Isolation and characterization of membrane proteins and their functional analysis-GFC/IEC/SDS-PAGE and MALDI-TOF-MS experiments
Prior to the identification of membrane proteins by matrix-assisted laser-resolved time-of-flight mass spectrometry (MALDI-TOF-MS), these proteins need to be separated for mass spectrometry analysis. After solubilization of membrane samples with the non-denaturing detergent dodecyl-β-D-maltoside, membrane proteins can be separated by ion-exchange chromatography (IEC) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This experiment was derived from the "Guide to Plant Proteomics Experiments" [France], H. Thielement, M. Zivi, C. Damerweil, and V. Michen, eds.
Operation method
Isolation, characterization and functional analysis of membrane proteins by GFC/IEC/SDS-PAGE and MALDI-TOF-MS methods
Materials and Instruments
Separation Buffer Solubilization Buffer Elution Buffer Laemmli Sample Buffer Segment Rinse Buffer Enzymatic Buffer Peptide Extraction Buffer Move 3.1 Membrane Separation For more product details, please visit Aladdin Scientific website.
Because the membrane components to be separated are mixed with soluble cytoplasmic proteins in the form of small vesicles when the material is milled, it is desirable to descale these cytoplasmic proteins before separating and characterizing the membrane proteins. To accomplish this, we need to separate the membrane components with a descaler solution that can penetrate these vesicles to a critical microcellular concentration (CMC) (see Note 1), allowing the encapsulated hydrophilic proteins to slowly diffuse outward. The solubilization buffer also contains a dissociation salt and a divalent metal chelator to dissociate proteins loosely bound to the membrane perimeter. The experimental procedure described here for isolating plant plasma membranes is shown in Figure 22-1.
( 1 ) Suspend 0.5-5 mg of the membrane fraction in the separation buffer (concentration: 0.2 mg/ml) and mix well at 4°C with stirring.
( 2 ) The test time can be up to 4 hours.
( 3 ) After treatment, centrifuge the membrane suspension at 4°C, 100000 g for 1h.
( 4 ) Determine the protein content of the supernatant and precipitate.
( 5 ) Analyze the bands of membrane peripheral protein fraction (supernatant) and membrane inlay protein fraction (precipitate) on SDS-PAGE.
3.2 Protein solubilization
Separation of membrane mosaic proteins first requires the use of desolvating agents to break their natural lipid environment [ 8, 9]. In the method presented here, we use neutral or amphoteric descaling agents because they do not modify the charge of the proteins isolated by IEC. Moreover, these two descaling agents are mild and maintain the natural conformation and activity of the protein. 
( 1 ) Solubilize 0.5-5 mg of the isolated membrane fraction with 1 ml of solubilization buffer at room temperature.
( 2 ) Add 1 ml of DM solution to the solubilization buffer to achieve a DM/protein ratio (m/m) of 3~7 (see Note 2).
( 3 ) Mix by vortexing for 2 min, stirring at low speed at room temperature, and solubilize for 2 hours.
( 4 ) Centrifuge at 190000 g for 1 h at room temperature.
( 5 ) Quantitatively analyze the protein in the supernatant and precipitate to obtain the solubilization yield.
( 6 ) Analyze the bands of solubilized proteins (supernatant) and insolubilized proteins (precipitate) on SDS-PAGE.
After these steps, SDS-PAGE profiles of solubilized proteins can be obtained that are very similar to the initial PM proteins (Figure 22-2), and about 85% of the initial proteins can be solubilized by the above experimental procedure.
3.3 Chromatography
1. IEC/SDS-PAGE separation
Anion-exchange chromatography chromatography (A EC, see Note 3) was performed using a Mono Q HR 5/5 column (Pharmacia/Amersham Biosciences) with protein elution monitored by 280 nm absorption throughout the chromatographic process and a fraction collection volume of 0.3 ml. The descaling agent in the sample and elution buffer tends to form bubbles in the piping system, so careful sonication (30 min ) of the buffer and adjustment of the elution rate (0-1 ml/min) are recommended. The sample and the descaling agent in the elution buffer tend to form bubbles in the piping system; therefore, careful sonication (30 min ) of the buffer and adjustment of the elution rate (0.5-1 ml/min) are recommended. 
