Attempting to distinguish between endogenous and contaminating cytokeratins in a corneal proteomic study

<p>Abstract</p> <p>Background</p> <p>The observation of cytokeratins (CK's) in mass spectrometry based studies raises the question of whether the identified CK is a true endogenous protein from the sample or simply represents a contaminant. This issue is especially important in proteomic studies of the corneal epithelium where several CK's have previously been reported to mark the stages of differentiation from corneal epithelial stem cell to the differentiated cell.</p> <p>Methods</p> <p>Here we describe a method to distinguish very likely endogenous from uncertain endogenous CK's in a mass spectrometry based proteomic study. In this study the CK identifications from 102 human corneal samples were compared with the number of human CK identifications found in 102 murine thymic lymphoma samples.</p> <p>Results</p> <p>It was anticipated that the CK's that were identified with a frequency of <5%, <it>i.e. </it>in less than one spot for every 20 spots analysed, are very likely to be endogenous and thereby represent a 'biologically significant' identification. CK's observed with a frequency >5% are uncertain endogenous since they may represent true endogenous CK's but the probability of contamination is high and therefore needs careful consideration. This was confirmed by comparison with a study of mouse samples where all identified human CK's are contaminants.</p> <p>Conclusions</p> <p>CK's 3, 4, 7, 8, 11, 12, 13, 15, 17, 18, 19, 20 and 23 are very likely to be endogenous proteins if identified in a corneal study, whilst CK's 1, 2e, 5, 6A, 9, 10, 14 and 16 may be endogenous although some are likely to be contaminants in a proteomic study. Further immunohistochemical analysis and a search of the current literature largely supported the distinction.</p

Prednisolone reduces the ability of serum to activate the IGF1 receptor in vitro without affecting circulating total or free IGF1

ObjectiveEnd-point bioassays based on thymidine or sulfate incorporation have demonstrated that glucocorticoid (GC) treatment inhibits serum IGF1 action, but the mechanism is unknown as serum IGF1 concentrations have been reported to either increase or remain unchanged.AimTo investigate whether GC treatment affects the ability of serum to activate the IGF1 receptor (IGF1R) in vitro (i.e. bioactive IGF1), using a specific cell-based IGF1 kinase receptor activation assay.Subjects and methodsTwenty children with stable asthma (age 7.7–13.8 years) treated for 1 week with 5 mg prednisolone in a randomized, double-blind, placebo-controlled crossover study. Non-fasting serum samples were collected in the afternoon after each 7-day period and assayed for bioactive IGF1, free IGF1, total IGFs, IGF-binding proteins (IGFBPs), and insulin.ResultsPrednisolone treatment reduced IGF1 bioactivity by 12.6% from 2.22±0.18 to 1.94±0.15 μg/l (P=0.01) compared with placebo. In contrast, no changes were observed for (μg/l; placebo vs prednisolone) total IGF1 (215±27 vs 212±24), free IGF1 (1.50±0.16 vs 1.43±0.17), total IGF2 (815±26 vs 800±31), IGFBP3 (3140±101 vs 3107±95), IGFBP2 (238±21 vs 220±19), IGFBP1 (32±6 vs 42±10), or IGFBP1-bound IGF1 (24±5 vs 26±7). Insulin remained unchanged as did IGFBP levels as estimated by western ligand blotting. Prednisolone had no direct effects on IGF1R phosphorylation.ConclusionsOur study gives evidence that GC treatment induces a circulating substance that is able to inhibit IGF1R activation in vitro without affecting circulating free or total IGF1. This may be one of the mechanisms by which GC inhibits IGF1 action in vivo. However, the nature of this circulating substance remains to be identified.</jats:sec