**Acronyms and abbreviations**

*Mass Spectrometry - Future Perceptions and Applications*

needed to be confirmed with MS/MS in future studies.

**4. Conclusions**

**Acknowledgements**

**Conflict of interest**

people or organizations.

**Author's contributions**

(Grant Nos. 81572278 and 81272798 to X.Z.).

For hPRL proteoforms, bioinformatics including NetPhos 3.1 Server (http:// www.cbs.dtu.dk/services/NetPhos) [24, 25] predicted 14 pS sites, 5 pT sites, and 3 pY sites in the hPRL (**Table 4**), NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/ services/NetNGlyc) [26] predicted ten significantly N-glycosylated sites (**Table 5**), and NetOGlyc 4.0 Server (http://www.cbs.dtu.dk/services/NetOGlyc) [27] predicted six significantly O-glycosylated sites in the hPRL (**Table 6**) in human pituitaries. These predicted PTM sites in hPRL proteoforms provided clues and

hGH and hPRL are two important hormones in human endocrine systems, which

are synthesized in the pituitary gland and secreted into the circulation system. Clarification of hGH proteoforms and hPRL proteoforms in human pituitary is essential step to elucidate their biological functions. Alternative splicing and PTMs are two important factors to cause proteoforms. 2DGE effectively presented 24 hGH proteoforms and 6 hPRL proteoforms with different p*I*-*Mr* distributions in 2DGE pattern of pituitary tissue proteome. MS/MS effectively identified their splicing variants and PTMs: (i) 24 hGH proteoforms in pituitary removed their signal peptide, whereas 6 hPRL proteoforms in human pituitary did not remove their signal peptide. (ii) 24 hGH proteoforms in human pituitary are derived from 4 types of alternative splicing variants, whereas 6 hPRL proteoforms do not exist any alternative splicing variants. (iii) PTMs pSer-77, pSer-132, and pSer-176 were identified in some of 24 hGH proteoforms, whereas although no PTMs were identified in hPRL proteoforms with MS/MS. However, phosphorylation, N-glycosylation, and O-glycosylation have been predicted with bioinformatics in hPRL proteoforms. Deamidation was presented in both hGH proteoforms and hPRL proteoforms. Therefore, 2DGE coupled with MS plays crucial roles in detection, identification, and quantification of hormone (hGH and hPRL) proteoforms, which benefits insight into the molecular mechanisms and discovery of effective biomarkers of hormone-related diseases.

The authors acknowledge the financial supports from the Xiangya Hospital Funds for Talent Introduction (to X.Z.), the Hunan Provincial "Hundred Talent Plan" program (to X.Z.), the Hunan Provincial Natural Science Foundation of China (Grant No. 14JJ7008 to X.Z.), China "863" Plan Project (Grant No. 2014AA020610-1 to X.Z.), and the National Natural Science Foundation of China

We declare that we have no financial and personal relationships with other

X.Z. conceived the concept, designed the book chapter, wrote and critically revised the book chapter, coordinated and was responsible for the correspondence

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