*2.5.5 STop and go extraction (STAGE) tips protocol*

Further desalting and cleaning of the peptides was performed by an extended protocol called STop and Go Extraction (STAGE) tips protocol [22]. The first step was to prepare Styrenedivinylbenzene-reverse phase sulfonated (SDB-RPS) for the process of solid phase extraction. Briefly, a large sized SDB-RPS disk was placed in a covered petri dish and from that a 47 mm disk core was used in this protocol as a solid phase extraction sorbent. These sorbents have a high affinity for polar organic compounds, and hence reduce the extraction time and solvent, used for analytical preparation of aqueous environmental samples.

## *2.5.6 Construction of STAGE tip and preparing of collection tubes*

STAGE tip (**Figure 3**) was prepared by using a flat-ended needle, to push the 47 mm SDB-RPS core carefully inside a 2 ml pipette-tip, keeping it 2–3 mm above its end. A hole was made in the middle of the lid of a 2 ml Eppendorf tube using a rotary cutting tool. The pipette tip with SDB-RPS disk was inserted into the Eppendorf tube through the hole. Double distilled water was used to make 10% v/v trifluoroacetic acid (TFA), then 90% of isopropanol was added to make 1% TFA and this was diluted 1:10 with water. Fresh elution solvent was made by mixing 700 μl of Acetonitrile, 71 μl of NH4OH stock and 229 μl of water.

#### *2.5.7 Desalting and washing the samples*

The sample was acidified by adding 10 μl of 10% TFA and was centrifuged for 5 minutes at maximum speed. Using one STAGE tip, the sample was pipetted into the top of the tip and centrifuged at 5000 g rpm for 1 minute. The tubes were repeatedly centrifuged until all the liquid passed through the STAGE tip. To wash the sample, 60 μl of 1% TFA was pipetted into the STAGE tip and centrifuged at 5000 g rpm for 2 minutes. Tubes were centrifuged three times to clear all the salts and contaminants from the bound peptides and the liquid in the collection tube was discarded.

#### *2.5.8 Setting up STAGE tip in autovial insert and elution of peptides*

For each sample, a separate autovial was used and a yellow spacer was placed between the STAGE tip and the collection tube to hold the STAGE tip in the center of the autovial. Elution solvent was prepared by mixing 30 ml of 1 M Ammonium Hydroxide with 70 ml Acetonitrile. To complete the reaction, 60 μl of elution solvent was pipetted into the STAGE tip and was microfuged for a few seconds to drag the liquid onto the disk, which was incubated on the bench for 10 minutes. The STAGE tip was placed in the insert of the autovial and was centrifuged at 5000 g rpm for 5 minutes. This step was repeated three times to allow all the fluid to pass through the disk into the autovial insert containing all the peptides. For removing all the liquid, the STAGE tip was discarded and the collection tubes with autovial inserts containing peptide samples were placed into the Speed Vac (Thermo Speed Vac Concentrator DNA 120), to vacuum-centrifuge. Then, the autovial inserts were removed from the Speed Vac and the springs were attached back to the bottom of the inserts, before placing them into the autovials. Samples

**63**

1x 106

 to 1x 107 .

observed in the following **Figures 4** and **5**.

*Early Predictive Biomarkers for Hypertension Using Human Fetal Astrocytes*

were prepared by adding 25 μl of MS Loading Solvent-A (2% Acetonitrile +0.1% Trifluoroacetic acid) into each autovial inserts with samples ready to be injected

*Shows a 2 ml pipette-tip (STAGE tip) inserted through a hole drilled into the top of a 2 ml collection tube. The pipette tip with SDB-RPS disk was inserted into the Eppendorf tube through the hole. Cell solution was* 

*pipetted from the top into tip so peptides could react with the SDB-RPS disk for 10 minutes.*

**2.6 Proteomic data of A1 and A2 HFAs using liquid chromatography – mass** 

grams (**Figures 4** and **5**), obtained from the proteomics laboratory, UTS.

(SDC) was used as lysis buffer, instead of reconstitution buffer.

