**2.6 Proteomic data of A1 and A2 HFAs using liquid chromatography – mass spectrometry (LC/MS/ MS) technique**

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


The optimisation of protein-elution in relation to the retention time was observed in the following **Figures 4** and **5**.

#### **Figure 4.**

*There were 4 panels in this first set of chromatograms obtained after LC–MS analysis of A1 and A2 astrocytes. Panel A, shows the retention time against frequency of the peptide-peaks, which were clumped together due to surfactant-like substances. Panel B, shows the relative abundance of peptide-spikes against time. Panel C, displays the expanded version of panel B. panel D, indicates mass-to-charge ratio (m/z), used for the standardisation of the other graphs.*

*Panels A, B and C with the same descriptions as for Figure 4, except displaying greater retention of protein spikes, because of the optimization of protein extraction method. Panel D, indicates mass-to-charge ratio (m/z), used for the standardisation of other graphs.*

#### *2.6.1 PEAKS software*

PEAKS (Bioinformatics Solutions Inc.) software was used for the data analysis of the proteins detected from LC–MS–MS, by integrating the area under the curve (**Figure 6**) and statistical comparison of the initial data (**Figure 7**), using Student's dependent t-test as mentioned below. Triplicates of each set of A1 and A2 samples (n = 9) were carefully analysed using either one of the two filters:

**65**

kinase (MAPK).

*Early Predictive Biomarkers for Hypertension Using Human Fetal Astrocytes*

scoring proteins as unique peptide count.

ii.False discovery rate (FDR) threshold (1%).

i.Peaks Peptide Score (-10lgP) threshold, only peptides with a score above this threshold were used to quantify the identified proteins, separating low

All values in this text are expressed as mean ± standard error mean (SEM). Differences between two groups were determined using Student's paired dependent t-test and were considered significant at P < 0.05. All statistical analyses were conducted using Microsoft Excel and GraphPad Prism version 8.0.1, GraphPad Software. Light microscopic micrographs were obtained on a Leica Microsystems, LAS version 4.4.0.

*Experimental error chart, indicating the minimal error (1%) in peptides-spectrum matches (PSMS) against* 

The results show that the HFAs increase in size and number with increasing time in tissue culture. Distinct differences were observed in the morphology of the cells present in A1 and A2 flasks, for example A1 astrocytes have more fibroblast-like structures as compared to A2 astrocytes, which look more like neurons (**Figure 7**). A noticeable difference was observed in the thickness of the processes between the two types of the HFAs. This thickening was due to the accumulation of filament proteins such as glial fibrillary acidic proteins (GFAP) as shown in **Figure 8**. Whereas **Figure 9** shows the astrocytes present in the spontaneously hypertensive rat (SHR) and normal Wistar Kyoto (WKY) rat brain slices. **Figure 9**, shows the similarities between A1 HFAs and the SHR astrocytes, implying that the A1 HFAs mimic the hypertensive condition.

**3.2 Identification of proteins in A1 and A2 HFAs, using LC/MS/MS technique**

protein (GFAP), calpain, calpastatin, cathepsin and mitogen activated protein

The present study was focused on specific proteins, such as glial fibrillary acidic

**3.1 A1 (reactive) and A2 (normal) HFAs in culture**

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

**2.7 Statistical analysis**

*part per million (ppm), using PEAKS software.*

**3. Results**

**Figure 6.**

i.Peaks Peptide Score (-10lgP) threshold, only peptides with a score above this threshold were used to quantify the identified proteins, separating low scoring proteins as unique peptide count.

ii.False discovery rate (FDR) threshold (1%).

#### **Figure 6.**

*Erythrocyte - A Peripheral Biomarker for Infection and Inflammation*

**64**

*2.6.1 PEAKS software*

*(m/z), used for the standardisation of other graphs.*

**Figure 5.**

**Figure 4.**

*standardisation of the other graphs.*

PEAKS (Bioinformatics Solutions Inc.) software was used for the data analysis

of the proteins detected from LC–MS–MS, by integrating the area under the curve (**Figure 6**) and statistical comparison of the initial data (**Figure 7**), using Student's dependent t-test as mentioned below. Triplicates of each set of A1 and A2 samples (n = 9) were carefully analysed using either one of the two filters:

*Panels A, B and C with the same descriptions as for Figure 4, except displaying greater retention of protein spikes, because of the optimization of protein extraction method. Panel D, indicates mass-to-charge ratio* 

*There were 4 panels in this first set of chromatograms obtained after LC–MS analysis of A1 and A2 astrocytes. Panel A, shows the retention time against frequency of the peptide-peaks, which were clumped together due to surfactant-like substances. Panel B, shows the relative abundance of peptide-spikes against time. Panel C, displays the expanded version of panel B. panel D, indicates mass-to-charge ratio (m/z), used for the* 

*Experimental error chart, indicating the minimal error (1%) in peptides-spectrum matches (PSMS) against part per million (ppm), using PEAKS software.*
