Comparison of different methods for the absolute quantification of harbour seal transferrin glycoforms using HPLC-ICP-MS

Abstract

Marine mammals such as harbour seals (Phoca vitulina) represent a valuable indicator for the state of their habitat or environmental changes in particular due to their key role as top predators within the marine food web. Therefore, the characterization (qualitative and quantitative) of target proteins of such organisms can be of potential use for environmental monitoring processes. In this regard, serum transferrin (Tf) is a key protein related to iron transport and its metabolism. Thus, the present work illustrates the development and comparison of different approaches for the accurate, absolute quantification of Tf isolated from blood samples of North Sea harbour seals with the aim to use possible changes in Tf glycoform patterns as an additional parameter in extended studies, focusing on the assessment of their immune status. For this purpose, different HPLC-ICP-MS approaches have been developed, which allow the highly resolved separation and detection of up to nine different Tf glycoforms in seal blood samples in less than 30 minutes, as well as their sensitive and specific absolute quantification on the basis of their characteristic iron content. One method is based on the application of a reversed gradient sheath flow to compensate gradient related effects during the iron specific detection of Tf. Here a simple flow injection with a certified element standard was used for calibration. The second developed methodology utilizes isotope dilution analysis, which was applied for the quantification of the separated Tf glycoforms. Such accurate protein quantification methods are urgently needed in particular when aiming on comparable long term health related investigations. However, since both methods can be applied independently of the availability of specific protein standards or antibodies they can be seen as universal quantification methods for the targeted biomarker.