Identification of Glioblastoma Phosphotyrosine-Containing Proteins with Two-Dimensional Western Blotting and Tandem Mass Spectrometry

Abstract

To investigate the presence of, and the potential biological roles of, protein tyrosine phosphorylation in the glioblastoma pathogenesis, two-dimensional gel electrophoresis- (2DGE-) based Western blotting coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis was used to detect and identify the phosphotyrosine immunoreaction-positive proteins in a glioblastoma tissue. MS/MS and Mascot analyses were used to determine the phosphotyrosine sites of each phosphopeptide. Protein domain and motif analysis and systems pathway analysis were used to determine the protein domains/motifs that contained phosphotyrosine residue and signal pathway networks to clarify the potential biological functions of protein tyrosine phosphorylation. A total of 24 phosphotyrosine-containing proteins were identified. Each phosphotyrosine-containing protein contained at least one tyrosine kinase phosphorylation motif and a certain structural and functional domains. Those phosphotyrosine-containing proteins were involved in the multiple signal pathway systems such as oxidative stress, stress response, and cell migration. Those data show 2DGE-based Western blotting, MS/MS, and bioinformatics are a set of effective approaches to detect and identify glioblastoma tyrosine-phosphorylated proteome and to effectively rationalize the biological roles of tyrosine phosphorylation in the glioblastoma biological systems. It provides novel insights regarding tyrosine phosphorylation and its potential role in the molecular mechanism of a glioblastoma.

Figure 1

Two-dimensional gel electrophoresis-based Western blot analysis of antiphosphotyrosine proteins in a glioblastoma tissue (160 μg protein per 2D gel). (a) Silver-stained image on a 2D gel before transfer of proteins to a PVDF membrane. (b) Silver-stained image on a 2D gel after transfer of proteins to a PVDF membrane. (c) Western blotting image of antiphosphotyrosine proteins (antiphosphotyrosine antibodies + secondary antibody). (d) Negative control of Western blotting to show the cross-reaction of the secondary antibody (only the secondary antibody, no antiphosphotyrosine antibody).

Figure 2

Tyrosine kinase phosphorylation motif and functional domains of putative phosphotyrosine-containing proteins in a glioblastoma tissue. INIT-Met, initiator methionine; HSP90, heat shock protein 90 family signature; GLU_RICH, glutamic acid-rich region profile; NLS_BP, bipartite nuclear localization signal profile; TPR, tetratricopeptide; ASN, N-glycosylation site; HSP70, heat shock protein 70 family signature; GTP, guanosine triphosphate; PLG, plasminogen.

Figure 3

Phosphotyrosine sites, tyrosine kinase phosphorylation motifs, and functional domains of phosphotyrosine-containing proteins in a glioblastoma tissue. SAM, the sterile α motif; PH, pleckstrin homology; Arf-GAP, ADP ribosylation factor GTPase-activating protein domain; Rho-GAP, Rho GTPase-activating proteins domain; SEMA, semaphorins; IPT/TIG, Ig-like, plexins, transcription factors/trigger factor-like protein; KRAB, Krueppel-associated box; GM2, the second monosialic ganglioside; HIS, histidine; MPN, domain at Mpr1p and Pad1p N-termini; EFTUD2, elongation factor Tu GTP-binding domain-containing protein 2; SNRNP200, small nuclear ribonucleoprotein 200 kDa.

Figure 4

Significant signaling pathway networks mined from phosphotyrosine-containing proteins in a glioblastoma tissue. Significant signaling pathway networks that are involved in human glioblastoma phosphotyrosine-containing proteins and that function in (a) cancer, organismal injury and abnormalities, reproductive system disease, and developmental disorder (merged Networks 1 and 3 in the Supplemental Table 3) and (b) cell morphology, cellular assembly and organization, cellular function, and maintenance (Network 2). A black solid edge denotes a direct relationship between two nodes (molecules: proteins; genes). A black unsolid edge denotes an indirect relationship between two nodes (molecules: proteins; genes). The various shapes of nodes denote the different functions. A curved line means intracellular translocation; a curved arrow means extracellular translocation.

Figure 5

Significant canonical pathways that are involved with phosphotyrosine-containing proteins in a glioblastoma tissue. Each significant canonical pathway was collected as in Supplemental Figure 1.

Figure 6

Significant disease biological events that are involved with phosphotyrosine-containing proteins in a glioblastoma tissue.

Figure 7

Experimental data-based diagram that rationalizes phosphotyrosine-containing proteins in the glioma biological system. The orange frame means identified phosphotyrosine-containing proteins. ANXA5, annexin A5; PLXD1, plexin-D1; TRAP1, TNFR-associated protein 1 (heat shock protein 75 kDa, mitochondrial); PRP8, pre-mRNA-processing-splicing factor 8; ACTB, actin, cytoplasmic 1; ZN569, zinc finger protein 569; GFAP, glial fibrillary acidic protein; HEXA, beta-hexosaminidase subunit alpha; ENOA, alpha-enolase; LDHB, L-lactate dehydrogenase B chain; 14-3-3, 14-3-3 protein; HSP90A, heat shock protein HSP 90-alpha; TBAIA, tubulin alpha-1A chain; CE192, centrosomal protein of 192 kDa; ACTBL, beta-actin-like protein 2; TBA8, tubulin alpha-8 chain; ARAP1, Arf-GAP with Rho-GAP domain, ANK repeat, and PH domain-containing protein 1; HXA1, homeobox protein Hox-A1; APOA1, apolipoprotein A-I; and HSP90B, heat shock protein HSP 90-beta.