**2.5 Protein extraction and digestion using single-pot, solid-phase-enhanced sample-preparation (SP3) protocol**

The proteins of interest were extracted from A1 and A2 HFAs using the SP3 protocol, before performing mass spectrometric analysis. All the chemicals and equipment used for the preparation of lysis buffer and for protein extraction and digestion using SP3 protocol are shown in **Table 2** and **Figure 1**.


#### **Table 2.**

*Reagents and equipment used for SP3 protocol.*

#### **Figure 1.**

*The magnetic rack used for placing the sample-vials containing magnetic beads. When vials are placed on the magnetic rack, the beads start to stick to the wall of the vial. This process allows the protein molecules to attach safely to the beads, so unwanted fluid could be easily discarded.*

## *2.5.1 Preparation of reconstitution (lysis) buffer for SP3*

Reconstitution buffer was freshly prepared by mixing 100 μl of each of the following chemicals, i.e., 10% SDS, 10% Triton x-100, 10% NP-40, 10% Deoxycholate and 10% Glycerol. In addition, 10 μl of 5 M NaCl, 20 μl of HEPES (pH 8.0) and 470 μl of water was added to the mixture.

#### *2.5.2 Extraction of the proteins from A1 and A2 HFA samples*

Eppendorf tubes containing pellets of A1 and A2 astrocytes were removed from −80°C and were resuspended in 50 μl of lysis buffer. To facilitate the extraction process, the tubes were heated to 90°C on the heating block for 10 minutes. To reduce the number of disulfide bonds, 1 μl Tris 2-carboxyethyl phosphine (TCEP) was added to the samples, followed by 1 μl of acrylamide monomer to alkylate the mixture. After an hour of incubation at room temperature, the alkylation reaction was stopped by adding 1 μl of dithiothreitol (DTT). Before extracting the proteins from both types of HFAs, a standard curve was constructed using an ampule of 2 mg/ml bovine serum albumin (BSA).

#### *2.5.3 Construction of the standard curve*

The standard curve was constructed using a 96 well microplate, and bovine serum albumin (BSA) was added at a concentration ranging from 0 to 2000 μg/ml diluted with double distilled water. Similarly, A1 and A2 sample solutions were also diluted with double distilled water at a ratio of 1:10 & 1:20. Working solution of BCA was prepared by adding reagent B (cupric sulfate) with reagent A (cuprous sulfate), at a ratio of 1:50. The plates were prepared according to **Table 3**. Nine wells of the plate were filled with 25 μl of BSA solution ranging from 0 to 2000 μg/ml along with the blank and two dilutions of A1 and A2 samples. Then 200 μl of working solution was added in each of the fourteen wells. The plates were covered with parafilm and were incubated at 37°C for 30 minutes, and the plates were read on the Tecan Infinite 200 Pro, spectrophotometer, at a wavelength of 562 nm, using Magellan software.

**61**

**Figure 2.**

*concentrations in the A1 and A2 astrocytes.*

*Early Predictive Biomarkers for Hypertension Using Human Fetal Astrocytes*

**S.N. Vial volume of diluent (**μ**l) Volume and source of BSA (**μ**l) BSA concentration (**μ**g/ml)**

A 0 300 of Stock 2000 B 125 375 of Stock 1500 C 325 325 of Stock 1000 D 175 175 of vial B dilution 750 E 325 325 of vial C dilution 500 F 325 325 of vial E dilution 250 G 325 325 of vial F dilution 125 H 400 100 of vial G dilution 25 I 400 0 Blank

Data obtained was used to plot a standard curve and the concentration of the

The cell-solution was diluted to a final volume of 48 μl by adding double distilled water and 2 μl of hydrophilic magnetic beads to the samples, giving a total volume of 50 μl of protein solution. Proteins were bonded with the beads by adding 50 μl of 100% ethanol to the above mixture and vortexed for 5 minutes. Hydrophilic magnetic beads, were coated with carboxylate functional groups to capture the proteins, making it easier to wash the cells in 100% ethanol. Surfactant was removed by rinsing the cells three times with 80% ethanol and the supernatant with unbound

*BCA (Bicinchoninic acid) protein assay was used to plot standard curve for quantitation of the protein* 

proteins in the A1 and A2 astrocyte samples were determined from **Figure 2**.

