University of Minnesota researchers found that Bio-Rad Laboratories' ProteoMiner protein enrichment kit enhanced identification of changes to low-abundance proteins and detection of post-translationally modified (PTM) proteins in human saliva. These findings offer promise for improving differential proteomic analyses and biomarker studies aimed at identifying disease-specific proteins and their PTM variants in various types of biological samples and fluids. The study was published in the Journal of Proteome Research.
Even when highly sensitive mass spectrometers are used to analyze complex biological samples and bodily fluids, high-abundance proteins obscure the detection of lower-abundance proteins and their post-translational modifications," said Sri Bandhakavi, who led the study at the University of Minnesota in 2010. (Bandhakavi is now a senior scientist at Bio-Rad.) "These lower-abundance proteins and PTMs are often of most interest to researchers, given their association with specific disease or physiological states."
Unlike immunodepletion methods that are sample-specific, the ProteoMiner kit can be used with a variety of samples. ProteoMiner technology consists of millions of unique hexapeptides immobilized on beads; each bead has potentially unique protein-binding properties. Treatment of samples with the ProteoMiner kit causes partial depletion of high-abundance proteins and simultaneous concentration of low-abundance proteins, resulting in dynamic range compression (DRC) of samples.
Study Findings
Bandhakavi and his former colleagues at the University of Minnesota compared saliva samples from healthy women with those with metastatic breast cancer, and processed the samples with and without using the ProteoMiner kit. The researchers' workflow enabled them to identify low-abundance proteins that were affected in breast cancer samples and that retained quantitative accuracy following treatment with the ProteoMiner kit.
"The workflow is readily transferable to diverse samples, signaling the appropriateness of including ProteoMiner technology in differential analyses of clinical samples or cellular lysates," said Bandhakavi. "Given our ability to identify proteins putatively associated with breast cancer, the study further highlights the systemic diagnostic potential of saliva as a noninvasive alternative to serum, long the gold standard, for biomarker discovery."
The researchers also demonstrated that DRC could serve as a general methodology for increasing identification of various protein PTMs of biological interest in complex samples. Knowledge of these PTMs is often critical to understanding a protein's functional state or potential role in disease. Glycosylated proteins were studied due to their large size and linkage to various diseases.
The researchers coupled the ProteoMiner technology with covalent glycopeptide enrichment and tandem mass spectrometry. With DRC, they identified two times more N-linked glycoproteins and their glycosylation sites than without DRC, increasing the known salivary glycoprotein catalog. The authors concluded that ProteoMiner technology should also boost detectability of other biologically interesting PTMs such as phosphorylation.
Source: Bio-Rad Laboratories