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.

Standardization approaches in absolute quantitative proteomics with mass spectrometry

“…This common application of the concept of on‐line IDA can be used for absolute quantification of peptides/proteins. Again, it can be applied to elements naturally present, such as iron or sulfur, or intentionally added after labeling with usually lanthanide‐containing species such as europium or lutetium . This controlled labeling enables quantification of any peptide species, unlike direct quantification with naturally present elements, in which only peptides or proteins that contain that target element can be quantified.…”

Inductively Coupled Plasma–Mass Spectrometry (ICP-MS) for Quantitative Analysis in Environmental and Life Sciences: A Review of Challenges, Solutions, and Trends

“…The potential of non-species-specific IDA has been demonstrated in particular for the quantification of proteins that naturally contain elements such as sulfur 59,110 or selenium 111 in a covalently bound form; however, the analysis of stable metal-containing proteins such as transferrin via iron IDA has also been recently demonstrated. 112,113 The non-species-specific IDA approach has been further refined to improve the mass-flow stability, which is a critical parameter influencing the accuracy of the quantification procedure. 114,115…”

“…112,113 The nonspecies-specific IDA approach has been further refined to improve the mass-flow stability, which is a critical parameter influencing the accuracy of the quantification procedure. 114,115 The main drawback of this IDA approach is that any chemical or physical loss of the analyte during the sample preparation as well as the separation procedure cannot be compensated.…”

Simultaneous quantification of proteins in human serum via sulfur and iron using HPLC coupled to post-column isotope dilution mass spectrometry

“…Different quantitative strategies of serum Tf based on HPLC-ICP-MS and species-specic or species-unspecic isotope dilution have been developed and the quantication results obtained by species-unspecic isotope dilution were in good agreement with species-specic isotope dilution using a synthesized 57 Fe-Tf spike. 18,19 However, preservation of the integrity of the metal-protein bond and conrmation of the uniform stoichiometry of the metal-protein complex are two prerequisites for an accurate quantication of transferrin. 20 Potential metal losses may happen in sample preparation and in liquid chromatography separation.…”