Enhanced Denaturation Reveals Hidden Information Within Serum Proteome

Characterization of the human serum proteome has the potential to reveal vital information regarding disease states; however, serum remains difficult to analyze. Liotta, Ferrari and Petricoin (2003) estimated that 10% of the serum proteome is made up of small molecules with low concentrations and that these are unidentifiable.¹ Denaturing agents are often employed to simplify complex protein mixtures found in serum samples. An Italian research group based in Rome and led by researcher Francesco Facchiano has now sought to determine whether a more precise denaturation technique can be identified, based on denaturation methods currently used.

In their recent publication (2013), the Facchiano group investigated 69 denaturation treatments.² Treatments were carefully analyzed and selected to optimize the solubility and the solvent accessibility of proteins. These treatments included changes in hydrophobicity, ionic strength, temperature and ultrasounds (shock waves). The Facchiano group found that the ability to identify small proteins, and proteins at very low concentrations, was highly dependent upon the particular denaturation technique used. Out of the 69 techniques reviewed, three of the most discriminating treatments were selected for a combined protocol, subsequently named “TRIDENT.”

Preliminary testing using the TRIDENT analysis was first applied to human and bovine sera. Samples were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by time-of-flight mass spectrometry (MALDI-TOF/MS) using a Voyager-DE STR (Applied Biosystems). The TRIDENT protocol was able to detect an additional set of at least 40 protein bands in the same serum sample, with the bands estimated to be lower than 5 ng/band. TRIDENT-SDS-PAGE was also able to reveal some proteins in human serum — namely, H2 MHC-I antigen, the IGHG1 protein and Q10 α-chain — that are undetectable or extremely difficult to identify in serum fractionated using conventional procedures and analyzed by MALDI-TOF/MS.

To demonstrate the effectiveness of TRIDENT, researchers injected mice with cutaneous melanoma, along with human sera from patients affected by early non-metastatic cutaneous melanoma. Sera from healthy mice and humans were used as controls. Following the TRIDENT analysis, proteins were identified using MALDI-TOF/MS and confirmed using liquid chromatography and tandem mass spectrometry on an LTQ Linear Ion Trap mass spectrometer (Thermo Scientific).

In mice with melanoma, nine proteins were differentially expressed. Eight proteins were differentially expressed in human melanoma. Three new proteins of interest were also identified: α2macroglobulin (downregulated in melanoma, p<0.001), Apolipoprotein-E and Apolipoprotein-A1 (both upregulated in melanoma, p<0.04). The differential expressions were also confirmed using western blot and dot blots.

The Facchiano group maintains that further investigation is required to optimize protein discrimination and identification in serum/plasma. Continuing to refine the denaturation process may be key to finding novel biomarkers of disease and gaining a better understanding of the proteomic differences between cancerous and healthy sera.

References

1. Liotta, L.A., Ferrari, M., and Petricoin, E. (2003) “Clinical proteomics: Written in blood,” Nature, 425 (p. 905), doi:10.1038/425905a.

2. Verdoliva, V., et al. (2013) “Differential denaturation of serum proteome reveals a significant amount of hidden information in complex mixtures of proteins,” PLoS One, 8(3), doi: 10.1371/journal.pone.0057104.

Post Author: Emily Humphreys. As an undergraduate studying biology at the University of Utah, Emily balanced a heavy class schedule while working long hours in a lab studying eye development. Following graduation, she became involved in infectious disease and aging research involving SNPS.

While she enjoyed the thrill of research, Emily has since traded bench work for science journalism.
She has spent the last year writing about new developments involved in proteomics research, and now food testing.

When she isn’t writing,she can be found playing outside with her kids.

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