode of MALDI-TOF-MS (7.8). Set the laser energy to the threshold level to generate a signal, and the accelerating voltage to 20 kV.

8.5.2 The mass spectrometry scanning frequency shall be at least

50 Hz. The values at three different locations shall be measured at each spot on the target plate, and the average of the three measurements shall be taken to ensure the repeatability of the experiment.

8.5.3 Characteristic peaks for animal species appear in the range of 2450 m/z~2750 m/z. Examples of characteristic peaks for different species are provided in Appendix B and Appendix C. Before quantitatively analyzing animal hair fibres, the species of animal hair fibres in the specimen must be determined based on the characteristic peaks.