An update on medium- and low-abundant blood plasma proteome of horse

Abstract

The main objectives of the study were to: (1) deeply analyse the serum protein composition of Equus caballus, (2) assess the effectiveness of the high-abundant protein depletion and improve the concentration of medium- and low-abundant proteins. The analysis were performed on the blood plasma of three healthy part-Arabian mares. The implementation of two-dimensional electrophoresis and matrix-assisted laser desorption/ionisation - time of flight mass spectrometry allowed us to establish a horse plasma proteome map. Serum proteins were resolved at pH 4 to 7, followed by 12% SDS-PAGE. As a result 136 spots were successfully identified, representing the products of 46 unique genes. Of these, 22 gene products have not been previously identified in horse serum/plasma samples using proteomic tools. Gene ontology analysis showed that almost 30% of all identified gene products belong to the coagulation and complement cascades. These results can undoubtedly serve as a useful and prospective prerequisite for the future analysis of horse plasma proteome changes in different physiological and pathophysiological conditions. The use of the medium- and low-abundant protein enrichment tool increased their abundance and allowed us to identify a higher number of protein gene products. The highest depletion efficiency was observed for the most abundant plasma proteins, that is albumin, IgG heavy chains and serotransferrin.

Label-Free Mass Spectrometry-Based Quantitative Proteomics Analysis of Serum Proteins During Early Pregnancy in Jennies (Equus asinus)

“…Thus far, several proteomics approaches have been widely and effectively applied to explore the blood proteome of equids. Two-dimensional (2D) gel electrophoresis is routinely used for assessing quantitative changes in serum and/or plasma proteins of horses, such as for establishing the serum protein map (9,10), evaluating changes in plasma proteins after prolonged physical exercise (11), and comparing differences in serum protein expression profiles between distinct breeds (12). Henze et al (13) evaluated genetic differences in the serum proteome of horses, donkeys, and mules using surface-enhanced laser desorption-ionization timeof-flight mass spectrometry (MS), and they found considerable differences in the proteome of horses and donkeys and that the proteome of mules showed a higher similarity to donkeys than to that of horses.…”

Vitamin K and equine osteocalcin: an enigma explored

“…α-2 macroglobulin is responsible for the inactivation of plasmin and is therefore key to blood coagulation cascade (Bundgaard et al 2016). Many of these proteins can be found in the plasma of healthy horses, especially those that are highly abundant therefore, deciphering what proteins are expressed due to a stimulus is one of the challenges of proteomics (Lepczyński et al 2018).…”

“…However, the findings of Chapter 5 imply that the osteocalcin peptides that were emulated by the synthetic peptides, exist at concentrations in plasma well below this. It is true that the most important and specific changes generally occur in proteins of low abundance, this therefore warrants their enrichment to enable detection (Lepczyński et al 2018). This is why an enrichment strategy was explored, to try and increase detection in plasma.…”

“…A major issue stems from the wide dynamic range of protein levels and the presence of a small group of highly -abundant proteins such as albumin and immunoglobulins (IgG) that constitutes a large fraction of the total protein mass (Zhang et al 2007;Lepczyński et al 2018). These proteins tend to dominate proteomic analyses of plasma and other biological fluids and therefore present a barrier for detection of less-abundant proteins (Lepczyński et al 2018). The very low plasma concentrations of VKDPs compared to highly abundant proteins, make an enrichment step absolutely crucial.…”

“…The blood plasma proteome is comprised predominately of albumins and globulins as well as the following proteins; fibrinogen, immunoglobulins, α1-antytrypsine, transferrin, α-2 macroglobulin, haptoglobin and complement C3 (Leeman et al 2018;Lepczyński et al 2018).…”