**3.2 MS-determination of signal peptide contained in each hGH or hPRL proteoform in human pituitaries**

If signal peptide is contained in the sequence of hGH or hPRL, then it means that hormone is a prohormone (**Figure 6**). It is necessary to determine whether signal

#### **Figure 3.**

*MALDI-TOF MS spectrum of hGH isoform 1 (spot 6). T = autodigested fragments of trypsin. M\* = oxidized Met. C# = carbamidomethyl-Cys. N@ = deamidated Asn. Reproduced from Zhan et al. [1], with permission from Wiley-VCH, copyright 2005.*

#### **Figure 4.**

*MALDI-TOT-TOF MS spectrum of hPRL (spot v6). Reproduced from Qian et al. [15], with permission from Frontiers publisher open access article, copyright 2018.*

peptide is contained in each hGH proteoform or hPRL proteoform. MS can identify the characteristic tryptic peptide sequences before and after removal of signal peptide in hGH or hPRL (**Table 1**). For 24 hGH proteoforms in human pituitaries, checking all MS data of those 24 hGH proteoforms, the tryptic peptide sequence FPTIPLSR (position 27–34) (**Figure 3**) was detected for each hGH proteoform, but no any characteristic tryptic peptide sequence before removal of signal peptide was found (**Table 1**). It clearly demonstrated that no signal peptide sequence was contained in those 24 hGH proteoforms in the 2DGE pattern (**Figure 1A**). For 6 hPRL proteoforms in human pituitaries, checking all MS data of those 6 hPRL proteoforms, the tryptic peptide sequence MNIKGSPWK (position 1–9) (**Figure 4**) was detected for each hPRL proteoform, but no any characteristic tryptic peptide sequence after removal of signal peptide was found (**Table 1**). It clearly demonstrated that signal peptide sequence was contained in those 6 hPRL proteoforms in the 2DGE pattern (**Figure 1B**).

**75**

**Figure 5.**

**Figure 6.**

*article, copyright 2018.*

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

*MALDI-TOF-TOF MS/MS spectrum of the tryptic peptide 118SWNEPLYHLVTEVR131 from hPRL in spot v6. Reproduced from Qian et al. [15], with permission from Frontiers publisher open access article, copyright 2018.*

**3.3 MS-identification of splicing variants of hGH in human pituitaries**

spot 13. hGH variant 1 existed in the rest 20 spots.

MS is an effective method to identify splicing variants of hGH. hGH splicing variant 2 is derived from deletion of positions 58–72 from hGH, hGH splicing variant 3 is derived from deletion of positions 111-148 from hGH, and hGH splicing variant 4 is derived from deletion of positions 117-162 from hGH (**Figure 6**). The characteristic tryptic peptides for each hGH splicing variant can be theoretically obtained and observed by MS (**Table 2**). The MS data of 24 hGH proteoforms were checked one-by-one. The results showed that hGH splicing variant 2 existed in spot 9, hGH splicing variant 3 existed in spots 2 and 23, and hGH splicing variant 4 existed in

*The amino acid sequences of hGH and hPRL prohormones. Modified from Zhan et al. [1], with permission from Wiley-VCH, copyright 2005; and from Qian et al. [15], with permission from Frontiers publisher open access* 

However, hPRL does not have splicing variants. The *Mr* of hPRL proteoforms was 26.0 kDa for v1, 26.1 kDa for v4, 25.9 kDa for v5, and 25.9 kDa for v6 in 2DGE pattern (**Figure 1**), which was almost equal to the *Mr* (25.9 kDa) of hPRL prohormone. Moreover, the *Mr* of hPRL proteoforms was 26.4 kDa for v2, and 27.9 kDa for v3, which is slightly greater than *Mr* (25.9 kDa) of hPRL prohormone. These data further confirmed that those six hPRL proteoforms were not generated from mature hPRL (position 29–227) but

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

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

#### **Figure 5.**

*Mass Spectrometry - Future Perceptions and Applications*

peptide is contained in each hGH proteoform or hPRL proteoform. MS can identify the characteristic tryptic peptide sequences before and after removal of signal peptide in hGH or hPRL (**Table 1**). For 24 hGH proteoforms in human pituitaries, checking all MS data of those 24 hGH proteoforms, the tryptic peptide sequence FPTIPLSR (position 27–34) (**Figure 3**) was detected for each hGH proteoform, but no any characteristic tryptic peptide sequence before removal of signal peptide was found (**Table 1**). It clearly demonstrated that no signal peptide sequence was contained in those 24 hGH proteoforms in the 2DGE pattern (**Figure 1A**). For 6 hPRL proteoforms in human pituitaries, checking all MS data of those 6 hPRL proteoforms, the tryptic peptide sequence MNIKGSPWK (position 1–9) (**Figure 4**) was detected for each hPRL proteoform, but no any characteristic tryptic peptide sequence after removal of signal peptide was found (**Table 1**). It clearly demonstrated that signal peptide sequence was

