Proteomics identifies differentially expressed proteins in neonatal murine thymus compared with adults

Abstract

Background: The thymus is an immune organ essential for life and plays a crucial role in the development of T cells. It undergoes a fetal to adult developmental maturation process occurring in mouse during the postnatal months. The molecular modifications underlying these ontogenic changes are essentially unknown. Here we used a differential proteomic-based technique (2D-Difference Gel Electrophoresis) coupled with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to search for key proteins in the postnatal development of the thymus. Eight different BALB/c mice were used in the study: four mice aged of 1 day (neonatal) and four mice aged of 60 days (adult). Protein samples derived from thymus were labeled and run in 2D-PAGE (Two-Dimensional Polyacrylamide Gel Electrophoresis). One whole-thymus tissue from each mouse was run on gels and each gel containing a pooled sample of the eight mice was run in parallel. The pooled sample was set as the internal pool, containing equal amount of each protein extract used in the experiment. Gels were matched and compared with Difference In-gel Analysis software. Differential spots were picked, in-gel digested and peptide mass fingerprints were obtained.

Results: Among the differentially regulated proteins in neonatal thymus group, 111 proteins were identified by mass spectrometry, of which 95 proteins were up-regulated and 16 proteins were down-regulated. The identified proteins belong to several functional categories, including cell proliferation, cycle and apoptosis, transcription regulation, signal transduction, nucleotide processing, proteolysis and translation, protein folding, metabolism, oxidoreduction, cytoskeleton, immune response, and embryonic development. The major interaction networks comprised of cellular function and maintenance, cellular assembly and organization, and metabolism were also identified by STRING analysis.

Conclusions: The demonstrated molecular changes are relevant for understanding thymus development as well as neonatal immune function, and they provide the diagnostic disease markers. Further studies will be required to describe in detail the role of the identified proteins in thymus maturation and in the specific functions of neonatal thymus.

Figure 1

Example of 2-D DIGE spectra in neonatal thymus and adult thymus. The neonatal thymus sample was labeled with Cy3 Dye (green spots) and the adult sample with Cy5 Dye (red spots). In all gels, internal standard was labeled with Cy2 Dye.

Figure 2

Classification of proteins by gene ontology according to their biological function (A) and subcellular localization (B). Assignments were made on the basis of information provided from the Swiss-Prot database.

Figure 3

The simulated functional network of proteins that were differentially displayed in the STRING database. (A) Related networks and interactions between the identified proteins were shown. (B) Tight interactions centered on cytoskeletal proteins and chaperones.

Figure 4

Validation of differentially displayed proteins between the neonatal and adult thymus. (A) 60 μg of protein extracts was used for protein detection. There were more abundant ACTN4, CH60 and ACTB expression, while less COF1 expression in neonates. GADPH was used as a normalization control. (B) A summary of densitometric analysis of Western blotting was shown as mean±SE calculated from three experiments. The results were normalized to individual GAPDH expression.