Materials and Reagents

1.5 ml tubes (Corning, Axygen^®, catalog number: MCT-150-C )
ZipTip^® pipette tip (Merck Millipore, catalog number: ZTC18S096 )
Pierce^TM Spin columns, screw cap (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 69705 )
C18 (FUJIGEL HANBAI, catalog number: MB 100 - 40/75 )
Tris (2-carboxyethyl) phosphine hydrochloride solution, 0.5 M, pH 7.0 (TCEP) (Sigma-Aldrich, catalog number: 646547 )
Formic acid (Sigma-Aldrich, catalog number: 94318 )
Acetonitrile (Sigma-Aldrich, catalog number: 34851 )
RapiGest SF surfactant (WATERS, catalog number: 186001861 )
Iodoacetamide (Sigma-Aldrich, catalog number: I1149 )
Trypsin (Roche Diagnostics, catalog number: 11418025001 )
Potassium phosphate dibasic trihydrate (K₂HPO₄·3H₂O)
Potassium phosphate monobasic (KH₂PO₄)
Phosphoric acid (H₃PO₄)
Ammonium chloride (Sigma-Aldrich, catalog number: A9434 )
Hydrochloric acid (HCl)
0.5 M iodoacetamide stock (see Recipes)
0.1 μg/μl trypsin stock (see Recipes)
50 mM potassium phosphate buffer, pH 7.8 (see Recipes)
100 ml solvent A (5 mM NH₄Cl, 25% [v/v] acetonitrile, pH 3.0) (see Recipes)
100 ml solvent B (500 mM NH₄Cl, 25% [v/v] acetonitrile, pH 3.0) (see Recipes)

Equipment

2 μl, 10 μl, 100 μl, 1,000 μl Pipetman (Gilson, France)
Optimal MAX-XP ultracentrifuge (Beckman Coulter, model: Optimal MAX-XP )
AKTA purifier-10 (GE Healthcare, model: AKTApurifier 10 )
ISS 110 SpeedVac System (Thermo Fisher Scientific, Thermo Scientific^TM, model: ISS 110 )
PolySULFOETHYL A^TM columns, 2.1 x 200 mm, 5 μm particles, 300 Å poresize (PolyLC, catalog number: 202SE05 )

Software

UNICORN 5.2 software (GE Healthcare, USA)

Procedure

Plasma membrane protein preparation
1. Suspend the plasma membrane (PM) pellets (isolated according to Han et al., 2010) with 100 μl 50 mM potassium phosphate buffer, pH 7.8.
  Note: The recommended PM protein concentration is 1 μg/μl. This protocol set 100 μg PM protein as an example, so use 100 μl buffer to suspend the pellets.
2. Add 25 μl 1% (w/v) RapiGest SF stock to the working concentration of 0.2% (w/v), mix well and then boil the mixture at ~100 °C for 5 min to dissolve proteins from PM.
  Note: RapiGest SF is an acid labile denaturant (Figure 1A) which could solubilize and unfold proteins to make them more amenable for cleavage (Figure 1B). The recommended RapiGest SF working concentration is 0.1% (w/v). For PM proteins increase the incubation time (at 37 °C, up to 1 h is safe; at 100 °C, 5 min is enough) or use higher RapiGest SF concentration (up to 0.5% [w/v] is safe) could make the following digestion more efficient.
  
  Figure 1. Working principle of RapiGest SF surfactant. A. RapiGest SF can be easily broken down by acidification and then removed by centrifugation. B. RapiGest SF is a mild denaturant which making proteins more amenable for cleavage without disrupting the endoproteases activity. Pictures were downloaded from the official website of Waters: (http://www.waters.com/waters/en_US/RapiGestSFSurfactant/nav.htm?locale=/&cid=1000941)
3. Cool down the mixture to room temperature (RT) for the following digestion.
Plasma membrane protein digestion
1. Reduction: add 2.5 μl 0.5 M TCEP, pH 7.0 to the protein mixture to a working concentration of 10 mM, mix them gently and then incubate at 56 °C for 1 h.
2. Alkylation: cool down the mixture to RT, and add 14 μl 0.5 M iodoacetamide stock to a working concentration of 50 mM, mix them gently and then incubate at RT, dark, for 45 min.
3. Digestion: add 20 μl 0.1 μg/μl trypsin stock to the protein mixture to a working concentration of 1:50 (w/w), mix them gently and then incubate at 37 °C for 12 h.
4. Acidification: add 0.5 μl formic acid to the mixture to a working concentration of 0.2-0.5% (v/v) (pH < 2), incubate at 37 °C for 30-45 min, then centrifuge at 4 °C, 20,000 x g for 20 min to remove RapiGest SF. Discharge the pellets, transfer the supernatant to a new tube.
5. Desalting: use C18 filled spin column to concentrate and purify the peptides mixture.
  Note: If the peptides mixture is less than 5.0 μg, please use ZipTip^® pipette tip to do the desalting.
6. Put the purified peptides mixture into SpeedVac system to lyophilize for the following fractionation use.
Peptides mixture fractionation
The following protocol was modified from Zhu et al. (2009). It performs on the AKTA purifier-10 system with the strong cation exchange (SCX) PolySULFOETHYL A^TM column.
1. Resuspend the lyophilized peptides mixture in solvent A.
2. Run solvent B for 20 min, then solvent A for 30 min through the AKTA purifier-10 system to balance the PolySULFOETHYL A^TM column.
3. Load the suspended peptides and run the following gradient at a flow rate of 200 μl/min, collect the products in every 2 min.
  0-5 min: 100% solvent A
  5-95 min: 0-60% solvent B
  95-115 min: 60%-100% solvent B
  115-145 min: 100% solvent B
  145-155 min: 100% solvent A
4. Run ddH₂O for 20 min, then 100% acetonitrile for 10 min to store the column.
5. Pool the products according to the 214 nm UV absorption peak (Figure 2).
  
