**3.3 PRL proteoform changes in human pituitary adenomas compared to controls**


The PRL proteoform pattern changed in different subtypes of pituitary adenomas compared to control pituitaries (**Table 3**). The ratio of each subtype of pituitary

#### **Table 1.**

*Human PRL prohormone amino acid sequence (position 1–227) and mature PRL (position 29–227). The signal peptide is position 1-28 in the bold letters.*

**51**

**Figure 1.**

*Prolactin Proteoform Pattern Changed in Human Pituitary Adenoma Relative to Control…*

adenoma relative to control pituitaries was decreased or unchanged. The proportional ratio of six PRL proteoforms among five subtypes of pituitary adenomas was

*PRL proteoform pattern in human pituitaries with a 2DGE gel image. Reproduced from Qian et al. [9], with* 

portion of PRL proteoform v5 is the largest. The PRL proteoform changes suggest

**3.4 Bioinformatics prediction of potential factors to form PRL proteoforms and** 

PRL is a hormone which is secreted by pituitary gland. PRL has a variety of biological functions. Only when it reaches a specific target organ and binds to its receptor can it play its biological function (**Figure 6**). PRL can bind to short PRL receptor or long PRL receptor and then plays its biological functions. The long or short PRL receptors definitely bind to different PRL proteoforms. PRL proteoforms are definitely derived from a PRL gene undergoing splicing, transcription, translation, PTMs, translocation/re-distribution, and interaction with other molecules, etc. Therefore, phosphorylation sites in hPRL (position 1–227) were predicted

/LH+

and PRL+

pituitary adenomas, the

pituitary adenoma, the pro-

changed (**Table 4** and **Figure 5**). In FSH+

their scientific merit for clinical application.

**pathway networks**

proportion of PRL proteoform v1 is the largest. In FSH+

*copyright permission from Frontiers in open access article, copyright 2018.*

*DOI: http://dx.doi.org/10.5772/intechopen.92836*

*Prolactin Proteoform Pattern Changed in Human Pituitary Adenoma Relative to Control… DOI: http://dx.doi.org/10.5772/intechopen.92836*

#### **Figure 1.**

*Proteoforms - Concept and Applications in Medical Sciences*

**3. Results and discussion**

(http://www.cbs.dtu.dk/services/NetPhos) [26, 27], NetOGlyc 4.0 Server (http:// www.cbs.dtu.dk/services/NetOGlyc) [28], and NetNGlyc 1.0 Server (http://www.cbs. dtu.dk/services/NetNGlyc) [29]. The PRL proteoform pattern changes were tested with the Chi-square test among different subtypes of pituitary adenomas (p < 0.05).

**3.1 The amino acid sequences of human PRL prohormone and mature PRL**

227 amino acids (position 1–227; 25.9 kDa), containing a signal peptide (position 1–28) (**Table 1**), which was assigned with Swiss-Prot accession No. P01236. However, the mature human PRL only contains 199 amino acids (position 29–227; 22.9 kDa), which removed the signal peptide (position 1–28), and secreted into the

circulation system to bind to its target organ for exerting PRL function.

that six PRL proteoforms are all PRL prohormone, but not mature PRL.

**3.3 PRL proteoform changes in human pituitary adenomas compared to** 

The PRL proteoform pattern changed in different subtypes of pituitary adenomas compared to control pituitaries (**Table 3**). The ratio of each subtype of pituitary

**MNIKGSPWKG SLLLLLVSNL LLCQSVAP**LP ICPGGAARCQ VTLRDLFDRA

VVLSHYIHNL SSEMFSEFDK RYTHGRGFIT KAINSCHTSS LATPEDKEQA

QQMNQKDFLS LIVSILRSWN EPLYHLVTEV RGMQEAPEAI LSKAVEIEEQ

TKRLLEGMEL IVSQVHPETK ENEIYPVWSG LPSLQMADEE SRLSAYYNLL

*Reproduced from Qian et al. [9], with copyright permission from Frontiers in open access article, copyright 2018.*

*Human PRL prohormone amino acid sequence (position 1–227) and mature PRL (position 29–227). The signal* 

