Article pubs.acs.org/JAFC
Highly Accurate Quantification of Hydroxyproline-Containing Peptides in Blood Using a Protease Digest of Stable Isotope-Labeled Collagen Yuki Taga,* Masashi Kusubata, Kiyoko Ogawa-Goto, and Shunji Hattori Nippi Research Institute of Biomatrix, 520-11 Kuwabara, Toride, Ibaraki 302-0017, Japan S Supporting Information *
ABSTRACT: Collagen-derived hydroxyproline (Hyp)-containing dipeptides and tripeptides, which are known to possess physiological functions, appear in blood at high concentrations after oral ingestion of gelatin hydrolysate. However, highly accurate and sensitive quantification of the Hyp-containing peptides in blood has been challenging because of the analytical interference from numerous other blood components. We recently developed a stable isotope-labeled collagen named “SIcollagen” that can be used as an internal standard in various types of collagen analyses employing liquid chromatography−mass spectrometry (LC-MS). Here we prepared stable isotope-labeled Hyp-containing peptides from SI-collagen using trypsin/ chymotrypsin and plasma proteases by mimicking the protein degradation pathways in the body. With the protease digest of SIcollagen used as an internal standard mixture, we achieved highly accurate simultaneous quantification of Hyp and 13 Hypcontaining peptides in human blood by LC-MS. The area under the plasma concentration−time curve of Hyp-containing peptides ranged from 0.663 ± 0.022 nmol/mL·h for Pro-Hyp-Gly to 163 ± 1 nmol/mL·h for Pro-Hyp after oral ingestion of 25 g of fish gelatin hydrolysate, and the coefficient of variation of three separate measurements was 97% for Hyp and all the peptides and >99% for peptides having two labeling sites), which enables highly accurate quantitative analysis. MRM chromatograms of the labeled Hyp and Hyp-containing peptides in the SI-digest are shown in Figure S2 in the Supporting Information. The total of the generated Hyp and Hyp-containing peptides was 10.8% (w/w) of the digested SI-collagen. Considering the Hyp content in 12100
dx.doi.org/10.1021/jf5039597 | J. Agric. Food Chem. 2014, 62, 12096−12102
Journal of Agricultural and Food Chemistry
Article
focused on peptides containing Hyp, SI-digest can be also applied to the analysis of other collagen-derived oligopeptides, such as Gly-Pro, which is reported to have an antihypertensive effect.38 Over the past decade, many studies have explored functional properties of the oral intake of collagen/gelatin hydrolyzed with enzyme.39 Tracing the collagen-derived bioactive peptides in the body is important in estimating the effects of the ingestion of gelatin hydrolysate. Our method is useful for quantification of the extent of the absorption and metabolism of orally ingested gelatin hydrolysate and, thus, can facilitate the investigation of biological activity of collagen-derived peptides and the development of functionally characterized gelatin hydrolysate.
in a 100 mm dish can be used to analyze approximately 100 plasma samples even by conventional LC-MS. Quantitative Analysis Using SI-Digest. To evaluate the usefulness of SI-digest as an internal standard mixture, we reanalyzed the plasma samples measured in Figure 1. The samples were first mixed with SI-digest and subjected to MRM analysis following ethanol deproteinization. As shown in Figure 3, 13 Hyp-containing peptides were detected with a wide range of plasma concentrations after the ingestion of gelatin hydrolysate. The AUC0−6 h of each Hyp-containing peptide was calculated as ranging from 0.663 ± 0.022 nmol/mL·h for Pro-Hyp-Gly to 163 ± 1 nmol/mL·h for Pro-Hyp (Table 2). The AUC0−6 h of Pro-Hyp was significantly higher than those of other Hyp-containing peptides, consistent with the previous observations.3,4,21 To quantify hard-to-detect minor peptides, we concentrated the samples after ethanol precipitation. Although the procedure increases the detection limit of the analytes, it potentially induces increased matrix effects leading to impaired quantitative accuracy. By using SI-digest as the internal standard mixture, we were able to overcome the quantitative limitations and detect previously unreported Hypcontaining peptides (Glu-Hyp, Ser-Hyp, Glu-Hyp-Gly). In addition, high reproducibility of the quantification of respective Hyp-containing peptides was shown by the triplicate measurements with
