**4. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and in-gel digestion**

## **4.1. SDS-PAGE**

All chemicals including Precision Plus Protein Unstained Standards (catalog number: 161- 0363EDU), Coomassie Brilliant Blue G-250 Staining Solution Kit (catalog number: 161- 0435EDU) using for SDS-PAGE, were purchased from Bio-Rad (Bio-Rad Laboratories, Inc., CA 94547, USA). The procedure was carried out according to Laemmli [14].

The following stock solutions were prepared: (i) 1.5 M Tris–HCl, pH 8.8; (ii) 0.5 M Tris–HCl, pH 6.8; (iii) 30% acrylamide/bisacrylamide solution (37.5:1); (iv) *N*,*N*,*N*,*N* tetramethylethylenediamine (TEMED); (v) freshly prepared 10% ammonium persulphate (APS) solution; (vi) 10% sodium dodecyl sulphate (SDS) solution; (vii) 10X SDS gel running buffer (30 g Tris-base, 144 g glycine, 10 g SDS, dissolved in MilliQ water and adjusted to a volume of one liter); (viii) 5X sample buffer (10% SDS, 50% glycerol, 300 mM Tris–HCl (pH 6.8), 0.05% bromphenol blue. Dithiothreitol (DTT) was added to a nal concentration of 100 mM prior to use).

The membrane fraction was solubilized in lysis buffer containing 3% SDS. Equal volumes containing approximately 25 μg/lane of MP were separated by 12% SDS-PAGE and were visualized by staining with Coomassie Brilliant Blue G-250.

**Figure 2.** The separation of membrane proteins (MPs) by SDS-PAGE. The gel was cut into 10 slices that covered known apparent mass ranges. Lane M, protein standard markers; lane 1 & lane 2: membrane protein fractions isolated from mouse brain; 1-10: slices to be cut for trypsin in-gel digestion, separation and analyses by nanoLC-MS/MS.

## **4.2. In-gel digestion**

66 Chromatography – The Most Versatile Method of Chemical Analysis

Assay Kit (Bio-Rad, Hercules, CA 94547 USA).

**3. Protein quantification** 

cuvettes were recommended.

**4.1. SDS-PAGE** 

mM prior to use).

**(SDS-PAGE) and in-gel digestion** 

centrifuged at 10,000 rpm for 15 min at 4oC to sediment large organelles. The obtained supernatant was used for recentrifugation again at 10,000 rpm for 15 min at 4oC. The supernatant was collected and centrifuged at 40,000 rpm at 4oC for 1 hr. After discarding the clear supernatant, the membrane pellets were retained and washed by resuspending in ice-cold 0.1 M Na2CO3 containing protease inhibitors for 1 hr. The mouse brain membrane protein fractions were obtained by centrifugation again at 40,000 rpm for 1 h at 4oC. The sample was divided and stored at −80oC until use. The protein concentration of the extracted membrane fractions was assessed using a Quick StartTM Bradford Protein

Protein concentration of the extracted membrane fractions was determined using Bio-Rad's Quick StartTM Bradford Protein Assay [5]. The assay is based on the observation that the maximum absorbance for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465nm to 595 nm when binding to protein occurs. Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible colour change. For the standard curve, bovine serum albumin over a wide range of concentrations (0.1 - 20 μg/μl) was used. The low concentration range assay was used in the test tube format. 2 μl of standard or sample was added to 798 μl of MilliQ water. 200 μL of Bio-Rad reagent was added, mixed, and incubated for 10 min at room temperature. The absorbance at the wavelength of 595 nm was measured in a spectrophotometer. Glass or polystyrene (cheap) cuvettes have been used, however the color reagent stained both. Disposable

**4. Sodium dodecyl sulfate polyacrylamide gel electrophoresis** 

CA 94547, USA). The procedure was carried out according to Laemmli [14].

