ABSTRACT
Spectral Counts approaches (SpCs) are largely employed for the comparison of protein expression profiles in label-free (LF) differential proteomics applications. Similarly, to other comparative methods, also SpCs based approaches require a normalization procedure before Fold Changes (FC) calculation. Here, we propose new Complexity Based Normalization (CBN) methods that introduced a variable adjustment factor (f), related to the complexity of the sample, both in terms of total number of identified proteins (CBN(P)) and as total number of spectral counts (CBN(S)). Both these new methods were compared with the Normalized Spectral Abundance Factor (NSAF) and the Spectral Counts log Ratio (Rsc), by using standard protein mixtures. Finally, to test the robustness and the effectiveness of the CBNs methods, they were employed for the comparative analysis of cortical protein extract from zQ175 mouse brains, model of Huntington Disease (HD), and control animals (raw data available via ProteomeXchange with identifier PXD017471). LF data were also validated by western blot and MRM based experiments. On standard mixtures, both CBN methods showed an excellent behavior in terms of reproducibility and coefficients of variation (CVs) in comparison to the other SpCs approaches. Overall, the CBN(P) method was demonstrated to be the most reliable and sensitive in detecting small differences in protein amounts when applied to biological samples.
Subject(s)
Biomarkers/metabolism , Cerebral Cortex/metabolism , Disease Models, Animal , Huntington Disease/metabolism , Proteome/analysis , Animals , Biomarkers/analysis , Cerebral Cortex/pathology , Escherichia coli Proteins/metabolism , Huntington Disease/pathology , Mice , Mice, Inbred C57BL , Models, Theoretical , Reference Standards , Reproducibility of Results , Tandem Mass SpectrometryABSTRACT
A mini-library of symmetrical and unsymmetrical diglycosyl (di)sulfides, containing d-galactose, l-fucose and N-acetyl glucosamine units, were synthesized and tested for the antiproliferative activity against cervix carcinoma (HeLa) and melanoma (A375) tumor cell lines as well as healthy fibroblasts (HDF). Comparative analysis of results seems to indicate that the most relevant antiproliferative effect is not primarily influenced by interactions with galectins, as the most cytotoxic compound observed for HeLa and A375 is not a ligand for such receptors. The most active molecules against HeLa and A375 lines also exhibited a good selectivity, showing a low toxicity to HDF cells. Obtained results offer useful indications for future design of structurally simple antitumor molecules based on sugar moieties with bridging sulfur atoms.
