Differential Denaturation of Serum Proteome Reveals a Significant Amount of Hidden Information in Complex Mixtures of Proteins

Abstract

Recently developed proteomic technologies allow to profile thousands of proteins within a high-throughput approach towards biomarker discovery, although results are not as satisfactory as expected. In the present study we demonstrate that serum proteome denaturation is a key underestimated feature; in fact, a new differential denaturation protocol better discriminates serum proteins according to their electrophoretic mobility as compared to single-denaturation protocols. Sixty nine different denaturation treatments were tested and the 3 most discriminating ones were selected (TRIDENT analysis) and applied to human sera, showing a significant improvement of serum protein discrimination as confirmed by MALDI-TOF/MS and LC-MS/MS identification, depending on the type of denaturation applied. Thereafter sera from mice and patients carrying cutaneous melanoma were analyzed through TRIDENT. Nine and 8 protein bands were found differentially expressed in mice and human melanoma sera, compared to healthy controls (p<0.05); three of them were found, for the first time, significantly modulated: α2macroglobulin (down-regulated in melanoma, p<0.001), Apolipoprotein-E and Apolipoprotein-A1 (both up-regulated in melanoma, p<0.04), both in mice and humans. The modulation was confirmed by immunological methods. Other less abundant proteins (e.g. gelsolin) were found significantly modulated (p<0.05).Conclusions: i) serum proteome contains a large amount of information, still neglected, related to proteins folding; ii) a careful serum denaturation may significantly improve analytical procedures involving complex protein mixtures; iii) serum differential denaturation protocol highlights interesting proteomic differences between cancer and healthy sera.

Iron homeostasis, complement, and coagulation cascade as CSF signature of cortical lesions in early multiple sclerosis

“…Six CSF samples obtained from three MS patients representative of the MSlow group and three MS patients representative of the MShigh patients, according to the immunoassay (BioPlex) CSF profiling as described above (Table S2), were selected and analyzed using TRIDENT analysis with denaturation by three different protocols as previously described and fully explained in Data S1. The obtained protein datasets for each experiment were analyzed by Database for Annotation, Visualization and Integrated Discovery (DAVID, v6.8) software (https://david.ncifcrf.gov), and gene ontology analyses and protein–protein interactions using STRING software as described in detail in Data S1.…”

Immunocapture-Selected Reaction Monitoring Screening Facilitates the Development of ELISA for the Measurement of Native TEX101 in Biological Fluids*

“…The endogenous TEX101 protein present in pooled seminal plasma was used to calibrate the assay. Because limit of detection (LOD) of the immunoassay without seminal plasma pretreatment was not high enough, we examined literature (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) to identify conditions that would improve the assay sensitivity in seminal plasma. Thus, seminal plasma was subjected to pretreatment with the following detergents, salts and solvents: 1% SDS, 2-5% Triton X-100, 1% CHAPS, 2% sodium deoxycholate, 1 M urea, 3 M guanidine hydrochloride, 10% dimethyl sulfoxide, 10% dimethylformamide, 10% glycerol, 10% methanol, 10% acetonitrile.…”

Inhibition of cell proliferation, migration and invasion of B16-F10 melanoma cells by α-mangostin

“…Data acquisition and analysis was performed as previously described [16][17]. Data were searched with 1.5 Da and 1 Da tolerance respectively for precursor and fragment ions.…”