( 1 ) Equilibrate the column with 15 ml of Elution Buffer at room temperature.
( 2 ) Up-sample the newly solubilized protein (0.5~2 mg protein, 2 ml solution) onto the chromatography column, and when the fractions are collected, rinse the column with 5 ml elution buffer.
( 3 ) Elute the proteins with a 10 to 15 ml linear salt gradient (0 to 1 mol/L NaCl salt concentration in elution buffer).
( 4 ) Wash the column with 5 ml of elution buffer containing 1 mol/L NaCl to wash out any residual sample from the column.
( 5 ) Add cold acetone [ -20°C, 80% ( V/V) ] to the collected fractions and leave overnight at -20°C to precipitate proteins.
( 6 ) Centrifuge at 17000 g for 30 min at 4°C in a microcentrifuge.
( 7 ) Add 50 μl of Laemmli Sample Buffer to the protein sample precipitated with acetone as described above, and resuspend the precipitated proteins by vortexing and ultrasonic shaking (15 min in an ultrasonic water bath).
( 8 ) Separate the protein bands by SDS-PAGE.
A representative IEC/SDS-PAGE method (starting from the solubilization of 0.5 mg of yeast plasma membrane) is shown in Figure 22-3A. We can see that 3-6 consecutively collected fractions contain the same band, and a protein can be identified from 70% of the electrophoretic bands by MALDI-TOF/MS, and 2 or 3 proteins can be identified from 5%-25% of the bands (Figure 22-3B). MALDI-TOF/MS could identify one protein from 70% of the bands and two or three proteins from 5%~25% of the bands (Figure 22-3B). The data were analyzed by comparing SDS-PAGE lanes of anion-exchange chromatography (AEC) fractions eluted from the same gradient in consecutive rows of different treatments. 
2. GFC/AEC/SDS-PAGE Separation
Separation of complex protein mixtures, such as membrane crude extracts, requires two consecutive chromatographic steps. Therefore, a larger amount (~5 mg) of solubilized protein sample must be obtained. This also facilitates the discrimination of low abundance proteins on the SDS-PAGE lane. Since molecular sieve chromatography (GFC) dilutes the sample, it is used in the first step of chromatography, and the diluted sample after GFC chromatography is concentrated by ion exchange chromatography (IEC) in the second step. The following is a GFC chromatography experiment on an Arabidopsis membrane solubilized protein using a Superdex 200 Highload 16/60 column (see Note 5).
( 1 ) Equilibrate the GFC column with 180 ml of elution buffer at a flow rate of 1 ml/min at room temperature.
( 2 ) Inject the newly solubilized protein sample onto the column.
( 3 ) Elute the protein with 180 ml of elution buffer (0.5 ml/min) and set a collection fraction volume of 0.3 ml.
( 4 ) Combine neighboring fractions that contain less than 500 μg of protein (amount estimated from 280 nm absorption) (see Note 6).
( 5 ) Take a 100 μg sample of the GFC separation fraction, add cold acetone [-20°C, 80% (V/V)], and leave overnight at -20°C to precipitate the protein.
( 6 ) Centrifuge at 17000 g for 30 min at 4°C in a microcentrifuge.
( 7 ) Add 50 μl of Laemmli Sample Buffer to the protein sample precipitated with acetone as described above, vortex and shake, and ultrasonicate (15 min in an ultrasonic water bath) to resuspend the precipitated protein.
( 8 ) Separate the proteins of the GFC-eluted fraction by SDS-PAGE as described above.
