*Application of Two-Dimensional Gel Electrophoresis in Combination with Mass Spectrometry… DOI: http://dx.doi.org/10.5772/intechopen.82524*

*Mass Spectrometry - Future Perceptions and Applications*

**76**

**Hormone**

hGH

(signal peptide sequence

1–26)

**Before and after removal of signal** 

**Position**

**Characteristic tryptic peptide sequence**

MATGSR

**Calc. [M + H]+**

622.3 (MSO: 1;

638.3)

3043.7

−

−

3100.7(Cys\_CAM:

17)

3646.9

−

−

−

3704.0 (Cys\_CAM:

17)

3662.9 (MSO: 1)

4004.2

−

−

−

4061.2 (Cys\_CAM:

17)

4020.1 (MSO: 40)

930.5 1891.0

1907.0 (MSO: 40)

+

±

−

**Observed** 

**[M + H]+**

−

**peptide**

Before

1–6 7–34 1–34 7–42

> After

27–34 27–42

FPTIPLSR FPTIPLSRLFDNAMLR

TSLLLAFGLLCLPWLQEGSAFPTIPLSRLFDNAMLR

MATGSRTSLLLAFGLLCLPWLQEGSAFPTIPLSR

TSLLLAFGLLCLPWLQEGSAFPTIPLSR


**Table 2.**

**79**

**Figure 7.** *MS2*

*copyright 2005.*

**Table 3.**

*Application of Two-Dimensional Gel Electrophoresis in Combination with Mass Spectrometry…*

1 1/2 pSer176

3 1 pSer132

6 1 pSer77, pSer132,

 1 pSer176, pSer132 2 pSer176 1 pSer176 1 pSer77, pSer132 1 pSer176; pSer132

14 1 pSer132 15 1 pSer176, pSer132

18 1 pSer176, pSer132

24 1 pSer132 *Modified from Zhan et al. [1], with permission from Wiley-VCH, copyright 2005.*

**hGH proteoforms (Spot No.) Splicing variants Phosphorylation (p) Deamidation (d)**

5 1 pSer176; dAsn178

16 1 pSer176 dAsn178

 *spectrum of a phosphopeptide ([M + 3H]3+: m/z = 899.47; retention time = 51.44 min; scan number 2167) derived from hGH proteoform 6 (Spot 6). Reproduced from Zhan et al. [1], with permission from Wiley-VCH,* 

pSer176

dAsn178

*DOI: http://dx.doi.org/10.5772/intechopen.82524*

2 3; 1/2

4 1

7 1

13 4; 3

17 1

19 1 20 1 21 1 22 1 23 3; 1

*PTMs were identified in hGH proteoforms.*

*Determination of the hGH splicing variants.*

**78**


*Application of Two-Dimensional Gel Electrophoresis in Combination with Mass Spectrometry… DOI: http://dx.doi.org/10.5772/intechopen.82524*

#### **Table 3.**

*Mass Spectrometry - Future Perceptions and Applications*

**78**

**GH variants**

1

**Tryptic peptide that covers the splicing site**

142DLEEGIQTLMGR153

121SVFANSLVYGASDSNVYDLLK141

46LHQLA FDTYQEFEEAYIPK64

68YSF LQNPQTSLCFSESIPTPSNR90

43AHRLHQLAFDTYQ EFEEAYIPK64

LHQLAFDTYQEF58NPQTSLCFSESIPTPSNR

AHRLHQLAFDTYQEF58NPQTSLCFSESIPTPSNR

LHQLAFDTYQEF58NPQTSLCFSESIPTPSNREETQQK

ISLLLIQ111TLMGR

ISLLLIQ111TLMGRLEDGSPR

SNLE LLRISLLLIQ111TLMGR

ISLLLIQSWLEPV117QIFK

ISLLLIQSWLEPV117QIFKQTYS K

SNLE LLRISLLLIQSWLEPV117QIFK

*Modified from Zhan et al. [1], with permission from Wiley-VCH, copyright 2005.*

**Table 2.**

*Determination of the hGH splicing variants.*

4

3

2

**Calculated [M + H] + ion (***m/z***)**

1361.7

1377.7 (MSO: 151)

2262.1

2342.1

2616.2

2673.2 (Cys-CAM: 79)

2706.3

3470.6

—

9

3527.4

—

—

—

—

1357.7

2; 23

—

—

—

—

—

2027.0

—

—

13

3527.7 (Cys-CAM: 64)

3834.8

3891.9 (Cys-CAM: 64)

4213.9

4271.0 (Cys-CAM: 64)

1357.8

1373.8 (MSO: 113)

2112.2

2128.2 (MSO: 113)

2183.3

2199.3 (MSO: 113)

