A multiplexed quantitative proteomics approach for investigating protein expression in the developing central nervous system.
Cell transplantation using stem cell-derived neurons is commonly viewed as a candidate therapy for neurodegenerative diseases. However, methods for differentiating stem cells into homogenous populations of neurons suitable for transplant remain elusive. This suggests that there are as yet unknown signalling factors working in vivo to specify neuronal cell fate during development. These factors could be manipulated to better differentiate stem cells into neural populations useful for therapeutic transplantation. Here a quantitative proteomics approach is described for investigating cell signalling in the developing central nervous system (CNS), using the embryonic ventral mesencephalon as a model. Briefly, total protein was extracted from embryonic ventral midbrain tissue before, during and after the birth of dopaminergic neurons, and digested using trypsin. Two-dimensional liquid chromatography, coupled with tandem mass spectrometry, was then used to identify proteins from the tryptic peptides. Isobaric tagging for relative and absolute quantification (iTRAQ) reagents were used to label the tryptic peptides and facilitate relative quantitative analysis. The success of the experiment was confirmed by the identification of proteins known to be expressed in the developing ventral midbrain, as well as by Western blotting, and immunolabelling of embryonic tissue sections. This method of protein discovery improves upon previous attempts to identify novel signalling factors through microarray analysis. Importantly, the methods described here could be applied to virtually any aspect of development.
Orme
Rowan P
RP
Institute for Science and Technology in Medicine, Department of Life Sciences, Huxley Building, Keele University, Keele, Staffordshire, ST5 5BG, England, United Kingdom.
Gates
Monte A
MA
Fricker-Gates
Rosemary A
RA
D000595:Amino Acid Sequence; D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D002490:Central Nervous System / Q000737:chemistry* / Q000196:embryology* / Q000378:metabolism; D019295:Computational Biology / Q000379:methods; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D008636:Mesencephalon / Q000737:chemistry / Q000196:embryology* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000502:physiology; D012984:Software / Q000639:trends; D013234:Stem Cells / Q000737:chemistry / Q000378:metabolism
D009419:Nerve Tissue Proteins; D010446:Peptide Fragments