iTRAQ-Based Quantitative Proteomic Analysis of the Potentiated and Dormant Antler Stem Cells

Abstract

As the only known organ that can completely regenerate in mammals, deer antler is of real significance in the field of regenerative medicine. Recent studies have shown that the regenerative capacity of the antlers comes from the pedicle periosteum and the cells resident in the periosteum possess the attributes of stem cells. Currently, the molecular mechanism of antler regeneration remains unclear. In the present study, we compared the potentiated and dormant antler stem cells using isobaric tags for the relative and absolute quantification (iTRAQ) labeling of the peptides, coupled with two-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) to compare the proteome profiles. Proteins were identified by searching against the NCBI nr database and our own Cervine transcriptome database, and bioinformatics analysis was conducted to identify the differentially expressed proteins. Based on this searching strategy, we identified 169 differentially expressed proteins in total, consisting of 70 up- and 99 down-regulated in the potentiated vs. dormant antler stem cells. Reliability of the iTRAQ was confirmed via quantitative real-time polymerase chain reaction (qRT-PCR) to measure the expression of selected genes. We identified transduction pathways through the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, such as HIF-1 and PI3K-AKT signaling pathways that play important roles in regulating the regeneration of antlers. In summary, the initiation stage of antler regeneration, a process from dormant to potentiated states in antler stem cells, is regulated by multiple proteins and complicated signal networks.

Proteomic Analysis of Plasma Membrane Proteins of Antler Stem Cells Using Label-Free LC–MS/MS

“…In the PPCs, plasma membrane proteins accounted for 40.9% (671 out of 1639), and in the FPCs for 40.6% (685 out of 1686). We evaluated the proteomics of PPCs reported by Dong et al [ 12 ] in the same way, and found that 16.3% (407 out of 2500) were plasma membrane protein.…”

“…For some signal pathways, such as PI3K/AKT and VEGF, although not significantly enriched, key node proteins in the pathways were differentially expressed ( Figure S1 ). Transcriptome data published by our laboratory [ 12 ] were used to map whole signal pathways.…”

“…Due to their interfacial position in cells, plasma membrane proteins play central roles in various cellular processes including signal transduction, cell adhesion, and transport of molecules [ 10 ]. While the proteome of ASCs has been studied previously [ 11 , 12 , 13 ], the majority of differentially expressed proteins (DEPs) and special markers identified were natively soluble proteins. Membrane proteins, characterized by heterogeneity, hydrophobicity, and low abundance, were discarded in these studies since no special enrichment was adopted.…”

A proteomic approach reveals the differential protein expression in Drosophila melanogaster treated with red ginseng extract (Panax ginseng)

“…Both wild type and RGE-treated D. melanogaster (21 d old, about 20 flies each) were resuspended in 600 μL lysis buffer (7M Urea, 2M Thiourea, 4% CHAPS, and 1% phenylmethylsulfonyl fluoride (PMSF)). Bullet Blender (Next Advance, Averill Park, NY, USA) was used to break the tissue as described previously [13] . Supernatants were collected after sharking in ice for 2 h and centrifugation at 12,000 g at 4°C for 10 min.…”

iTRAQ-Based Identification of Proteins Related to Muscle Growth in the Pacific Abalone, Haliotis discus hannai

“…In contrast, recently developed proteomic techniques, such as isobaric tags for relative and absolute quantitation (iTRAQ), provide more reliable quantitative measurements and allow for comparisons among samples [ 11 ]. Indeed, iTRAQ, used in combination with two-dimensional liquid chromatography and tandem mass spectrometry (LC-MS/MS) is one of the most reliable methods of quantitative proteomic analysis available to date [ 12 , 13 , 14 ], especially in aquatic animals, such as medaka fish [ 15 ], Urobatis jamaicensis [ 16 ], zebrafish [ 17 ], Crassostrea gigas [ 18 ], Portunus trituberculatus [ 19 ], and Apostichopus japonicus [ 20 ].…”