The optimisation of protein-elution in relation to the retention time was

The process of LC/MS/MS technique was optimised with multiple steps using different procedures. The first step of optimisation was the technique used to extract the protein from A1 and A2 HFAs. The following steps were taken to improve the protein extraction from the astrocytes, after analysing the chromato-

i.Clumping of the peptide-peaks was observed due to the effect of surfactantlike substances, as shown in **Figure 4**. Therefore, 1% sodium deoxycholate

ii.The number of astrocytes for extraction of the proteins was increased from

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

and analysed on mass spectrometer.

**Figure 3.**

**spectrometry (LC/MS/ MS) technique**

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

#### **Figure 3.**

*Erythrocyte - A Peripheral Biomarker for Infection and Inflammation*

*2.5.5 STop and go extraction (STAGE) tips protocol*

preparation of aqueous environmental samples.

*2.5.7 Desalting and washing the samples*

*2.5.6 Construction of STAGE tip and preparing of collection tubes*

700 μl of Acetonitrile, 71 μl of NH4OH stock and 229 μl of water.

with 1 μl of trypsin at 37°C.

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

Further desalting and cleaning of the peptides was performed by an extended protocol called STop and Go Extraction (STAGE) tips protocol [22]. The first step was to prepare Styrenedivinylbenzene-reverse phase sulfonated (SDB-RPS) for the process of solid phase extraction. Briefly, a large sized SDB-RPS disk was placed in a covered petri dish and from that a 47 mm disk core was used in this protocol as a solid phase extraction sorbent. These sorbents have a high affinity for polar organic compounds, and hence reduce the extraction time and solvent, used for analytical

STAGE tip (**Figure 3**) was prepared by using a flat-ended needle, to push the 47 mm SDB-RPS core carefully inside a 2 ml pipette-tip, keeping it 2–3 mm above its end. A hole was made in the middle of the lid of a 2 ml Eppendorf tube using a rotary cutting tool. The pipette tip with SDB-RPS disk was inserted into the Eppendorf tube through the hole. Double distilled water was used to make 10% v/v trifluoroacetic acid (TFA), then 90% of isopropanol was added to make 1% TFA and this was diluted 1:10 with water. Fresh elution solvent was made by mixing

The sample was acidified by adding 10 μl of 10% TFA and was centrifuged for 5 minutes at maximum speed. Using one STAGE tip, the sample was pipetted into the top of the tip and centrifuged at 5000 g rpm for 1 minute. The tubes were repeatedly centrifuged until all the liquid passed through the STAGE tip. To wash the sample, 60 μl of 1% TFA was pipetted into the STAGE tip and centrifuged at 5000 g rpm for 2 minutes. Tubes were centrifuged three times to clear all the salts and contaminants

For each sample, a separate autovial was used and a yellow spacer was placed between the STAGE tip and the collection tube to hold the STAGE tip in the center of the autovial. Elution solvent was prepared by mixing 30 ml of 1 M Ammonium Hydroxide with 70 ml Acetonitrile. To complete the reaction, 60 μl of elution solvent was pipetted into the STAGE tip and was microfuged for a few seconds to drag the liquid onto the disk, which was incubated on the bench for 10 minutes. The STAGE tip was placed in the insert of the autovial and was centrifuged at 5000 g rpm for 5 minutes. This step was repeated three times to allow all the fluid to pass through the disk into the autovial insert containing all the peptides. For removing all the liquid, the STAGE tip was discarded and the collection tubes with autovial inserts containing peptide samples were placed into the Speed Vac (Thermo Speed Vac Concentrator DNA 120), to vacuum-centrifuge. Then, the autovial inserts were removed from the Speed Vac and the springs were attached back to the bottom of the inserts, before placing them into the autovials. Samples

from the bound peptides and the liquid in the collection tube was discarded.

*2.5.8 Setting up STAGE tip in autovial insert and elution of peptides*

**62**

*Shows a 2 ml pipette-tip (STAGE tip) inserted through a hole drilled into the top of a 2 ml collection tube. The pipette tip with SDB-RPS disk was inserted into the Eppendorf tube through the hole. Cell solution was pipetted from the top into tip so peptides could react with the SDB-RPS disk for 10 minutes.*

were prepared by adding 25 μl of MS Loading Solvent-A (2% Acetonitrile +0.1% Trifluoroacetic acid) into each autovial inserts with samples ready to be injected and analysed on mass spectrometer.
