Materials and Reagents

Seven-day old protonemata of the moss P. patens
Liquid nitrogen
Tris-HCl (pH 7.5)-saturated phenol (AMRESCO, catalog number: K168 )
Ammonium acetate (Sigma-Aldrich, catalog number: A1542 )
Methanol (Sigma-Aldrich, catalog number: 34860 )
DTT (Promega Corporation, catalog number: V3155 )
Acetone (Sigma-Aldrich, catalog number: 650501 )
Iodoacetamide (Sigma-Aldrich, catalog number: I6125 )
Ammonium bicarbonate (pH 8.5) (Sigma-Aldrich, catalog number: 40867 )
Trypsin (Promega Corporation, catalog number: V5111 )
Sucrose (Sigma-Aldrich, catalog number: S5390 )
Tris (Sigma-Aldrich, catalog number: T1378 )
EDTA (Sigma-Aldrich, catalog number: E6635 )
1,4-dithiothreitol (DTT) (Promega Corporation, catalog number: V3155)
Protease inhibitors cocktail (Sigma-Aldrich, catalog number: P9599 )
Phosphatase inhibitors cocktail 2 (Sigma-Aldrich, catalog number: P0044 )
NH₄OH (Sigma-Aldrich, catalog number: 320145 )
Urea (Sigma-Aldrich, catalog number: U6504 )
CHAPS (Sigma-Aldrich, catalog number: C9426 )
Acetonitrile (ACN) (Sigma-Aldrich, catalog number: 34851 )
Trifluoroacetic acid (TFA) (Sigma-Aldrich, catalog number: 302031 )
Glutamic acid (Sigma-Aldrich, catalog number: G0355000 )
Protein extraction buffer (see Recipes)
Protein resuspension buffer (see Recipes)
Loading buffer (see Recipes)
Washing buffer I (see Recipes)
Washing buffer II (see Recipes)
Elution buffer (see Recipes)

Equipment

Porcelain Mortar and Pestle (90 mm)
Tubes and tube holder (2 ml)
Titanium dioxide micro-columns (320 µm x 50 mm) (Column Technology, Freemont)
Centrifuge
Vortex
Freeze dryer (Christ, model: Alpha 1-4 LSC )

Procedure

This procedure includes proteins extraction, protein digestion and phosphopeptide purification. The following should all be carried out at cold room temperature except for special instructions.

Add protease inhibitor mix (protease inhibitors and phosphatase inhibitors) to the ice-cold protein extraction buffer according to the manufacturer’s instructions.
Pour liquid nitrogen into mortar until nearly full in order to pre-cool the mortar and pestle.
Add the fresh or frozen (-80 °C freezer) tissue (about 1 g) into mortar, grind them to fine powder in liquid nitrogen, and then thaw on ice for 10-15 min.
Add 2 ml ice-cold protein extraction buffer into mortar, homogenize by grinding on ice for 10 min, and then transfer 0.8 ml to each tube.
Add an equal volume of ice-cold Tris-HCl (pH 7.5)-saturated phenol to each tube, vortex tubes for 5-10 min.
Centrifuge samples at 13,000 rpm for 15 min at 4 °C.
Collect phenol phase and mixed with three volumes of 100 mM ammonium acetate in methanol.
Precipitate proteins from mixture of phenol phase and 100 mM ammonium acetate overnight at -20 °C.
Centrifuge proteins at 13,000 rpm for 15 min at 4 °C.
Rinse proteins 3 times with ice-cold acetone containing 13 mM DTT and then lyophilize by freeze dryer for 5-10 min.
The protein concentration was determined according to Peterson (1977) using BSA as a standard.
Dissolve 500 µg protein power in 100 µl protein resuspension buffer at room temperature.
Reduce protein (500 µg protein in 100 µl resuspension buffer) with 20 mM DTT at 37 °C for 2.5 h and alkylate protein with 40 mM iodoacetamide for 40 min at room temperature in the dark.
Precipitate the protein mixture with 1 ml 100% acetone overnight at -20 °C.
Collect protein by centrifugation, dissolve protein in 100 mM ammonium bicarbonate (pH 8.5), and digest protein with trypsin (50:1 w/w, protein to trypsin ratio) at 37 °C for 16 h.
Lyophilize the digested peptide mixture, and then dilute in a 200 µl loading buffer.
Rinse the titanium dioxide micro-columns (TiO₂ columns, 320 µm x 50 mm) with 100 µl loading buffer.
Load the 200 µl samples onto TiO₂ columns by applying air pressure created with a plastic syringe.
Wash the TiO₂ columns with 100 µl loading buffer, 100 µl washing buffer I, and finally with 100 µl washing buffer II.
Elute the columns which bound phosphopeptides with 100 µl of elution buffer.
Pool and lyophilize the eluates which is purification of phosphopeptides [Representative data is shown in Wang et al. (2014a) and Wang et al. (2014b)].

Recipes

Protein extraction buffer
250 mM sucrose
20 mM Tris-HCl (pH 7.5)
10 mM EDTA 1 mM 1,4-dithiothreitol
Protease inhibitors cocktail
Phosphatase inhibitors cocktail 2
Protein resuspension buffer
8 M urea
4% CHAPS
65 mM DTT
40 mM Tris-HCl (pH 7.5)
Loading buffer
65% ACN and 2% TFA solution saturated with glutamic acid
Washing buffer I
65% ACN
0.5% TFA
Washing buffer II
65% ACN
0.1% TFA
Elution buffer
300 mM NH₄OH
50% ACN

Acknowledgments

This work was supported by grants from Project of Ministry Science & Technology of China (2013CB967300, 2007CB948201) to Prof. He, grant from National Natural Science Foundation of China (No. 31371243, 30970250), China Postdoctoral Science Foundation and State Education Ministry Scientific Research Foundation for the Returned Overseas Chinese Scholars to Dr. Wang.

References

Peterson, G. L. (1977). A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem 83(2): 346-356.
Wang, X., Qi, M., Li, J., Ji, Z., Hu, Y., Bao, F., Mahalingam, R. and He, Y. (2014a). The phosphoproteome in regenerating protoplasts from Physcomitrella patens protonemata shows changes paralleling postembryonic development in higher plants. J Exp Bot 65(8): 2093-2106.
Wang, X. Q., Yang, P. F., Liu, Z., Liu, W. Z., Hu, Y., Chen, H., Kuang, T. Y., Pei, Z. M., Shen, S. H. and He, Y. K. (2009). Exploring the mechanism of Physcomitrella patens desiccation tolerance through a proteomic strategy. Plant Physiol 149(4): 1739-1750.
Wang, X., Zhou, S., Chen, L., Quatrano, R. S. and He, Y. (2014b). Phospho-proteomic analysis of developmental reprogramming in the moss Physcomitrella patens. J Proteomics 108: 284-294.