**3.3. Identification of differentially expressed proteins associated with spontaneous unexplained preterm labor with intact membrane by MALDI-TOF/TOF MS**

Identified spots with significant protein scores are shown in **Tables 2** and **3**. The spot numbers in **Tables 2** and **3** corresponded to the numbers in **Figure 1C**.

As indicated in **Tables 2** and **3**, S100-A9 and Ig kappa chain C (IGKC) region were overexpressed (**Table 2**), while vimentin, calpain small subunit 1 (CAPNS1), protein PP4-X, ferritin light subunit (FTL), septin 2, peroxiredoxin 3 (Prdx 3), cytokeratin 8, cytokeratin 1, and tropomodulin 3 (Tmod-3) were underexpressed in the fetal side of the placenta obtained from sPTL-IM compared to the control (two different spots were identified as the same protein;

**Figure 1.** Representative 2D gels of placenta proteome. (AI and AII) Representative 2D gels of placenta tissue samples from sPTL-IM and (BI and BII) sTL-IM. The differentially expressed protein spots are circled on the gels and labeled with unique spot numbers. Dotted circles indicate overexpressed proteins in the gels analyzed using ImageMaster Software, while solid circles indicate underexpressed proteins. The spot number on the gels corresponds to the numbers in **Tables 2** and **3**. (C) A closer view of differentially expressed protein spots from 2D gels and their 3D images. The gray background boxes correspond to the sPTL-IM gels and the white background boxes correspond to the sTL-IM gels.

Spontaneous Unexplained Preterm Labor with Intact Membrane: Finding Protein Biomarkers…

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9

Spontaneous Unexplained Preterm Labor with Intact Membrane: Finding Protein Biomarkers… http://dx.doi.org/10.5772/intechopen.74925 9

**3. Results**

8 Electrophoresis - Life Sciences Practical Applications

**its corresponding control**

ImageMaster 2D platinum software program.

**3.1. Characteristics of the research population and body weight of fetus**

lighter babies (at birth) and placentas than their control (*P* < 0.05, **Table 1**).

**3.2. Comparative two-dimensional protein profile analysis of placenta tissues obtained from spontaneous unexplained preterm labor with intact membrane and** 

parallel for the maternal side of the placenta tissues (**Figure 1AII**, **BII**, and **C**).

fold changes (solid circle in **Figure 1AII** and **BII**, *P* < 0.05) for the same comparison.

**3.3. Identification of differentially expressed proteins associated with spontaneous** 

Identified spots with significant protein scores are shown in **Tables 2** and **3**. The spot numbers

As indicated in **Tables 2** and **3**, S100-A9 and Ig kappa chain C (IGKC) region were overexpressed (**Table 2**), while vimentin, calpain small subunit 1 (CAPNS1), protein PP4-X, ferritin light subunit (FTL), septin 2, peroxiredoxin 3 (Prdx 3), cytokeratin 8, cytokeratin 1, and tropomodulin 3 (Tmod-3) were underexpressed in the fetal side of the placenta obtained from sPTL-IM compared to the control (two different spots were identified as the same protein;

**unexplained preterm labor with intact membrane by MALDI-TOF/TOF MS**

in **Tables 2** and **3** corresponded to the numbers in **Figure 1C**.

Five pregnant women who were experiencing sPTL-IM were recruited in the study group while 10 pregnant women experiencing sTL-IM were recruited as the control group. Clinical data of these pregnant women were compared between both groups. As shown in **Table 1**, there were no statistical significant differences between sPTL-IM and sTL-IM groups in maternal age and parity. Pregnant women resulted in a PTB due to sPTL-IM have significantly

To further investigate the proteomic alterations in the sPTL-IM, the spot intensities in placenta samples (maternal and fetal sides) from sPTL-IM and sTL-IM were compared using

