Susan DiPietro (Presenter)
Thermo Fisher Scientific
Authorship: P Humphryes, V Barattini, S DiPietro*
Thermo Fisher Scientific, Chromatography Consumables, Runcorn, UK; *Thermo Fisher Scientific, West Palm Beach, Florida, USA
| Short Abstract Identification and quantification of biomarkers is often hindered by poor sample solubility and incomplete protein digestion. Clean-up and enrichment strategies can improve analytical sensitivity, however these approaches cannot enhance the rate of trypsin digestion or its reproducibility. A novel digestion technique has been developed to overcome these barriers by increasing the efficiency and reproducibility of digestion, whilst significantly improving the workflow efficiency. |
Long Abstract
Introduction
Identification and quantification of biomarkers is often hindered by poor sample solubility and incomplete protein digestion. Clean-up and enrichment strategies can improve analytical sensitivity, however these approaches cannot enhance the rate of trypsin digestion or its reproducibility.
Typically protein digestion is performed in the presence of chaotropes, surfactants, salts and organic solvents to improve the proteins solubility and thereby make more active sites available to the enzyme. Additionally sample pre-treatment such as protein reduction and alkylation is required. However these steps can cause unwanted chemically induced post translational modifications which can often impede downstream analysis.
A novel digestion technique has been developed to overcome these barriers by increasing the efficiency and reproducibility of digestion, whilst significantly improving the workflow efficiency.
Methods (80 words)
Thyroglobulin, insulin and bovine serum albumin (BSA) were digested using the novel process (SMART DigestTM kit) in the presence of a range of buffering agents to determine the optimal digestion conditions.
The resulting peptides were then separated by HPLC using a solid core C18 column with a linear gradient. Data was collected using both UV and mass spectrometry detection.
Results
A comparison between conventional in-solution digestion protocols and the novel protocol demonstrated that complete digestion could be achieved in just 3.5 hours for thyroglobulin which represents a 6 fold increase in speed. Additionally, the resultant digested peptides showed improved quantitative linearity compared to standard digestion approaches. The novel protocol produced larger peak response for the insulin tryptic peptide quantified when compared to in solution digestion, which indicated more efficient digestion than conventional techniques.
The novel buffer was also shown to solubilise much higher concentrations of protein for longer periods of time than standard buffering agents. Fewer steps were required to achieve complete digestion of sample and no additional clean-up was required prior to analysis representing a significant time saving.
Conclusions
The novel process was shown to outperform conventional in-solution digestion protocols and provided a fast, simple and clean methodology, making it ideal for workflows requiring high throughput, clean tryptic digestions.
References & Acknowledgements:
Clarke, N J, et al, A novel mass spectrometry-based assay for the accurate measurement of thyroglobulin from patient samples containing antithyroglbulin antobodies, Journal of Investigative Medicine, 2012, 60, 1157-1163.
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