Lewis Couchman (Presenter)
King's College Hospital
Bio: Lewis works in the departments of toxicology and clinical biochemistry at King's College Hospital, London. He is primarily involved with development, validation, implementation and running of routine mass spectrometry-based methods for clinical applications. He is particularly interested in automated and on-line sample preparation methods, and the implementation of high-resolution mass spectrometry in the clinical laboratory. Lewis is also completing a PhD using mass spectrometry for assessment of 'true' vitamin D status.
Authorship: Lewis Couchman (1,2), Don Jones (2), Caje Moniz (1)
(1) Department of Clinical Biochemistry, King’s College Hospital, London, UK; (2) Cancer studies and molecular medicine, University of Leicester, Leicester, LE2 7LX, UK
| Short Abstract Analysis of vitamin D binding protein (VDBP) may be a useful adjunct to measuring total serum 25-hydroxyvitamin D concentrations in assessing vitamin D status. We have developed a method for the measurement of ‘total’ VDBP using ‘non-specific’ tryptic peptides with qualitative identification of isoforms using isoform-specific tryptic peptides. Whole serum samples were digested using SMART DigestTM kits at 70°C (30 min), and digests diluted with isotope-labelled peptides prior to direct analysis by TurboFlow LC-MS. This method also allows for simultaneous quantitation of ‘total’ serum albumin, which may be an additional factor useful in calculating a ‘free vitamin D’ index. |
Long Abstract
Analysis of vitamin D binding protein (Gc globulin; VDBP), especially when one considers the relative prevalence of the three main VDBP isoforms (Gc2, Gc1f and Gc1s) in different ethnic groups, may be a very useful adjunct to measuring total serum 25-hydroxyvitamin D concentrations in assessing ‘true’ vitamin D status [1]. Immunoassays for VDBP measurement may not distinguish between different isoforms, and furthermore, may show differential cross-reactivity with different isoforms of the protein, leading to confusion for quantitative analysis. Additionally, the well-known ‘small-molecule’ biomarkers of vitamin D status (primarily 25-hydroxyvitamins D) can bind in the circulation to other plasma proteins, notably albumin.
Serum concentrations of VDBP (and albumin) are high, meaning that proteomic analysis can be easily carried out following digestion of whole-serum samples, without the need for any prior depletion and/or immunoenrichment strategies. Furthermore, it is serendipitous that the three main isoforms of VDBP give rise to three different isoform-specific tryptic peptides (LPDATPK, LPDATPTELAK, and LPEATPTELAK for Gc2, Gc1f and Gc1s isoforms, respectively), each of which are well-suited to analysis by LC-MS.
We have developed a rapid, simple method using heat-assisted digestion and on-line sample clean-up for the simultaneous quantitative analysis of ‘total’ VDBP and ‘total’ albumin using selected tryptic peptides which are not subject to isoform-dependent sequence changes or other modifications (e.g. glycosylation), plus in addition, qualitative identification of the VDBP isoforms present. Samples (50 µL) were diluted with internal standard solution (isotope-labelled VDBP and albumin tryptic peptides, 200 µL) and thoroughly mixed. A portion of the mixture was diluted with SMART DigestTM buffer (ThermoScientific), mixed, and incubated (70 °C, 30 min). After incubation, the digests were diluted with LC eluent and directly analysed by TurboFlow LC-MS (analysis was carried out using (i) LC-MS/MS, TSQ Vantage, ThermoFisher Scientific, and (ii) LC-HR-MS, Q Exactive, ThermoFisher Scientific) [2]. Total analysis time was 10 minutes. Total VDBP and albumin were quantified by calibration using standards prepared containing gel-purified VDBP (mixture of all three isoforms, Sigma Aldrich, 50-500 mg/L) and human serum albumin (also Sigma Aldrich, 10-60 g/L) and using measured peak-area ratios to the relevant isotope-labelled peptides.
The described method is ideally suited to high-throughput clinical laboratories and has the capacity for automation. The digestion process is dramatically simplified using the SMART DigestTM kit approach, and gives comparable data to traditional reduction/denaturation/alkylation protocols.
References & Acknowledgements:
References
[1] Powe CE, Evans MK, Wenger J, et al. Vitamin D-binding protein and vitamin D status of black Americans and white Americans. N. Engl. J. Med. 2014; 369: 1991-2000.
[2] Couchman L, Jones DJL, Moniz CF. The use of turbulent flow chromatography for rapid, on-line analysis of tryptic digests. Rapid Commun. Mass Spectrom. 2015; 29 (22): 2140-6.
| Description | Y/N | Source |
| Grants | yes | ThermoFisher Scientific |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |