Proteomic analysis of rare molecular species of translated polypeptides from a mouse fetal thymus cDNA library

• Published in: Proteomics (Weinheim) 2001-04, Vol.1 (4), p.560-573
• Main Authors: Lefkovits, Ivan, Kettman, John R., Frey, Johann Rudolf
• Format: Article
• Language: English
• Subjects: Animals; cDNA array; Cloning, Molecular; DNA, Complementary - genetics; Electrophoresis, Gel, Two-Dimensional; Fetal Proteins - genetics; Fetal Proteins - isolation & purification; Gene Library; Lymphocyte proteins; Mice; Mice, Inbred BALB C; Plasmids - genetics; Protein Biosynthesis; Proteome - genetics; Proteome - isolation & purification; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; RNA, Messenger - genetics; RNA, Messenger - isolation & purification; Thymus Gland - metabolism; Two-dimensional gel electrophoresis
• ISSN: 1615-9853; 1615-9861
• DOI: 10.1002/1615-9861(200104)1:4<560::AID-PROT560>3.0.CO;2-S

Proteomic patterns from an ordered cDNA library of mouse fetal thymus origin of an overall complexity of 1536 clones are described. Patterns have been analyzed at 96, 12, or 8 clones in a mixture, or as individual clones. Clones yield in some instances a single spot, in other cases a complex cluster or family of spots is formed. The determination of the clonal address (a six‐digit number, indicating the interception of three pooling dimensions) by inspection of pools of 8, 12 or 16 clones is a reliable approach; nevertheless a complete proof consists in retrieving the clone and then submitting to transcription, translation and proteomic analysis. The spot clusters are meaningful clone identifiers; cluster components (families of polypeptides) are characteristic of individual clones and are independent of clones coexisting (and being co‐expressed) in a given pool. A ‘cluster’ originates from a single cloned message and might be due to post‐translational modification (offered by the reticuleocyte machinery) or as a result of programmed degradation. Thirteen clones or families of clonal products are shown, and the heterogeneity of the ‘appearance’ of clones is documented. In about half, the assignment of a clonal polypeptide product to a storage well position has failed; this might be due to a variety of considerations elaborated herein. The arguments are presented, that analysis of abundant proteins of a cell (activated lymphocyte) comprises ‘classical proteomics’, but for the analysis of the rare molecular species of proteins, Poissonian approaches of replicable material have to be used.