Phosphorylation-Mediated Molecular Pathway Changes in Human Pituitary Neuroendocrine Tumors Identified by Quantitative Phosphoproteomics

Abstract

To investigate the biological role of protein phosphorylation in human nonfunctional pituitary neuroendocrine tumors (NF-PitNETs), proteins extracted from NF-PitNET and control tissues were analyzed with tandem mass tag (TMT)-based quantitative proteomics coupled with TiO₂ enrichment of phosphopeptides. A total of 595 differentially phosphorylated proteins (DPPs) with 1412 phosphosites were identified in NF-PitNETs compared to controls (p < 0.05). KEGG pathway network analysis of 595 DPPs identified nine statistically significant signaling pathways, including the spliceosome pathway, the RNA transport pathway, proteoglycans in cancer, SNARE interactions in vesicular transport, platelet activation, bacterial invasion of epithelial cells, tight junctions, vascular smooth muscle contraction, and protein processing in the endoplasmic reticulum. GO analysis revealed that these DPPs were involved in multiple cellular components (CCs), biological processes (BPs), and molecule functions (MFs). The kinase analysis of 595 DPPs identified seven kinases, including GRP78, WSTF, PKN2, PRP4, LOK, NEK1, and AMPKA1, and the substrate of these kinases could provide new ideas for seeking drug targets for NF-PitNETs. The randomly selected DPP calnexin was further confirmed with immunoprecipitation (IP) and Western blot (WB). These findings provide the first DPP profiling, phosphorylation-mediated molecular network alterations, and the key kinase profiling in NF-PitNET pathogenesis, which are a precious resource for understanding the biological roles of protein phosphorylation in NF-PitNET pathogenesis and discovering effective phosphoprotein biomarkers and therapeutic targets and drugs for the management of NF-PitNETs.

Keywords: TMT; TiO2; biomarkers; liquid chromatography; molecular network; phosphoprotein; phosphoproteome; phosphoproteomics; phosphorylation; pituitary neuroendocrine tumor (PitNET); signaling pathway; tandem mass spectrometry; therapeutic target.

Figure 1

MS/MS spectrum of the representative peptide. The representative peptide ⁶³³TPEELDDS*DFETEDFDVR⁶⁵² derived from catenin alpha-1 protein (P35221). The observed b- and y-ions were labeled in the MS/MS spectrum. S* = phosphorylated amino acid residue.

Figure 2

Distribution profile of phosphoproteins based on the number of phosphosites in NF-PitNETs and control pituitary tissues.

Figure 3

The volcano plot for 595 differentially phosphorylated proteins (DPPs) between NF-PitNETs and controls. The abscissa represents the logarithm of the expression level difference from a certain protein in NF-PitNETs and controls, namely log2(FC). The greater the absolute value of abscissa, the greater the expression with multiple differences between NF-PitNETs and controls. The y-coordinate represents the negative log of p-value, namely the −log10(p-value). The larger the ordinate value, the more significant DPPs were, and the more reliable the screened DPPs were.

Figure 4

Kinases identified with phosphoproteins.

Figure 5

Semiquantitative analysis of phosphorylated calnexin between NF-PitNETs and controls. The total proteins were extracted from NF-PitNETs (T) and control pituitary tissues (N). Calnexin was immunoprecipitated from the total proteins (T: n = 1.5 mg; N: n = 1.5 mg) with anti-calnexin antibodies (6 μg). For the negative control experiment to test the specificity of anti-calnexin antibodies, IgG (6 μg) was used to replace anti-calnexin antibodies for immunoprecipitation. (A). Half of the IP product was used to detect the expression level of calnexin in NF-PitNETs (T) and control pituitary tissues (N) with another different anti-calnexin antibody. A portion of anti-calnexin antibodies (Ab: n = 1 μg) and total proteins (N: n = 20 μg; T: n = 20 μg) were used as the control to immunoblot with another different anti-calnexin antibody. (B). Half of the IP product was used to detect the phosphorylation level of calnexin in NF-PitNETs (T) and control pituitary tissues (N) with anti-phosphoserine antibodies. A portion of anti-calnexin antibodies (Ab: n = 1 μg) and total proteins (N: n = 20 μg; T: n = 20 μg) were used as control to immunoblot with anti-phosphoserine antibody.