**10 20 30 40 50**

60 70 80 90 100

110 120 130 140 150

160 170 180 190 200

**3.2 PRL proteoform pattern in human pituitaries**

210 220 HCLRRDSHKI DNYLKLLKCR IIHNNNC

*peptide is position 1-28 in the bold letters.*

In human pituitary, the PRL prohormone is synthesized in the ribosome, with

The PRL proteoform pattern was found in human pituitaries. Qian et al. [9] found six PRL proteoforms with 2DGE in human pituitaries and then verified four of six PRL proteoforms with 2DGE-based Western blot in human pituitaries (**Figures 1** and **2**). The p*I* and *Mr* of these PRL proteoforms are slightly different. Each PRL proteoform was digested with trypsin, and followed by MS and MS/MS analysis (**Figures 3** and **4**). The characteristic tryptic peptide are calculated to determine whether the signal peptide (position 1–28) in each PRL proteoform (**Table 2**), which was compared to the observed ions of each PRL proteoform. It found all PRL proteoforms all contained the tryptic peptide sequence MNIKGSPWK (position 1–9), which clearly demonstrated

**50**

**Table 1.**

**controls**

*PRL proteoform pattern in human pituitaries with a 2DGE gel image. Reproduced from Qian et al. [9], with copyright permission from Frontiers in open access article, copyright 2018.*

adenoma relative to control pituitaries was decreased or unchanged. The proportional ratio of six PRL proteoforms among five subtypes of pituitary adenomas was changed (**Table 4** and **Figure 5**). In FSH+ /LH+ and PRL+ pituitary adenomas, the proportion of PRL proteoform v1 is the largest. In FSH+ pituitary adenoma, the proportion of PRL proteoform v5 is the largest. The PRL proteoform changes suggest their scientific merit for clinical application.

#### **3.4 Bioinformatics prediction of potential factors to form PRL proteoforms and pathway networks**

PRL is a hormone which is secreted by pituitary gland. PRL has a variety of biological functions. Only when it reaches a specific target organ and binds to its receptor can it play its biological function (**Figure 6**). PRL can bind to short PRL receptor or long PRL receptor and then plays its biological functions. The long or short PRL receptors definitely bind to different PRL proteoforms. PRL proteoforms are definitely derived from a PRL gene undergoing splicing, transcription, translation, PTMs, translocation/re-distribution, and interaction with other molecules, etc. Therefore, phosphorylation sites in hPRL (position 1–227) were predicted

#### **Figure 2.**

*Verification of PRL proteoforms with 2DGE-based Western blot in human pituitaries. (A) Western blot image. (B) Silver-stained image. Reproduced from Qian et al. [9], with copyright permission from Frontiers in open access article, copyright 2018.*

#### **Figure 3.**

*PMF analysis of hPRL that was contained in spot v6. Reproduced from Qian et al. [9], with copyright permission from Frontiers in open access article, copyright 2018.*

with NetPhos 3.1 Server with a score more than 0.5. It obtained 22 statistically significantly phosphorylation sites in hPRL (position 1–227). N-glycosylation sites in hPRL (position 1–227) were predicted with NetNGlyc 1.0 Server with score more than 0.5. It obtained 10 statistically significant N-glycosylation sites in hPRL (position 1–227). O-glycosylation sites in hPRL (position 1–227) were predicted with NetOGlyc 4.0 Server with score more than 0.5. It obtained six statistically significant O-glycosylation sites in hPRL (position 1–227). These data suggest that PTMs such as phosphorylation and glycosylation might be the important reason to cause the PRL proteoforms.

**53**

**Table 2.**

*human pituitary.*

**Figure 4.**

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

*2018.*

*Prolactin Proteoform Pattern Changed in Human Pituitary Adenoma Relative to Control…*

*MS/MS analysis of the tryptic peptide 118SWNEPLYHLVTEVR131 that was derived from PRL in spot v6. Reproduced from Qian et al. [9], with copyright permission from Frontiers in open access article, copyright* 

505.2803 1–4 MNIK − 1060.5608 1–9 MNIKGSPWK + 3930.1893 1–38 MNIKGSPWKGSLLLLLVSNLLLCQSVAPLPICPGGAAR − 574.2983 5–9 GSPWK − 3443.9268 5–38 GSPWKGSLLLLLVSNLLLCQ SVAPLPICPGGAAR − 2888.6463 10–38 GSLLLLLVSNLLLCQSVAPL PICPGGAAR − 3589.0154 10–44 GSLLLLLVSNLLLCQSVAPL PICPGGAARCQVTLR − 954.5189 29–38 LPICPGGAAR − 1654.8879 29–44 LPICPGGAARCQVTLR − 2301.1954 29–49 LPICPGGAARCQVTLRDLFD R −