*MALDI-TOT-TOF MS spectrum of hPRL (spot v6). Reproduced from Qian et al. [15], with permission* 

*from Frontiers publisher open access article, copyright 2018.*

*MALDI-TOF MS spectrum of hGH isoform 1 (spot 6). T = autodigested fragments of trypsin. M\* = oxidized Met. C# = carbamidomethyl-Cys. N@ = deamidated Asn. Reproduced from Zhan et al. [1], with permission from* 

contained in those 6 hPRL proteoforms in the 2DGE pattern (**Figure 1B**).

**74**

**Figure 3.**

**Figure 4.**

*Wiley-VCH, copyright 2005.*

*MALDI-TOF-TOF MS/MS spectrum of the tryptic peptide 118SWNEPLYHLVTEVR131 from hPRL in spot v6. Reproduced from Qian et al. [15], with permission from Frontiers publisher open access article, copyright 2018.*


#### **Figure 6.**

*The amino acid sequences of hGH and hPRL prohormones. Modified from Zhan et al. [1], with permission from Wiley-VCH, copyright 2005; and from Qian et al. [15], with permission from Frontiers publisher open access article, copyright 2018.*

#### **3.3 MS-identification of splicing variants of hGH in human pituitaries**

MS is an effective method to identify splicing variants of hGH. hGH splicing variant 2 is derived from deletion of positions 58–72 from hGH, hGH splicing variant 3 is derived from deletion of positions 111-148 from hGH, and hGH splicing variant 4 is derived from deletion of positions 117-162 from hGH (**Figure 6**). The characteristic tryptic peptides for each hGH splicing variant can be theoretically obtained and observed by MS (**Table 2**). The MS data of 24 hGH proteoforms were checked one-by-one. The results showed that hGH splicing variant 2 existed in spot 9, hGH splicing variant 3 existed in spots 2 and 23, and hGH splicing variant 4 existed in spot 13. hGH variant 1 existed in the rest 20 spots.

However, hPRL does not have splicing variants. The *Mr* of hPRL proteoforms was 26.0 kDa for v1, 26.1 kDa for v4, 25.9 kDa for v5, and 25.9 kDa for v6 in 2DGE pattern (**Figure 1**), which was almost equal to the *Mr* (25.9 kDa) of hPRL prohormone. Moreover, the *Mr* of hPRL proteoforms was 26.4 kDa for v2, and 27.9 kDa for v3, which is slightly greater than *Mr* (25.9 kDa) of hPRL prohormone. These data further confirmed that those six hPRL proteoforms were not generated from mature hPRL (position 29–227) but


**77**

**Hormone**

hPRL (signal peptide sequence 1–28)

**Before and after removal of signal peptide**

Before

1–4 1–9 1–38

5–9 5–38 10–38 10–44 29–38 29–44 29–49 *Modified from Zhan et al. [1], with permission from Wiley-VCH, copyright 2005; and from Qian et al. [15], with permission from Frontiers publisher open access article, copyright 2018. Note: +: this peptide* 

*ion was observed in each MS spectrum. −: this peptide ion was not observed by MS. ±: tryptic peptide ion was detected in some but not all PMF data from 24 hGH proteoforms.*

**Table 1.**

*Characteristic tryptic peptides before or after removal of signal peptide sequence in hGH or in hPRL.*

After

MNIK MNIKGSPWK MNIKGSPWKGSLLLLLVSNL LLCQSVAPLPICPGGAAR

GSPWK GSPWKGSLLLLLVSNLLLCQ SVAPLPICPGGAAR

GSLLLLLVSNLLLCQSVAPL PICPGGAAR

GSLLLLLVSNLLLCQSVAPL PICPGGAARCQVTLR

LPICPGGAAR LPICPGGAARCQVTLR

LPICPGGAARCQVTLRDLFD R

**Position**

**Characteristic tryptic peptide sequence**

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

505.2803 1060.5608 3930.1893

574.2983 3443.9268 2888.6463 3589.0154

954.5189 1654.8879 2301.1954

−

−

−

−

−

−

−

−

+ −

**Observed [M + H]+**

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

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


