**3.1 MS-identification of open reading frames (ORFs) of hGH and hPRL proteoforms within 2DGE pattern, and confirmed with Western blot**

Over 1000 protein spots were found in each pituitary 2D gel (n = 30). MS is an effective method to identify ORFs of a gene across 2DGE map, generally cannot identify proteoforms with the common procedure because MS only detects a partial amino acid sequence but not its complete sequence of a protein. However, each proteoform has its specific *pI* and *Mr*, which can be separated with 2DGE. 2DGE coupled with MS can effectively array and identify proteoforms that are derived from the same gene. Thus, 24 hGH proteoforms (**Figure 1A**) [1] and 6 hPRL proteoforms (**Figure 1B**) [15] were MS-characterized within pituitary 2DGE map.

Some hGH proteoforms and hPRL proteoforms within 2DGE map (**Figure 1**) were also validated with Western immunoblot. For example, four hPRL proteoforms were confirmed with 2DGE-Western blot (**Figure 2**). It demonstrated that 2DGE-MS was an effective and reliable approach to detect and identify proteoforms derived from hGH and hPRL genes. The other hGH proteoforms and hPRL proteoforms were not be validated with immnuoblot, which might be due to no-reactivity of antibody to a specific proteoform.

The hGH proteoforms or hPRL proteoforms in each 2D spot were identified with MS including PMF and MS/MS. For example, the protein in spot 6 in **Figure 1A** was identified as hGH isoform 1 (P01241) with MALDI-TOF PMF data (**Figure 3**). The protein in spot v6 in **Figure 1B** was identified as hPRL (P01236) with MALDI-TOF-TOF PMF data (**Figure 4**), and MS/MS data (**Figure 5**).

Therefore, 2DGE-MS clearly identified 24 hGH proteoforms and 6 hPRL proteoforms with different p*I*-*Mr* distributions on a 2DGE pattern. Furthermore,

**73**

glycosylation.

**Figure 2.**

**Figure 1.**

**proteoform in human pituitaries**

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

*2DGE patterns of hGH proteoforms (A) and hPRL proteoforms (B) in human pituitaries. The total proteins extracted from pituitary tissues were separated with IPG strip pH 3–10 NL and 12% SDS-PAGE gel, and stained with silver-staining. 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.*

one investigated the reasons to form those hormone proteoforms, including signal peptide, splicing variation, and PTMs such as deamination, phosphorylation, and

*hPRL proteoforms were validated with 2DGE-Western blot analysis. (A) Western blot image of hPRL proteoforms (rabbit anti-hPRL antibodies + goat anti-rabbit alkaline phosphatase-conjugated IgG). (B) Silver-stained image on a 2D gel after proteins were transferred to a PVDF membrane. Reproduced from* 

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

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

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

*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 1.**

*Mass Spectrometry - Future Perceptions and Applications*

The tryptic peptide mixture was purified with ZipTipC18, and analyzed with LC-ESI-Q-IT MS on an LCQDeca mass spectrometer (Thermo Finnigan, San Jose, CA, USA). The MS/MS data were obtained to search Swiss-Prot database for protein

The tryptic peptide mixture was purified with ZipTipC18, and analyzed with MALDI-TOF-TOF MS on Perspective Biosystems UltraFlex III MALDI-TOF-TOF (Bruker Daltonics). The MS and MS/MS data were obtained to search Swiss-Prot database for protein identification. The detailed procedure was described

Many phosphorylation sites of hGH proteoforms were identified with MS/MS, and deamidation was found in many hGH proteoforms. The PTM sites of hPRL in human pituitary adenoma and control tissues, including phosphorylation sites, N-glycosylation sites, and O-glycosylation sites were predicted with NetPhos 3.1 Server (http://www.cbs.dtu.dk/services/NetPhos) [24, 25], NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc) [26], and NetOGlyc 4.0 Server (http://

**3.1 MS-identification of open reading frames (ORFs) of hGH and hPRL proteoforms within 2DGE pattern, and confirmed with Western blot**

Over 1000 protein spots were found in each pituitary 2D gel (n = 30). MS is an effective method to identify ORFs of a gene across 2DGE map, generally cannot identify proteoforms with the common procedure because MS only detects a partial amino acid sequence but not its complete sequence of a protein. However, each proteoform has its specific *pI* and *Mr*, which can be separated with 2DGE. 2DGE coupled with MS can effectively array and identify proteoforms that are derived from the same gene. Thus, 24 hGH proteoforms (**Figure 1A**) [1] and 6 hPRL proteoforms (**Figure 1B**) [15] were MS-characterized within pituitary 2DGE map. Some hGH proteoforms and hPRL proteoforms within 2DGE map (**Figure 1**) were also validated with Western immunoblot. For example, four hPRL proteoforms were confirmed with 2DGE-Western blot (**Figure 2**). It demonstrated that 2DGE-MS was an effective and reliable approach to detect and identify proteoforms derived from hGH and hPRL genes. The other hGH proteoforms and hPRL proteoforms were not be validated with immnuoblot, which might be due to no-reactivity

The hGH proteoforms or hPRL proteoforms in each 2D spot were identified with MS including PMF and MS/MS. For example, the protein in spot 6 in **Figure 1A** was identified as hGH isoform 1 (P01241) with MALDI-TOF PMF data (**Figure 3**). The protein in spot v6 in **Figure 1B** was identified as hPRL (P01236) with MALDI-TOF-

Therefore, 2DGE-MS clearly identified 24 hGH proteoforms and 6 hPRL proteoforms with different p*I*-*Mr* distributions on a 2DGE pattern. Furthermore,

identification. The detailed procedure was described previously [22].

www.cbs.dtu.dk/services/NetOGlyc) [27], respectively.

*2.3.2 LC-ESI-Q-IT MS*

*2.3.3 MALDI-TOF-TOF MS*

previously [15].

**2.4 Bioinformatics**

**3. Results and discussion**

of antibody to a specific proteoform.

TOF PMF data (**Figure 4**), and MS/MS data (**Figure 5**).

**72**

*2DGE patterns of hGH proteoforms (A) and hPRL proteoforms (B) in human pituitaries. The total proteins extracted from pituitary tissues were separated with IPG strip pH 3–10 NL and 12% SDS-PAGE gel, and stained with silver-staining. 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.*

#### **Figure 2.**

*hPRL proteoforms were validated with 2DGE-Western blot analysis. (A) Western blot image of hPRL proteoforms (rabbit anti-hPRL antibodies + goat anti-rabbit alkaline phosphatase-conjugated IgG). (B) Silver-stained image on a 2D gel after proteins were transferred to a PVDF membrane. Reproduced from Qian et al. [15], with permission from Frontiers publisher open access article, copyright 2018.*

one investigated the reasons to form those hormone proteoforms, including signal peptide, splicing variation, and PTMs such as deamination, phosphorylation, and glycosylation.