**Position Characteristic tryptic peptide sequence Observed** 

**[M+H]+**

**3.5 Potential clinical application of PRL proteoform pattern**

*+, this peptide ion was observed with mass spectrometry in each MS spectrum.* 

*−, this peptide was not observed with mass spectrometry.* 

Prolactin synthesized in the ribosome in the pituitary secretes into blood circulation to reach its target organ and exert its biological roles, which is closely associated with multiple physiological and pathological processes, including pituitary adenomas. This study found six PRL proteoforms with different with differential isoelectric point (p*I*) and relative mass (*Mr*) in control pituitary tissues, which were identified with 2DGE coupled with Western blot and MS. This prolactin

*Reproduced from Qian et al. [9], with copyright permission from Frontiers in open access article, copyright 2018.*

*Characteristic tryptic peptides to determine signal peptide (position1–28) within human PRL proteoforms in* 

*DOI: http://dx.doi.org/10.5772/intechopen.92836*

*Prolactin Proteoform Pattern Changed in Human Pituitary Adenoma Relative to Control… DOI: http://dx.doi.org/10.5772/intechopen.92836*

#### **Figure 4.**

*Proteoforms - Concept and Applications in Medical Sciences*

with NetPhos 3.1 Server with a score more than 0.5. It obtained 22 statistically significantly phosphorylation sites in hPRL (position 1–227). N-glycosylation sites in hPRL (position 1–227) were predicted with NetNGlyc 1.0 Server with score more than 0.5. It obtained 10 statistically significant N-glycosylation sites in hPRL (position 1–227). O-glycosylation sites in hPRL (position 1–227) were predicted with NetOGlyc 4.0 Server with score more than 0.5. It obtained six statistically significant O-glycosylation sites in hPRL (position 1–227). These data suggest that PTMs such as phosphorylation and glycosylation might be the important reason to

*PMF analysis of hPRL that was contained in spot v6. Reproduced from Qian et al. [9], with copyright* 

*permission from Frontiers in open access article, copyright 2018.*

*Verification of PRL proteoforms with 2DGE-based Western blot in human pituitaries. (A) Western blot image. (B) Silver-stained image. Reproduced from Qian et al. [9], with copyright permission from Frontiers in open* 

**52**

**Figure 3.**

**Figure 2.**

*access article, copyright 2018.*

cause the PRL proteoforms.

*MS/MS analysis of the tryptic peptide 118SWNEPLYHLVTEVR131 that was derived from PRL in spot v6. Reproduced from Qian et al. [9], with copyright permission from Frontiers in open access article, copyright 2018.*


*+, this peptide ion was observed with mass spectrometry in each MS spectrum.* 

*−, this peptide was not observed with mass spectrometry.* 

*Reproduced from Qian et al. [9], with copyright permission from Frontiers in open access article, copyright 2018.*

#### **Table 2.**

*Characteristic tryptic peptides to determine signal peptide (position1–28) within human PRL proteoforms in human pituitary.*

#### **3.5 Potential clinical application of PRL proteoform pattern**

Prolactin synthesized in the ribosome in the pituitary secretes into blood circulation to reach its target organ and exert its biological roles, which is closely associated with multiple physiological and pathological processes, including pituitary adenomas. This study found six PRL proteoforms with different with differential isoelectric point (p*I*) and relative mass (*Mr*) in control pituitary tissues, which were identified with 2DGE coupled with Western blot and MS. This prolactin

#### *Proteoforms - Concept and Applications in Medical Sciences*


*Modified from Qian et al. [9], with copyright permission from Frontiers in open access article, copyright 2018. a Characterized with LC-ESI MS/MS.*

*b Characterized with LC-ESI-MS/MS and MALDI-TOF PMF.*

*All other proteins were characterized with MALDI-TOF PMF. Con, control; −, decreased relative to controls; −100, lost relative to controls; 1, no change relative to controls; Mr, kDa.*

