An Integrated Approach of Differential Mass Spectrometry and Gene Ontology Analysis Identified Novel Proteins Regulating Neuronal Differentiation and Survival

Abstract

MS-based quantitative proteomics is widely used for large scale identification of proteins. However, an integrated approach that offers comprehensive proteome coverage, a tool for the quick categorization of the identified proteins, and a standardized biological study method is needed for helping the researcher focus on investigating the proteins with biologically important functions. In this study, we utilized isobaric tagging for relative and absolute quantification ( MS-based quantitative proteomics strategies such as iTRAQ 1 (1) and stable isotope labeling with amino acids in cell culture (2) are powerfully effective for the comprehensive characterization of biological phenomena (1-5). Although these methods have been applied for cancer biomarker (6, 7) and drug target (8) discovery, their use in the elucidation of biological and functional processes has been limited because of certain technical problems that arise when attempting to meaningfully process the immense amount of data obtained from such experiments. The following four main issues are typically the sources of such difficulties. 1) Quantitative identification by one type of MS system may fail to cover the total proteome because of ionization efficiency differences, such as those between ESI and MALDI, for certain peptides, leading to theoretical limitations in proteome coverage. 2) The public protein databases are often insufficient for searching non-human species because of the limited available genomic information.3) The identification of the functions and biological processes of thousands of proteins is a formidable task because of the lack of simple and user-friendly software to automate gene ontology (GO) annotation. Furthermore it is 1 The abbreviations used are: iTRAQ, isobaric tagging for relative and absolute quantitation; NGF, nerve growth factor; GO, gene ontology; PC12PRS, PC12 proteome reference set; ICC, immunocytochemistry; siRNA, short interfering RNA; TCTP, translationally controlled tumor protein; ProT␣, prothymosin ␣; TrkA, tropomyosin-related kinase A; 2-D, two-dimensional; RP, reverse phase; GOA, gene ontology annotation; PCNA, proliferating cell nuclear antigen; PI, propidium iodide; PAIRBP1, plasminogen activator inhibitor 1 RNA-binding protein; PAI, plasminogen activator inhibitor; mESC, mouse embryonic stem cell; MAGE, melanoma antigen; p75NTR, p75 neurotrophin receptor; QqTOF, quadrupole/quadrupole/time-of-flight mass spectrometers. Research

Integrated Proteomics Identified Novel Activation of Dynein IC2-GR-COX-1 Signaling in Neurofibromatosis Type I (NF1) Disease Model Cells

“…In our previous study, molecules related to NGF-inducible neurite outgrowth in PC12 cells were analyzed using software called MANGO (8), which was reported as a novel, unique, and useful strategy in the global study of neural cells (8,19). We identified 39 up-regulated-proteins and 33 down-regulated-proteins in 48 h of NGF-treated PC12 cells in that study, and unique anti-apoptotic proteins were extracted as NGF induced proteins in the PC12 cells.…”

“…iTRAQ Sample Preparation, Fractionation, and Desalting-Protein samples (100 g) were precipitated using a 2-D Clean-Up kit (GE Healthcare), and the precipitates were dissolved in 10 l of 6 M urea. iTRAQ sample labeling was performed according to the manufacturer's protocol with minimum modification (8). The digests were incubated with eight different iTRAQ reagents (113-121) for 2 h as follows: iTRAQ 113 for NGF 0 h and control siRNA, 114 for 24-h control, 115 for 48-h control, 116 for 72-h control, 117 for 0-h NF1 siRNA, 118 for 24-h NF1 siRNA, 119 for 48-h NF1 siRNA, and 121 for 72-h NF1 siRNA.…”

“…Our previous research has examined specific genes and proteins related to phenotypic changes in neural tumors using integrated proteomic techniques such as 2D-DIGE combined with Pro-Q Diamond staining to identify phosphorylated proteins, isobaric tagging for relative and absolute quantitation (iTRAQ) (7,8), which provides information on peptide and protein quantitative expression levels from different sources in a single experiment by liquid chromatography combined with tandem mass spectrometry (LC-MS/MS), as well as DNA array technology, using the original data mining tool, MANGO (Molecular Annotation by Gene Ontology) (8). Processing the voluminous data arising from each analysis highlighted the need for a mining system for data integration.…”

Quantitative Proteomics with siRNA Screening Identifies Novel Mechanisms of Trastuzumab Resistance in HER2 Amplified Breast Cancers

“…Notable proteins that showed a decrease in protein ratio (Table III) included MAGED1 (ratio ϭ 0.24) and HER4/ERBB4 (ratio ϭ 0.11). Melanoma antigen family D1 (MAGED1) exhibits anti-proliferative, anti-invasive, and anti-migratory effects in MCF-7 and MDA-MB-231 breast cancer cell lines and causes morphological changes and inhibition of neurite outgrowths in neuronal cells (64,65). Recent genomic sequencing found MAGED1 to be mutated in 5% of multiple myeloma patients (66).…”

Translationally Controlled Tumor Protein Is a Novel Biological Target for Neurofibromatosis Type 1-associated Tumors

“…In our previous studies, using an integrated proteomics approach in neurofibromin-deficient PC12 cells (6), translationally controlled tumor protein (TCTP) was identified as an antiapoptotic factor uniquely regulated in response to NGF stimulation in PC12 cells (7). TCTP has been found in many eukaryotes, reported as multifunctional, and implicated in diverse processes, including growth, apoptosis, survival, development, protein synthesis, and transcription regulation (8).…”