**2. Methods**

*Proteoforms - Concept and Applications in Medical Sciences*

molecules, etc. [12].

PRL, and there are cases where dopamine is used to treat hyperprolactinemia [1]. PRL's biological functions include production, growth, development, immunoregulation, and metabolism [5, 6]. PRL can exert its biological functions only when it binds to its receptor and activates some signaling pathways [7]. According to the concept of proteoforms, a protein is defined as a set of proteoforms, due to different splicing, post-translational modifications (PTMs), and even unknown factors. Each proteoform has its own specific isoelectric point (p*I*) and molecular weight (*Mr*). For human PRL in the UniProt protein database, its p*I* is 6.5 and *Mr* is 25.88 kDa. However, Ben-Jonathan et al. found that human serum contained PRLs with *Mr* > 100, 40–60 and 16 kDa, besides the PRL with 25.88 kDa [8]. Qian et al. found six PRLs with different p*I* and *Mr* in human pituitary tissues by two-dimensional gel electrophoresis (2DGE) and mass spectrometry (MS) [9]. Similarly, Zhan et al. found 24 growth hormone (GH) with different p*I* and *Mr* in human pituitary tissues by 2-DGE and MS [10]. A possible reason of this difference of p*I* and *Mr* in human PRL and GH is that they undergo PTMs or splicing [11]. A proteoform is a specific form that protein exerts its final functions, which is derived from a gene undergoing splicing, transcription, translation, PTMs, translocation/re-distribution, and interaction with other

Recently 2DGE and MS have been recognized as high throughput and useful tool to study proteoforms [13–15]. 2DGE is able to separate each proteoform in the first dimension—isoelectric focusing (IEF) based on proteoform charge difference, and in the second dimension—sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) based on proteoform relative mass difference [16]. Therefore, 2DGE achieves proteoform separation based on the difference in p*I* and *Mr* of proteoforms. And then the protein (exactly proteoforms) on the 2D gel is transferred to a polyvinylidene fluoride membrane for detection with a specific antibody. The immunoreactive positive 2D gel spots represent the proteoforms of a protein. The proteoform in each immunoreactive positive spot was subjected to in-gel digestion with trypsin, and identification with MS [17, 18]. MS is a key technique to identify organic molecules and analyze the extreme structure of certain substances [19]. Especially, top-down MS can quickly and extremely accurately determine the molecular weight of biomacromolecules, which enables proteomics research from protein general identification to advanced structural studies and protein-protein interaction studies. Moreover, with the development of MS technology, the accuracy and sensitivity of mass spectrometers have been greatly improved. MS has its absolute advantages in the use of less sample, faster analysis, and simultaneous separation and identification. Therefore, 2DGE in combination with MS is presenting as a super-high approach in separation and identification of largescale human proteoforms [14]. If the stable isotope labeling is introduced to prepare the protein sample prior to 2DGE-MS, then 2DGE-MS can also quantify the abundance of a proteoform between two given conditions such as tumors

Pituitary adenomas are the common disease occurred in pituitary organ to severely impact on the human endocrine system. PRL is an important pituitary hormone. It has important scientific merit in clarification of PRL proteoform pattern changed in different subtypes of pituitary adenomas compared to control pituitaries. This book chapter focuses on the PRL proteoforms in human pituitary and investigates the PRL proteoform pattern alterations in pituitary adenomas relative to controls, with 2DGE and MS. These findings provide the scientific data to in-depth study the PRL functions and to discover PRL proteoform biomarker for

**48**

vs. controls [20].

PRL-related adenomas.

### **2.1 Pituitary tissue samples and preparation of protein samples**

Eight human post-mortem control pituitary tissues, five PRL-positive prolactinoma tissues, three non-hormone expressed nonfunctional pituitary adenoma (NF-NFPA) tissues, three luteinizing hormone (LH)-positive NFPA tissues, three follicle-stimulating hormone (FSH)-positive NFPA tissues, and three LH/FSH-both positive NFPA tissues were used to extract proteins, with the previously described procedure [21, 22]. The extracted protein of each tissue sample was used for 2DGE and MS analysis.

### **2.2 2DGE**

A amount (70 μg) of proteins was diluted into 350 μL of protein sample buffer (7 mol/L urea, 2 mol/L thiourea, 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 rehydration (18 h, 20°C) of precast IPG strips pH 3–10 NL (180 x 3 × 0.5 mm) in 18-cm IPG strip holder on an IPGphor instrument, and IEF (25°C) with parameters (Gradient 250 V and 1 h for 125 Vh, gradient at 1000 V and 1 h for 500 Vh, gradient at 8000 V and 1 h for 4000 Vh, step-and-hold at 8000 and 4 h for 32,000 Vh, step-and-hold at 500 V and 0.5 h for 250 Vh to achieve a total of 36,875 Vh). After IEF, the proteins on IPG strip were reduced (15 min) with DTT, and alkylated with iodoacetamide, followed by separation with 12% SDS-PAGE (250 × 215 × 1.0 mm) in an Ettan DALT II system (GE Healthcare, up to 12 gels at a time) with a constant voltage (250 V, 360 min). All 2DGE-arrayed proteins were stained with silver-staining [23], and then digitized and analyzed with Discovery Series PDQuest 2D Gel Analysis software [24, 25]. Each sample was performed for 3–5 times.

#### **2.3 2DGE-based Western blot**

The proteins in the 2D gel were partially transferred to a polyvinylidene fluoride (PVDF) membrane (0.8 mA/cm2 for 80 min) using Amersham Pharmacia Biotech Nova Blot semi-dry transfer instrument, followed by blocking (1 h, room temperature) with bovine serum albumin (BSA), incubation (1 h, room temperature) with rabbit anti-hPRL antibodies, incubation (1 h, room temperature) with goat anti-rabbit alkaline phosphatase conjugated IgG, and visualization with 5-bromo-4 chloro-3-indolyl phosphate. The detailed procedure was described previously [9].

#### **2.4 In-gel digestion with trypsin and MS identification of PRL**

The proteins in each Western blot-positive spot was performed in-gel digestion with trypsin, purification of tryptic peptides with ZipTipC18, followed by analysis with three types of MS instruments, including MALDI-TOF MS [24], LC-ESI-Q-IT MS [24], and MALDI-TOF-TOF MS [9]. The detailed procedure was described previously [9, 24]. The obtained peptide mass fingerprint (PMF) and tandem mass spectrometry (MS/MS) data were used to search Swiss-Prot human database for protein determination and PTM analysis.

#### **2.5 Bioinformatics and statistical analysis**

The phosphorylation sites, O-glycosylation sites, and N-glycosylation sites in the PRL amino acid sequence were predicted with NetPhos 3.1 Server (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).
