**2. Materials and methods**

*Mass Spectrometry - Future Perceptions and Applications*

major regulators are growth hormone-releasing hormone (GHRH) synthesized in the hypothalamus, somatostatin derived from various tissues of the body, and ghrelin produced in the gastrointestinal tract. The mechanism of the effect of hGH is to directly bind with target organs for response of stimulation or it is indirectly influenced by the role of insulin-like growth factor-1 (IGF-1). The IGF-1 secreted from hepatocytes responds to the binding of hGH to surface receptors. If the Janus activating tyrosine kinases (JAKs) 1 and 2 are activated and bound to the cytoplasmic transcriptions factors STAT1, STAT3, and STAT5, and transported into the nucleus, thus enhanced gene transcription and metabolism would be induced, and the corresponding IGF-1 is generated and released into circulation. IGF-1 affects the growth and metabolism of peripheral tissues, which is able to understand as a combined impact of IGF-1 and hGH [2, 3]. The deficiency of hGH leads to growth deficit in children and the GH deficiency syndrome in adults, conversely, hGH hypersecretion results in gigantism or acromegaly [4–7]. It is obvious that hGH plays an important role in regulation of human life activities. In addition, hPRL should be paid more attention to its relevant functional study. The hPRL, a four long α-helix protein hormone, is mainly secreted by lactotrophs in the anterior pituitary gland except placental PRL [8]. The secretion of hPRL is usually regulated by PRL inhibitors such as dopamine from hypothalamus [9]. The hPRL is a polypeptide hormone with a wide variety of functions such as controls osmotic pressure and vascularization, participates in the immune response, and promotes neurogenesis in maternal and fetal brains [10, 11]. Moreover, the hPRL transports to the target organs and tissues via the blood circulation to bind to two different types of long or short hPRL receptors (hPRLRs) to activate signal pathways including JaK2 activation, Ras-Raf-MAPK pathway, modulatory pathways, PI3K and downstream pathways, and Stats [12]. However, hGH and hPRL exist multiple structural and functional formats, namely different proteoforms. The proteoform can clarify the function and effect of specific protein in human body and is an intuitive and visual expression of the gene. Therefore, the clarification of hormone proteoforms is necessary to understand its biological roles. Protein proteoforms are primarily derived from alternative splicing, post-translational modifications (PTMs), translocation, redistribution, and spatial conformation alterations [13]. The protein proteoforms can be identified with two-dimensional gel electrophoresis (2DGE) and mass

The isoelectric point (*pI*) and relative mass (*Mr*) are the basic characteristics of a proteoform. 2DGE is depended on different *pI* and *Mr* values to separate proteoforms, which first separates proteoforms by different *pI* values in the isoelectric focusing (IEF) direction, and second separates proteoforms by different *Mr* values in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) direction [14]. 2DGE-based Western blotting coupled with specific antibody can detect a proteoform of a specific protein. Thus, the proteoforms of hGH and hPRL are able to be separated and arrayed with 2DGE. MS is an effect method to characterize protein proteoforms and identify PTMs with an analysis of amino acid sequence and determination of PTM sites [15]. The combination of 2DGE and MS plays an important role in detection, identification, and quantification of proteoforms of hGH and hPRL. The MS methods that were used to study the proteoforms of hGH and hPRL were commonly divided into peptide fingerprint (PMF) and tandem mass spectrometry (MS/MS) analyses. PMF data are commonly obtained with matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) mass spectromety (MS), and MS/MS data can be obtained from MALDI-TOF-TOF MS or liquid chromatography (LC)-electrospray ionization (ESI)-quadruple-ion

**70**

trap (Q-IT) MS.

spectrometry (MS).

#### **2.1 Tissues and protein extraction**

The human control pituitary tissue samples were post-mortem tissues, and human pituitary adenoma tissue samples were obtained from neurosurgery. The detailed information of those tissue samples were described previously [15, 16]. The collected tissues were immediately frozen in liquid nitrogen, and stored at −80°C until used. The protein extraction of pituitary control and adenoma tissues was performed as described previously [17, 18]. In brief, the contaminated blood in each tissue sample was washed with 0.9% NaCl (3 mL, 5×). The proteins were extracted with protein extraction buffer that consists of 2 mol/L thiourea, 7 mol/L urea, 40 g/L CHAPS, 100 mmol/L dithiothreitol (DTT), 5 mol/L immobilized pH gradient (IPG) buffer pH 3–10 NL, and a trace of bromophenol blue, followed by centrifugation (15,000×*g*, 15 min, 4°C). The supernatant was collected as protein sample, and its protein concentration was determined with a Bio-Rad 2D Quant kit (Bio-Rad) [15].

## **2.2 2DGE and 2DGE-based Western blot**

Each protein sample was first separated by IEF with pH 3–10 NL IPG strips (180 mm × 3 mm × 0.5 mm), under the IEF condition that was a gradient from 0 to 250 V within 1 h (125 Vh), a gradient from 125 to 1000 V within 1 h (500 Vh), a gradient from 1000 to 8000 V within 1 h (4000 Vh), a step-and-hold at 8000 V for 4 h (32,000 Vh), and a step-and-hold at 500 V for 0.5 h (250 Vh) to achieve a total of 36,875 Vh within ~7.5 h [19]. After IEF, proteins were reduced with DTT, and alkylated with iodoacetamide, and then were separated with the 12% SDS-PAGE resolving gel (250 mm × 215 mm × 1.0 mm) in the Tris-glycine-SDS electrophoresis buffer that contained 192 mmol/L glycine, 25 mmol/L Tris-base, and 1 g/L SDS with an electrophoresis condition (250 V, 360 min, 25°C) [20]. The 2DGE-separated proteins were visualized with silver-staining [21].

After 2DGE, the proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane, blocked with bovine serum albumin (BSA), and incubated with hormone antibodies and secondary antibody. The proteins on the membrane were visualized with 5-bromo-4-chloro-3-indolyl phosphate [15].

The silver-stained 2D gels and 2DE-Western blot images were digitized and analyzed with Discovery Series PDQuest 2D Gel Analysis software [22, 23].

#### **2.3 MS-characterization**

### *2.3.1 MALDI-TOF MS*

The tryptic peptide mixture was purified with ZipTipC18, and analyzed with a Perspective Biosystems MALDI-TOF Voyager DE-RP MS (Framingham, MA, USA). The PMF data were obtained to search Swiss-Prot database with PeptIdent software for protein identification. The detailed procedure was described previously [22].