#### **Table 3.**

*Prolactin proteoform pattern changed in different subtypes of pituitary adenomas compared to control pituitaries.*


*Reproduced from Qian et al. [9], with copyright permission from Frontiers in open access article, copyright 2018. \* Chi-square test = 360.606, p = 0.000 (p < 0.01) among five subtypes of pituitary adenomas.*

#### **Table 4.**

*Proportional ratio changes of PRL proteoforms among five subtypes of pituitary adenomas.*

#### **Figure 5.**

*Proportional ratio changes of PRL proteoforms among five subtypes of pituitary adenomas. Reproduced from Qian et al. [9], with copyright permission from Frontiers in open access article, copyright 2018.*

**55**

*Prolactin Proteoform Pattern Changed in Human Pituitary Adenoma Relative to Control…*

proteoform pattern was significantly changed among different hormone-subtypes

*PRL proteoform-driven signaling pathway via the short or long PRL receptors. Reproduced from Qian et al.* 

serum PRL proteoforms. The reason is that pituitary tissues are impossible to obtain for clinical diagnosis, and prolactin must secrete into blood to exert its biological roles, we strongly believe serum PRL proteoforms exist and the serum PRL proteoform pattern changes among different pituitary adenomas. Therefore, we will further analyze serum PRL proteoform pattern changes among different subtypes of pituitary adenomas, and develop the PRL proteoform pattern as biomarker for prediction, diagnosis, or prognostic assessment of pituitary adenoma occurrence,

Six PRL proteoforms were identified in human pituitary tissue with 2DGE and MS analyses, and four of six PRL proteoforms were validated with 2DGE-based Western blot, MS, and MS/MS analyses. There were significant differences in PRL proteoform pattern among five different subtypes of pituitary adenomas (LH+

six PRL proteoforms are PRL prohormone. PRL proteoforms might be derived from PTMs such as phosphorylation, deamidation, and glycosylation. Further, different PRL proteoforms might bind to different PRL receptors to produce different physiological functions. These findings provide scientific basis for in-depth understanding of pituitary adenomas, and help develop biomarkers for treatment of pituitary adenoma patients. The serum PRL proteoform pattern has important clinical application value for prediction, diagnosis, and prognostic assessment of pituitary

The authors acknowledge the financial supports from the Hunan Provincial Hundred Talent Plan (to X.Z.), National Natural Science Foundation of China

, FSH<sup>+</sup>

) tissues. This result suggests the potentially important clinical value of

, and LH<sup>+</sup>

) (P < 0.05). Moreover, MS analysis revealed that

/FSH+

) and prolacti-

,

*DOI: http://dx.doi.org/10.5772/intechopen.92836*

of nonfunctional pituitary adenoma (NF<sup>−</sup>, LH<sup>+</sup>

*[9], with copyright permission from Frontiers in open access article, copyright 2018.*

noma (PRL<sup>+</sup>

**Figure 6.**

progression, and prognosis.

, FSH+

/LH+

, and PRL+

**4. Conclusions**

NF<sup>−</sup>, FSH+

adenomas.

**Acknowledgements**

*Prolactin Proteoform Pattern Changed in Human Pituitary Adenoma Relative to Control… DOI: http://dx.doi.org/10.5772/intechopen.92836*

#### **Figure 6.**

*Proteoforms - Concept and Applications in Medical Sciences*

**Swiss-Prot no.**

**PRL proteoform NF<sup>−</sup> (%) FSH+**

*Characterized with LC-ESI-MS/MS and MALDI-TOF PMF.*

*lost relative to controls; 1, no change relative to controls; Mr, kDa.*

*Characterized with LC-ESI MS/MS.*

**/LH+**

*Prolactin proteoform pattern changed in different subtypes of pituitary adenomas compared to control* 

*p***I** *M<sup>r</sup>* **Ratio** 

**(NF<sup>−</sup>: Con)**

V1 P01236 6.1 26.0 −8.3 −99.9 −46.2 −12.6 −3.4 V2a P01236 6.3 26.4 −4.9 −3.8 1 −4.1 1 V3 P01236 6.3 27.9 1 −12.3 −14.6 −26.2 1 V4b P01236 6.5 26.1 −100 −19.0 −17.6 −20.1 1 V5 P01236 6.8 25.9 −100 −19.7 −100 −36.7 1 V6 P01236 6.7 25.9 −100 −32.6 −11.3 −33.6 1 *Modified from Qian et al. [9], with copyright permission from Frontiers in open access article, copyright 2018.*

