Relative Quantification of Protein Posttranslational Modifications
Posttranslational modifications (PTMs) play critical roles in protein function and are emerging as an important theme in the etiology of numerous human diseases. Using a technique known as mass spectrometric immunoassay, we are able to extract specific target proteins out of complex human biomatrices then analyze them by mass spectrometry to identify PTMs and quantify them in terms of their relative abundance.
Most proteins exist as at least one or two molecular variants (i.e. postttranslationally modified forms), but some exist in a great variety of different forms. One such protein, osteocalcin, plays an important role in bone physiology. Clinically, circulating concentrations of osteocalcin are employed as a marker of bone formation and overall bone turnover. We have found that osteocalcin circulates in over 20 differentially truncated forms. Each one of these forms can be modified by 0 to 3 vitamin K dependent gamma-carboxyl groups which affect the ability of osteocalcin to bind metals, including calcium. Existing clinical assays are blind to these modifications. The assay that we have developed gives researchers the ability to study the function of osteocalcin in ways that have previously not been possible. As such, it promises to provide new insights into its biological function.Figure 1: Charge deconvoluted electrospray ionization (ESI)-mass spectrum of osteocalcin extracted from human blood plasma. Numerous truncated forms of the protein with variable numbers of Vitamin K dependent gamma-carboxylated glutamic acid residues are evidenced and readily quantified in terms of their relative fractional abundance. Adapted with permission from Rehder et al Mol Cell Proteomics 14(6): 1546-1555, 2015.
For example, we have used our new assay to quantify the precise fractions of circulating osteocalcin protein bearing 0, 1, 2, or 3 gamma-carboxylated glutamate residues in a double-blind controlled trial of nominally healthy 60-80 year olds maintained on their normal diet or supplemented with 500 micrograms of vitamin K1 (phylloquinone) per day. Results (shown in Figure 2), reveal that most older Americans on a typical diet consume inadequate quantities of vitamin K (which is found primarily in green leafy vegetables) to fully gamma-carboxylate their osteocalcin (yellow bars). This can be improved with vitamin K supplements (green bars). Interestingly about 80% of cow, rat and mouse osteocalcin bears 3 gamma-carboxyl groups—suggesting that humans (older Americans at least) are vitamin K deficient—even though adequate vitamin K is consumed to maintain proper blood coagulation processes. The health consequences of maintaining such vitamin K levels are currently unknown, but may, according to vitamin K experts, have a role to play in Alzheimer’s Disease
Figure 2: Detailed molecular comparison provided by ESI-MSIA of circulating osteocalcin gamma-carboxylation in 60-80 yr. old volunteers receiving a 500 µg/day oral vitamin K1 (phylloquinone) supplement or placebo. The relative percent abundance of circulating osteocalcin molecules bearing 0, 1, 2, and 3 gamma-carboxylated glutamate residues is shown for each group. Samples were collected after 24 months of treatment. Error bars represent 95% confidence intervals. * Indicates statistical significance as determined by a t-test (p < 0.001). Figure adapted with permission from Rehder et al Mol Cell Proteomics 14(6): 1546-1555, 2015. @ symbols indicate Dr. Borges’ personal osteocalcin profile—which is similar with regard to its degree of gamma-carboxylation to bovine and rat Oc—and maintained by a healthy diet and juicing regimen.