( 9 ) A sample of the GFC-eluted fraction (containing at least 500 μg of protein) was subjected to the second step of AEC chromatography using Mono Q HR 5/5 (Pharmacia/Amersham Biosciences) as described above.
After separation of the membrane crude extract by GFC/SDS-PAGE, a more complex protein profile was obtained, especially in the 40-70 kDa region (Fig. 22-4). When separated by GFC/AEC/SDSPAGE, a simpler protein profile is obtained (Figure 22-5), which is easy to identify by MALDI-TOF/MS (the cut protein bands contain less protein). 
3.4 Tryptic in-gel protein digestion
( 1 ) Cut off the strips stained with Caulmers Brilliant Blue.
( 2 ) Rinse the cut pellet with 2 ml (in a 2 ml centrifuge tube) of gel wash solution until the color is completely removed. In general, two 4-h decolorizing rinses can completely remove the color of the pellet.
( 3 ) Dehydrate the discolored pellets in pure acetonitrile solution for 1 hour.
( 4 ) Dry the pellet completely (30 min) in a vacuum desiccator.
( 5 ) Prepare fresh trypsin solution (0.02 μg/μl) with digestion buffer at 4°C to prevent self-digestion of the enzyme and set aside on ice.
( 6 ) Place the dried pellet in a small centrifuge tube, drop in 15 μl of trypsin solution, insert the tube into ice, and hydrate the pellet. 30 min later, there is only a thin layer of trypsin solution on the pellet. After the initial droplet of trypsin has been completely absorbed by the pellet, 15 mmol/L ammonium bicarbonate buffer is added to coat the pellet with a thin layer of ammonium bicarbonate. If the initial addition of trypsin solution is not completely absorbed by the pellet, aspirate the excess trypsin solution so that only a thin layer of trypsin surrounds the pellet (see Note 7).
( 7 ) Seal the centrifuge tube containing the pellet and hold at 37°C for at least 5 h, or overnight.
( 8 ) Add 150 μl of Peptide Extraction Buffer and hold at 20°C for 3 hours.
( 9 ) Remove the pellet and concentrate the peptide extract in a 500 μl tube to 5 μl using a vacuum desiccator; do not dry the peptide extract completely, as the peptide will be adsorbed on the wall of the tube and will not be washed off.
3.5 MALDI-TOF/MS
After the generic steps described in this book (see Chapter 19), the proteins will be crystallized on the target, as briefly described below:
( 1 ) Freshly prepare α-cyano-4-hydroxycinnamic acid from a semi-saturated 1:1 acetonitrile/water solution and acidify with 0.1% TFA.
( 2 ) Mix 0.8 μl of protein sample with 0.8 μl of substrate and spot sample onto the target immediately.
( 3 ) Allow the sample to dry and crystallize on the target.
( 4 ) Rinse the target with ultrapure water and let it dry.
When the spectrum is stabilized, read the spectrum (200~300 laser blasts, see Note 8).
3.6 Database Search
Many different search engines are now available for the identification of proteins for which peptide masses have been obtained, and ProFound [10] can be well used for the identification of mixed samples of proteins (up to 4 proteins). In our analyses, mixed samples were always assumed (although it also happens that the final identification results indicate only one protein). The parameters used for the search were as follows: appropriate taxonomic category of the species (Arabidopsis thaliana or Saccharomyces cerevisiae); mass error of 0.1 Da (maximum allowed peptide mass deviation from the calibration range); 1 zymosan deletion site permitted; protein molecular mass range: 0-1000 kDa; pI range: 0-14; and amino acids not chemically modified. Considering the number of protein species that may be present in the mixture, it is possible to make up to 4 settings for changing the number of possible proteins (maximum setting is 4). Access the results of the highest score obtained in the retrieval run. For each identified protein, the experimental match quality is used in the final ProFound " Single Protein" search. Finally, proteins with a likelihood close to 1 and a Z-score higher than 1.65 are considered.