A1(1:10) 9 1 38 A1(1:20) 19 1 26 A2(1:10) 9 1 22 A2(1:20) 19 1 18

*2.5.4 Reconstitution of the proteins from A1 and A2 samples*

*Dilution scheme protocol for standard in vials (work-range, 25–2000* μ*g/mL).*

*DOI: http://dx.doi.org/10.5772/intechopen.98561*

Samples

**Table 3.**


E 325 325 of vial C dilution 500 F 325 325 of vial E dilution 250 G 325 325 of vial F dilution 125 H 400 100 of vial G dilution 25 I 400 0 Blank

A1(1:10) 9 1 38 A1(1:20) 19 1 26 A2(1:10) 9 1 22 A2(1:20) 19 1 18

*Early Predictive Biomarkers for Hypertension Using Human Fetal Astrocytes DOI: http://dx.doi.org/10.5772/intechopen.98561*

#### **Table 3.**

Samples

*Erythrocyte - A Peripheral Biomarker for Infection and Inflammation*

*2.5.1 Preparation of reconstitution (lysis) buffer for SP3*

*safely to the beads, so unwanted fluid could be easily discarded.*

*2.5.2 Extraction of the proteins from A1 and A2 HFA samples*

470 μl of water was added to the mixture.

**Figure 1.**

2 mg/ml bovine serum albumin (BSA).

*2.5.3 Construction of the standard curve*

Reconstitution buffer was freshly prepared by mixing 100 μl of each of the following chemicals, i.e., 10% SDS, 10% Triton x-100, 10% NP-40, 10% Deoxycholate and 10% Glycerol. In addition, 10 μl of 5 M NaCl, 20 μl of HEPES (pH 8.0) and

*The magnetic rack used for placing the sample-vials containing magnetic beads. When vials are placed on the magnetic rack, the beads start to stick to the wall of the vial. This process allows the protein molecules to attach* 

Eppendorf tubes containing pellets of A1 and A2 astrocytes were removed from −80°C and were resuspended in 50 μl of lysis buffer. To facilitate the extraction process, the tubes were heated to 90°C on the heating block for 10 minutes. To reduce the number of disulfide bonds, 1 μl Tris 2-carboxyethyl phosphine (TCEP) was added to the samples, followed by 1 μl of acrylamide monomer to alkylate the mixture. After an hour of incubation at room temperature, the alkylation reaction was stopped by adding 1 μl of dithiothreitol (DTT). Before extracting the proteins from both types of HFAs, a standard curve was constructed using an ampule of

The standard curve was constructed using a 96 well microplate, and bovine serum albumin (BSA) was added at a concentration ranging from 0 to 2000 μg/ml diluted with double distilled water. Similarly, A1 and A2 sample solutions were also diluted with double distilled water at a ratio of 1:10 & 1:20. Working solution of BCA was prepared by adding reagent B (cupric sulfate) with reagent A (cuprous sulfate), at a ratio of 1:50. The plates were prepared according to **Table 3**. Nine wells of the plate were filled with 25 μl of BSA solution ranging from 0 to 2000 μg/ml along with the blank and two dilutions of A1 and A2 samples. Then 200 μl of working solution was added in each of the fourteen wells. The plates were covered with parafilm and were incubated at 37°C for 30 minutes, and the plates were read on the Tecan Infinite 200 Pro, spectrophotometer, at a wavelength of 562 nm, using Magellan software.

**60**

*Dilution scheme protocol for standard in vials (work-range, 25–2000* μ*g/mL).*

Data obtained was used to plot a standard curve and the concentration of the proteins in the A1 and A2 astrocyte samples were determined from **Figure 2**.

#### *2.5.4 Reconstitution of the proteins from A1 and A2 samples*

The cell-solution was diluted to a final volume of 48 μl by adding double distilled water and 2 μl of hydrophilic magnetic beads to the samples, giving a total volume of 50 μl of protein solution. Proteins were bonded with the beads by adding 50 μl of 100% ethanol to the above mixture and vortexed for 5 minutes. Hydrophilic magnetic beads, were coated with carboxylate functional groups to capture the proteins, making it easier to wash the cells in 100% ethanol. Surfactant was removed by rinsing the cells three times with 80% ethanol and the supernatant with unbound

#### **Figure 2.**

*BCA (Bicinchoninic acid) protein assay was used to plot standard curve for quantitation of the protein concentrations in the A1 and A2 astrocytes.*

compounds was discarded in a waste container. Proteins were extracted in aqueous solution by adding 100 μl of 200 mM ammonium bicarbonate to the protein bonded beads. Trypsinisation of proteins was achieved by incubating the samples overnight with 1 μl of trypsin at 37°C.