2027.2 2634.5

2852.7

**Observed [M + H]+ ion (***m/z***) in PMF** 

**hGH proteoforms (Spot No.)**

1; 3–8; 10–12; 14–22; 24

**data**

One or two ions were detected to

exclude splicing variants 3 and 4

One, two or three ions were detected to

exclude splicing variant 2

*PTMs were identified in hGH proteoforms.*

#### **Figure 7.**

*MS2 spectrum of a phosphopeptide ([M + 3H]3+: m/z = 899.47; retention time = 51.44 min; scan number 2167) derived from hGH proteoform 6 (Spot 6). Reproduced from Zhan et al. [1], with permission from Wiley-VCH, copyright 2005.*


*Modified from Qian et al. [15], with permission from Frontiers publisher open access article, copyright 2018. \*Score > 0.5 means a statistically significant result.*

#### **Table 4.**

*Prediction of phosphorylation sites in hPRL prohormone (positions 1–227) with NetPhos server with a score more than 0.5.*


*Modified from Qian et al. [15], with permission from Frontiers publisher open access article, copyright 2018. Note: Asparagines predicted to be N-glycosylated are highlighted in bold font.*

#### **Table 5.**

*Prediction of N-glycosylation sites in hPRL prohormone (positions 1–227) with NetNGlyc 1.0 server with score more than 0.5.*

**81**

**Table 6.**

*more than 0.5.*

*Application of Two-Dimensional Gel Electrophoresis in Combination with Mass Spectrometry…*

from hPRL prohormone (positions 1–227) in human pituitary tissues. These data coupled with MS data clearly demonstrated six hPRL proteoforms were not derived from splicing.

MS/MS was an effective method to identify the PTMs and their modification sites of hGH and hPRL to clarify the reasons of formation of hGH proteoforms and hPRL proteoforms. MS/MS identified phosphorylation at sites Ser-77, Ser-132, and Ser-176 in many hGH proteoforms (**Table 3**). A representative MS/MS spectrum was shown to determine phosphorylation site Ser-77 in hGH proteoform 6 (Spot 6) (**Figure 7**). Also, deamidation was found in many hGH proteoforms. In addition, there would be other PTMs in hGH proteoforms that need to be further characterized.

**#Seq name Source Feature Start End Score Strand Frame Comment**

CARBOHYD 25 25 0.134588 . .

CARBOHYD 42 42 0.190888 . .

CARBOHYD 54 54 0.194926 . .

CARBOHYD 66 66 0.176466 . .

CARBOHYD 73 73 0.111052 . .

CARBOHYD 80 80 **0.613645** . .

CARBOHYD 85 85 **0.618483** . .

CARBOHYD 88 88 **0.602886** . .

CARBOHYD 89 89 **0.717093** . .

CARBOHYD 90 90 **0.928857** . .

CARBOHYD 93 93 **0.778272** . .

CARBOHYD 128 128 0.181904 . .

CARBOHYD 151 151 0.11243 . .

CARBOHYD 163 163 0.424529 . .

CARBOHYD 169 169 0.122664 . .

CARBOHYD 179 179 0.380532 . .

CARBOHYD 183 183 0.309982 . .

CARBOHYD 191 191 0.1589 . .

CARBOHYD 194 194 0.249957 . .

*Modified from Qian et al. [15], with permission from Frontiers publisher open access article, copyright 2018.* 

*Prediction of O-glycosylation sites in hPRL prohormone (position 1–227) with NetOGlyc 4.0 server with score* 

#POSITIVE

#POSITIVE

#POSITIVE

#POSITIVE

#POSITIVE

#POSITIVE

**3.4 MS/MS-identification of PTMs in hormone proteoforms**

*DOI: http://dx.doi.org/10.5772/intechopen.82524*

SEQUENCE netOGlyc

SEQUENCE netOGlyc

SEQUENCE netOGlyc

SEQUENCE netOGlyc

SEQUENCE netOGlyc

SEQUENCE netOGlyc

SEQUENCE netOGlyc

SEQUENCE netOGlyc

SEQUENCE netOGlyc

SEQUENCE netOGlyc

SEQUENCE netOGlyc

SEQUENCE netOGlyc

SEQUENCE netOGlyc

SEQUENCE netOGlyc

SEQUENCE netOGlyc

SEQUENCE netOGlyc

SEQUENCE netOGlyc

SEQUENCE netOGlyc

SEQUENCE netOGlyc




















*Note: The bold value means a statistically significantly positive result.*

*Application of Two-Dimensional Gel Electrophoresis in Combination with Mass Spectrometry… DOI: http://dx.doi.org/10.5772/intechopen.82524*

from hPRL prohormone (positions 1–227) in human pituitary tissues. These data coupled with MS data clearly demonstrated six hPRL proteoforms were not derived from splicing.