A normalized amount of protein extracted from placenta tissues of sPTL-IM and sTL-IM was fixed on 13-cm pH 3–10 IPG strips. Second dimension separation was performed on 10% widerange PAGE, as described under "Materials and Methods" section. Silver-stained 2D gels representing sPTL-IM and sTL-IM (fetal and maternal sides) are shown in **Figure 1A** and **B**, respectively. A comparison of the fetal side of the placenta tissues obtained from both the sPTL-IM and sTL-IM proteomes revealed highly similar protein profiles (**Figure 1AI** and **BI**) despite important differences being observed in the relative levels of several proteins as illustrated by the closer view of differentially expressed protein profiles (**Figure 1C**). These results were in

Student's *t*-test analysis revealed 12 differentially expressed proteins that are significant (two spots with greater staining and 10 spots with decreased staining; dotted circles in **Figure 1** indicate upregulated proteins, whereas solid circles indicate downregulated proteins, AI and BI, *P* < 0.05) in the fetal side of sPTL-IM with 100% reproducibility across the sample set. Whereas on the maternal side, only two gel spots with decreased staining showed at least 2.0-

**Figure 1.** Representative 2D gels of placenta proteome. (AI and AII) Representative 2D gels of placenta tissue samples from sPTL-IM and (BI and BII) sTL-IM. The differentially expressed protein spots are circled on the gels and labeled with unique spot numbers. Dotted circles indicate overexpressed proteins in the gels analyzed using ImageMaster Software, while solid circles indicate underexpressed proteins. The spot number on the gels corresponds to the numbers in **Tables 2** and **3**. (C) A closer view of differentially expressed protein spots from 2D gels and their 3D images. The gray background boxes correspond to the sPTL-IM gels and the white background boxes correspond to the sTL-IM gels.


Accession #: accession numbers from SWISS-PROT database; A: arbitrary spot number supplied by Delta 2D. Numbers correspond to numbers in **Figure 1C**; B: fold change was calculated using ImageMaster 2D platinum software program; C: protein score generated by MS-MS identification platform, with a score higher than 67 being considered as statistically significant.

> cytokeratin 1) (**Table 3**), whereas two proteins namely annexin A1 (ANXA1) and cytokeratin 8 were underexpressed (**Table 3**) in the maternal side of the placenta obtained from sPTL-IM

> Accession #: accession numbers from SWISS-PROT database; A: arbitrary spot number supplied by Delta 2D. Numbers correspond to numbers in **Figure 1C**; B: fold change was calculated using ImageMaster 2D platinum software program; C: protein score generated by MS-MS identification platform, with a score higher than 67 being considered as statistically significant.

> Interestingly, in both the fetal and maternal sides of the placentas, cytokeratin 8 was reduced in expression in relation to sPTL-IM (**Table 3**). There were no overexpressed proteins found

> Of the total 12 proteins identified, four functional groupings emerge (**Figure 2**). Cytokeratin 8, cytokeratin 1, vimentin, Tmod-3, septin 2, and CAPNS1 are grouped under structural and cytoskeletal components (53.8%), S100-A9, IGKC, and ANXA1 are grouped under immunomodulatory proteins (23.1%), FTL and Prdx 3 are grouped under placenta-modulatory proteins (15.4%), and lastly, protein PP4-X is grouped under anticoagulation cascade (7.7%).

> **Figure 2.** Functional grouping of differentially expressed proteins identified in the placentas obtained from pregnant

compared to the control.

women experiencing sPTL-IM.

**Spot numberA**

Maternal side

**NCBI Accession #** **Protein identified** **Relative fold changeB**

85 1AIN\_A Annexin A1 −2.003 7.77/35.25 0.005 95 Anti-

213 AAA35748 Cytokeratin 8 −2.896 5.30/53.66 0.005 118 Filament

**Table 3.** Biochemical information of the underexpressed proteins in the placenta tissues of sPTL-IM.

**Calculated pI/Mw (kDa)**

Spontaneous Unexplained Preterm Labor with Intact Membrane: Finding Protein Biomarkers…

**P value** **Mascot scoreC**

**Function Sequence** 

7

9

inflammatory properties

http://dx.doi.org/10.5772/intechopen.74925

reorganization

**coverage (%)**

11

in the maternal side of the placenta.