All chemicals including Precision Plus Protein Unstained Standards (catalog number: 161- 0363EDU), Coomassie Brilliant Blue G-250 Staining Solution Kit (catalog number: 161- 0435EDU) using for SDS-PAGE, were purchased from Bio-Rad (Bio-Rad Laboratories, Inc.,

The following stock solutions were prepared: (i) 1.5 M Tris–HCl, pH 8.8; (ii) 0.5 M Tris–HCl, pH 6.8; (iii) 30% acrylamide/bisacrylamide solution (37.5:1); (iv) *N*,*N*,*N*,*N* tetramethylethylenediamine (TEMED); (v) freshly prepared 10% ammonium persulphate (APS) solution; (vi) 10% sodium dodecyl sulphate (SDS) solution; (vii) 10X SDS gel running buffer (30 g Tris-base, 144 g glycine, 10 g SDS, dissolved in MilliQ water and adjusted to a volume of one liter); (viii) 5X sample buffer (10% SDS, 50% glycerol, 300 mM Tris–HCl (pH 6.8), 0.05% bromphenol blue. Dithiothreitol (DTT) was added to a nal concentration of 100 In-gel digestion of proteins isolated by gel electrophoresis was carried out according to the protocol published by Shevchenko *et al* [25] with some modifications described in our previous study [3, 28, 29]. All chemicals including DTT, iodoacetamide (IAA), ammonium bicarbonate, ammonium acetate, trypsin (proteomics sequencing grade), sodium bicarbonate and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO, USA) prepared using deionized filter-sterilised MilliQ water.

Upon electrophoresis, proteins were fixed within a polyacrylamide matrix by incubating the entire gel in 5% (vol/vol) acetic acid in 1:1 (vol/vol) water:methanol. Coomassie blue-stained protein bands were excised from gels and placed into 1.5 ml eppendorf tubes, destained with 50% ACN in 25 mM NH4HCO pH 8.0 at room temperature with occasional vortexing, until gel pieces became white and shrank, and then acetonitrile was removed. The gel pieces

were then reduced by incubating with 5 mM DTT solution at 56oC for 45 min and alkylated for 1 hr with 20 mM IAA solution in darkness at room temperature. The membrane proteins were digested by adding trypsin buffer (0.03 μg/μl in 10 mM ammonium bicarbonate containing 10% (vol/vol) acetonitrile) and incubating overnight at 37oC. Check if all solution was absorbed and add more trypsin buffer, if necessary. Gel pieces should be completely covered with trypsin buffer (typically, 50 μl or more).

2D-NanoLC-ESI-MS/MS for Separation and Identification of Mouse Brain Membrane Proteins 69

PepMap nano RP Trapping column

Pre-concentrating sample

300 μm i.d. x 0.5 cm, PepMapTM C18, 5 μm, 100Å (LC Parking, Dionex, P/N 160454)

**Table 1.** The type of columns with their physico-chemical properties, functions and the mobile phase, loading/eluted solvents that were used for basic experimental setup of an online two-dimensional nano liquid chromatography system (2D-nanoLC, UltiMateTM/FAMOS/SwitchosTM, LC Parking, Dionex, The

For in-gel digest samples, as the rst step, the peptide mixture was re-dissolved in 30 μl of 0.1% FA and directly loaded onto a strong cation exchange (SCX) column (500 μm i.d.×1.5 cm, 5 μm, 300 Å) at a ow rate of 30 μl/min. Bound peptides were eluted by following ammonium acetate gradients from 10 mM to 2 M: 10 mM, 20 mM, 40mM, 60mM, 80mM, 100 mM, 200 mM, 500mM, 1M and 2M and then desalted and concentrated independently on a C18 trap column (300 μm i.d. ×0.5 cm, 5 μm, 100 Å). The eluted peptides were further separated onto a reversed phase C18 column (75 μm i.d.×15 cm, 5 μm, 100 Å), for the second dimension. The ow rate was maintained at 200 nl/min with solvent A (0.1% FA in LC-MS grade water). With 10 different concentrations of ammonium acetate (plugs), there should

2nd Dimension-Reversed Phase C18 column

Separation of a protein/peptide on a reversed phase

75 μm i.d. x 15 cm, packed with PepMapTMC18, 100 Å, 3 μm, (LC Parking, Dionex, P/N 160321)

A (0.1% FA in LC-MS grade water); B (0.1% FA in 85% LC-MS grade ACN)

1 2 3 4

1st Dimension - Exchange Ion Chromatography-SCX column

Separation of a protein/peptide on an *ion exchange*

500 μm i.d. x 1.5 cm, 300 Å, 5 μm (LC Parking, Dionex, P/N 161395)

Loading flow 30 μl/min Loading solvent 0.1% FA, pH2.9

> Ammonium acetate solutions: 10 mM, 20 mM, 40mM, 60mM, 80mM, 100 mM, 200 mM, 500mM, 1M, 2M

Type of column

Function

Physical properties of column

Eluted solvent (Elute positively charged peptides on SCX)

Mobile phase

be 10 identical runs.

Netherlands).

Injected volumn 20 μl Flow rate 200 nl/min
