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 (iTRAQ)-based quantitative differential LC/MS/MS, functional annotation with a proprietary gene ontology tool (Molecular Annotation by Gene Ontology (MANGO)), and standard biochemical methods to identify proteins related to neuronal differentiation in nerve growth factor-treated rat pheochromocytoma (PC12) cells, which serve as a representative model system for studying neuronal biological processes. We performed MS analysis by using both nano-LC-MALDI-MS/MS and nano-LC-ESI-MS/MS for maximal proteome coverage. Of 1,482 non-redundant proteins semiquantitatively identified, 72 were differentially expressed with 39 up- and 33 down-regulated, including 64 novel nerve growth factor-responsive PC12 proteins. Gene ontology analysis of the differentially expressed proteins by MANGO indicated with statistical significance that the up-regulated proteins were mostly related to the biological processes of cell morphogenesis, apoptosis/survival, and cell differentiation. Some of the up-regulated proteins of unknown function, such as PAIRBP1, translationally controlled tumor protein, prothymosin alpha, and MAGED1, were further analyzed to validate their significant functions in neuronal differentiation by immunoblotting and immunocytochemistry using each antibody combined with a specific short interfering RNA technique. Knockdown of these proteins caused abnormal cell morphological changes, inhibition of neurite formation, and cell death during each course of the differentiation, confirming their important roles in neurite formation and survival of PC12 cells. These results show that our iTRAQ-MANGO-biological analysis framework, which integrates a number of standard proteomics strategies, is effective for targeting and elucidating the functions of proteins involved in the cellular biological process being studied.
Highlights
MS-based quantitative proteomics is widely used for large scale identification of proteins
MCP Papers in Press, June 13, 2009, DOI 10.1074/ mcp.M900179-MCP200 rite formation and survival of PC12 cells. These results show that our isobaric tagging for relative and absolute quantification (iTRAQ)-MANGO-biological analysis framework, which integrates a number of standard proteomics strategies, is effective for targeting and elucidating the functions of proteins involved in the cellular biological process being studied
Identification of Differentially Expressed Proteins in Response to nerve growth factor (NGF) in PC12 Cells—For the fourplex iTRAQ labeling, four independent, separately cultured cell lysates were prepared in parallel
Summary
For quantification of each protein identified in the MALDI or ESI analysis, a -fold change of each protein expression was calculated by comparing the average iTRAQ ratio of 116 and 117 as NGF-treated groups with the average ratio of 114 and 115 as control groups. After being washed with PBS, cells were incubated in primary antibodies diluted in PBS containing 5% bovine serum albumin followed by anti-mouse or -rabbit Alexa Fluorா 488-conjugated IgG (Invitrogen) for 60 min at room temperature. Cells transfected with siRNAs were cultured onto 35-mm collagen-coated culture dishes (Iwaki) under the condition of 10% horse serum and 5% fetal bovine serum and stimulated with 50 ng/ml 2.5 S NGF (Wako) at 48 h. The camera, shutters, and filter wheel were controlled by MetaMorph imaging software (Molecular Devices), and the images were collected every 10 min with an exposure time of 50 ms
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