**Ratio (FSH+ /**

**Ratio (FSH+ : Con)**

**Ratio (LH+ : Con)**

**Ratio (PRL: Con)**

**LH+ : Con)**

*Chi-square test = 360.606, p = 0.000 (p < 0.01) among five subtypes of pituitary adenomas.*

*Proportional ratio changes of PRL proteoforms among five subtypes of pituitary adenomas.*

*Proportional ratio changes of PRL proteoforms among five subtypes of pituitary adenomas. Reproduced from* 

*Qian et al. [9], with copyright permission from Frontiers in open access article, copyright 2018.*

V1 2.64 53.34 24.24 9.45 40.48 V2 1.56 2.03 0.52 3.08 11.90 V3 0.31 6.57 7.66 19.65 11.91 V4t 31.83 10.14 9.18 15.08 11.90 V5 31.83 10.52 52.47 27.53 11.91 V6 31.83 17.40 5.93 25.21 11.90 Total 100.00 100.00 100.00 100.00 100.00 *Reproduced from Qian et al. [9], with copyright permission from Frontiers in open access article, copyright 2018.*

*All other proteins were characterized with MALDI-TOF PMF. Con, control; −, decreased relative to controls; −100,* 

 **(%) FSH+**

 **(%) LH+**

 **(%) PRL+**

 **(%)**

**54**

**Figure 5.**

*\**

**Table 4.**

**PRL proteoform no.**

*a*

*b*

**Table 3.**

*pituitaries.*

*PRL proteoform-driven signaling pathway via the short or long PRL receptors. Reproduced from Qian et al. [9], with copyright permission from Frontiers in open access article, copyright 2018.*

proteoform pattern was significantly changed among different hormone-subtypes of nonfunctional pituitary adenoma (NF<sup>−</sup>, LH<sup>+</sup> , FSH<sup>+</sup> , and LH<sup>+</sup> /FSH+ ) and prolactinoma (PRL<sup>+</sup> ) tissues. This result suggests the potentially important clinical value of serum PRL proteoforms. The reason is that pituitary tissues are impossible to obtain for clinical diagnosis, and prolactin must secrete into blood to exert its biological roles, we strongly believe serum PRL proteoforms exist and the serum PRL proteoform pattern changes among different pituitary adenomas. Therefore, we will further analyze serum PRL proteoform pattern changes among different subtypes of pituitary adenomas, and develop the PRL proteoform pattern as biomarker for prediction, diagnosis, or prognostic assessment of pituitary adenoma occurrence, progression, and prognosis.

### **4. Conclusions**

Six PRL proteoforms were identified in human pituitary tissue with 2DGE and MS analyses, and four of six PRL proteoforms were validated with 2DGE-based Western blot, MS, and MS/MS analyses. There were significant differences in PRL proteoform pattern among five different subtypes of pituitary adenomas (LH+ , NF<sup>−</sup>, FSH+ , FSH+ /LH+ , and PRL+ ) (P < 0.05). Moreover, MS analysis revealed that six PRL proteoforms are PRL prohormone. PRL proteoforms might be derived from PTMs such as phosphorylation, deamidation, and glycosylation. Further, different PRL proteoforms might bind to different PRL receptors to produce different physiological functions. These findings provide scientific basis for in-depth understanding of pituitary adenomas, and help develop biomarkers for treatment of pituitary adenoma patients. The serum PRL proteoform pattern has important clinical application value for prediction, diagnosis, and prognostic assessment of pituitary adenomas.

#### **Acknowledgements**

The authors acknowledge the financial supports from the Hunan Provincial Hundred Talent Plan (to X.Z.), National Natural Science Foundation of China

(Grant No. 81572278 and 81272798 to X.Z.), China "863" Plan Project (Grant No. 2014AA020610-1 to X.Z.), the Hunan Provincial Natural Science Foundation of China (Grant No. 14JJ7008 to X.Z.), and the Xiangya Hospital Funds for Talent Introduction (to X.Z.).