  Figure 2. SCX fractionation of peptides mixture from rice mature pollen grain. According to the 214 nm UV absorption peak, the peptides mixture can be pooled into 14 fractions for LC-MS/MS analysis.
6. Use C18 spin column or ZipTip^® pipette tip to purify the combined products.
7. Lyophilize the purified products for mass spectrometry identification.

Data analysis

The SCX separated peptides were monitored by their 214 nm UV absorption peak through the UNICORN 5.2 software (GE Healthcare, USA).

Notes

After SCX fractionation, the repooled peptides contains a high concentration of salts. Don’t forget to do the desalting procedure through C18 spin column or ZipTip^® pipette tip, or the following mass spectrometry identification will be affected.

Recipes

1% (w/v) RapiGest SF stock
Dissolve 1 mg of lyophilized RapiGest SF powder in 100 μl ddH₂O, store at -20 °C
0.5 M iodoacetamide stock
Dissolve 46 mg of iodoacetamide powder in 500 μl ddH₂O, use immediately
0.1 μg/μl trypsin stock
Dissolve 25 μg of trypsin powder in 250 μl ddH₂O, store at -20 °C
200 ml 50 mM potassium phosphate buffer, pH 7.8
2.072 g K₂HPO₄·3H₂O
0.125 g KH₂PO₄
Fill up to 200 ml with ddH₂O
Adjust pH to 7.8 with H₃PO₄
100 ml solvent A (5 mM NH₄Cl, 25% [v/v] acetonitrile, pH 3.0)
0.027 g NH₄Cl
25 ml acetonitrile
Fill up to 100 ml with ddH₂O
Adjust pH to 3.0 with HCl
100 ml solvent B (500 mM NH₄Cl, 25% [v/v] acetonitrile, pH 3.0)
2.675 g NH₄Cl
25 ml acetonitrile
Fill up to 100 ml with ddH₂O
Adjust pH to 3.0 with HCl

Acknowledgments

This protocol was mainly modified from Han et al. (2010) and Yang and Wang (2017). This work was supported by the Chinese Ministry of Science and Technology (grant No. 2013CB945101) and the China Postdoctoral Science Foundation (grant No. 2016M591284).

References

Alexandersson, E., Gustavsson, N., Bernfur, K., Karlsson, A., Kjellbom, P. and Larsson, C. (2008). Purification and proteomic analysis of plant plasma membranes. Methods Mol Biol 432: 161-173.
Han, B., Chen, S., Dai, S., Yang, N. and Wang, T. (2010). Isobaric tags for relative and absolute quantification- based comparative proteomics reveals the features of plasma membrane-associated proteomes of pollen grains and pollen tubes from Lilium davidii. J Integr Plant Biol 52(12): 1043-1058.
Wu, C. C. and Yates, J. R., 3rd (2003). The application of mass spectrometry to membrane proteomics. Nat Biotechnol 21(3): 262-267.
Yang, N. and Wang, T. (2017). Comparative proteomic analysis reveals a dynamic pollen plasma membrane protein map and the membrane landscape of receptor-like kinases and transporters important for pollen tube growth and interaction with pistils in rice. BMC Plant Biol 17(1): 2.
Yu, Y. Q., Gilar, M., Lee, P. J., Bouvier, E. S. and Gebler, J. C. (2003). Enzyme-friendly, mass spectrometry-compatible surfactant for in-solution enzymatic digestion of proteins. Anal Chem 75(21): 6023-6028.
Zhang, X. (2015). Less is more: Membrane protein digestion beyond urea-trypsin solution for next-level proteomics. Mol Cell Proteomics 14(9): 2441-2453.
Zhu, M., Dai, S., McClung, S., Yan, X. and Chen, S. (2009). Functional differentiation of Brassica napus guard cells and mesophyll cells revealed by comparative proteomics. Mol Cell Proteomics 8(4): 752-766.