**Table 2.** Biochemical information of the overexpressed proteins in the placenta tissues of sPTL-IM.



Accession #: accession numbers from SWISS-PROT database; A: arbitrary spot number supplied by Delta 2D. Numbers correspond to numbers in **Figure 1C**; B: fold change was calculated using ImageMaster 2D platinum software program; C: protein score generated by MS-MS identification platform, with a score higher than 67 being considered as statistically significant.

**Table 3.** Biochemical information of the underexpressed proteins in the placenta tissues of sPTL-IM.

**Spot numberA**

**Spot numberA**

Fetal side

Fetal side

**NCBI Accession #**

**NCBI accession** 

10 Electrophoresis - Life Sciences Practical Applications

13 NP\_002956 Protein

0 P01834 Ig kappa

**#**

48 NP\_001740 Calpain small

221 AAA52440 Ferritin light

380 EAW49398 Peroxire-

270 AAF31670 Tropomo-

**Protein identified**

**Protein identified**

S100-A9

chain C region

subunit 1

subunit

doxin 3

dulin 3

**Relative fold changeB**

**Table 2.** Biochemical information of the overexpressed proteins in the placenta tissues of sPTL-IM.

**Relative fold changeB**

365 AAA61279 Vimentin −9.454 5.03/53.74 0.035 188 Stabilization

Accession #: accession numbers from SWISS-PROT database; A: arbitrary spot number supplied by Delta 2D. Numbers correspond to numbers in **Figure 1C**; B: fold change was calculated using ImageMaster 2D platinum software program; C: protein score generated by MS-MS identification platform, with a score higher than 67 being considered as statistically significant.

252 AAC41689 Protein PP4-X −2.356 5.65/36.26 0.022 150 Membrane

276 NP\_004395 Septin-2 −2.311 6.15/41.69 0.015 70 Filament-

250 CAA31376 Cytokeratin 8 −2.104 4.91/30.84 0.026 162 Filament

369 AFA52002 Cytokeratin 1 −6.141 8.15/66.18 0.011 207 Filament

362 AFA52002 Cytokeratin 1 −2.759 8.15/66.18 132 Filament

**Calculated pI/Mw (kDa)**

**Calculated pI/Mw (kDa)**

> **P value**

−2.397 5.05/28.47 0.004 142 Stabilized

−2.494 5.65/16.44 0.080 227 Iron homeo-

−2.025 6.06/11.16 <0.001 103 Mitochon-

−3.083 5.08/39.73 0.027 161 Maintaining

**Mascot scoreC**

**P value Mascot scoreC**

16.202 5.71/13.29 <0.001 70 Calcium

11.658 5.58/11.77 <0.001 120 Antigen

of cytoskeletal interactions

calpain heterodimers

fusion

stasis and inflammatory response

forming cytoskeletal GTPase

drial dysfunction

reorganization

reorganization

cytoskeletal architecture

13

reorganization **Function Sequence** 

**Function Sequence** 

24

32

binding protein

binding agent

**coverage (%)**

9

16

9

30

7

13

12

13

17

**coverage (%)**

cytokeratin 1) (**Table 3**), whereas two proteins namely annexin A1 (ANXA1) and cytokeratin 8 were underexpressed (**Table 3**) in the maternal side of the placenta obtained from sPTL-IM compared to the control.

Interestingly, in both the fetal and maternal sides of the placentas, cytokeratin 8 was reduced in expression in relation to sPTL-IM (**Table 3**). There were no overexpressed proteins found in the maternal side of the placenta.

Of the total 12 proteins identified, four functional groupings emerge (**Figure 2**). Cytokeratin 8, cytokeratin 1, vimentin, Tmod-3, septin 2, and CAPNS1 are grouped under structural and cytoskeletal components (53.8%), S100-A9, IGKC, and ANXA1 are grouped under immunomodulatory proteins (23.1%), FTL and Prdx 3 are grouped under placenta-modulatory proteins (15.4%), and lastly, protein PP4-X is grouped under anticoagulation cascade (7.7%).

**Figure 2.** Functional grouping of differentially expressed proteins identified in the placentas obtained from pregnant women experiencing sPTL-IM.
