This paper reports the first proteomic application of 2-DE combined with MS analysis to characterise the total protein profile of sheep serum. Our 2-DE gels can be considered the first reference maps of sheep serum. Most of the serum proteins that are usually used as animal clinical biomarkers were identified. Even though specific new biomarkers are needed for the management of the main diseases that affect sheep, this farm species has been investigated less than others, such as cattle [1
]. In some cases, therapeutic protocols and diagnostic range values for sheep are borrowed from closely related species such as other ruminants. 2-DE gel reference maps of human, rat, bovine, swine, chicken and horse have been defined [1
] highlighting species-specific differences in serum protein profiles.
In the present study, many of the proteins we identified are found in different species, with different pI and molecular weights, and different isoforms and phenotypes [6
]. Considering the central role that serum plays in clinical diagnostics, the characterization of 2-DE gel serum sheep protein profiles increases the tools available for ovine medicine, both for identification of new disease markers, and for improved understanding of the physiological and pathological functions of proteins in diseased and healthy sheep.
SELDI-TOF MS has recently been applied to analyse ovine serum [25
]. It might be considered complementary to 2-D electrophoresis, because it is able to detect proteins and peptides with a very low molecular mass (< 10–20
kDa). However, many of the proteins with a high molecular weight that could be useful as serum tissue markers remain unrevealed. On the contrary, 2D-electrophoresis cannot detect proteins with a low molecular mass or pI values <4 or >9.
The map presented here is an integral serum 2-DE map which was obtained from whole serum protein content. Serum is rich in proteins such as albumin and immunoglobulin, that can mask less abundant proteins. Nonetheless, we decided to avoid sample pre-treatments, such as dealbumination, that could change the protein composition of our specimens. Our first goal was to provide a map for use in clinical diagnostic and prognostic purposes without pre-fractionating as it might have changed protein quantity or quality. Major serum proteins might be modified in physiological conditions or disease, and consequently, all sample treatments that could mask these changes should be avoided.
Since minor protein components might be involved in metabolism and regulation, one future challenge might be the definition of good depletion strategies or pre-fractioning methods for sheep serum to enhance the identification of low-abundance proteins without altering sample characteristics as has recently been applied to other animal species [26
The usefulness of the serum reference 2-DE map was confirmed by two different comparative investigations. The first ascertained serum protein pattern differences between healthy sheep and sheep affected by RTB, a common sheep disease. The second one, using a different group of animals, determined the serum protein changes during the peripartum period of four weeks before, and two-three weeks after lambing. The goal was to demonstrate the possible application of this proteomic approach in a mild pathological situation and in the physiological stressful peripartum condition, because in both situations it is important to recognise early signs of sub-clinical conditions.
Similar to previous studies on bovine and swine species [4
], we observed variations in proteins associated with the acute phase in both cases. Both in RTB affected and in lambing sheep, we saw a decrease in TTR a globular non-glycosylated protein, which is considered to be both a thyroxin- and a holo-retinol-binding protein. TTR is synthesized by different tissues such as the liver, retina, pancreas and placental trophoblast [27
]. It performs a multiplicity of biological functions [28
]. In particular, it plays a major and critical role in transport of maternal thyroid hormone to the developing fetus [27
]. TTR involvement in various central nervous system disorders, diabetes melitus, preclampsia has been described [29
]. Reduced TTR serum concentrations are often associated with acute phase response, which may be due to inflammation, trauma or other disorders like malnutrition [32
]. A decrease in TTR during inflammation has been reported as an effect of proinflammatory cytokines such as IL-1 and IL-6 [34
]. These findings also explain the decrease in Apo A-1 that we observed in serum from sheep affected by RTB. Apo A-1 is the major protein fraction of high-density lipoproteins, and is considered a negative acute-phase protein [35
]. During acute inflammation, IL-6, IL-1β, and TNF-α upregulate the expression of the pentraxins (C-reactive protein and serum amyloid-P), which in turn inhibit the synthesis of Apo A-1 [37
In contrast, we observed a significant increase in Apo A-1 after lambing, which can be explained by the effect of oestrogens. In females, these hormones enhance Apo A-1 levels. In sheep and other ruminants, delivery is induced by an increase in biosynthesis of oestrogens related to progesterone, and an enhancement of the oestrogen/progesterone ratio [38
]. A similar situation occurs in humans, but not in other animal species such as horses, and this again confirms the necessity to determine species differences. The above considerations also confirm that some proteins of the acute phase have other functions more important in specific situations. The hormonal changes that occur during lambing and influence lipid transport for lactation are the probable causes of the observed increase in Apo A-1. In sheep affected by RTB, Apo A-1 behaves as a negative acute-phase protein. In these animals, the decrease in negative acute phase proteins TTR and Apo A-1, was related to the increase in Hp [39
], a serum protein with several functions. Its main role is to bind free haemoglobin to allow hepatic recycling of haeme iron, and to prevent iron loss and renal damage. As a haemoglobin scavenger, this protein reduces circulating iron and inhibits enzymatic and non-enzymatic reactions catalysed by iron [40
]. Hp also acts as an antioxidant, has antibacterial activity, and is involved in modulating aspects of the acute phase response [41
]. Its blood level is associated with the prevalence and clinical evolution of many inflammatory diseases including infections, degenerative disorders and autoimmune disorders. Hp is considered a major positive acute-phase protein [42
], and its increase was more evident in diseased sheep than in lambing sheep.
The appropriate response to lambing, a critical situation with rapid, intense homeostatic changes, depends on good welfare conditions and an adequate nutritional status. Thus, management of this period is crucial for the health of the ewe, and to prevent disease or infections like mastitis [43
]. Increased α1AGP after lambing confirmed the stress response during delivery. This acute phase protein has been reported to be involved in similar stressful conditions, inflammation or tissue destruction [45
Although variations in Hp, α1AGP and TTR confirm lambing induces an acute phase reaction, the increase in Apo A-1 indicates a metabolic modification may also be taking place. These proteins may be suggested as putative bio-markers to monitor the adaptive response and metabolic changes in peripartum.
However, in both disease and lambing conditions, no changes were found in other acute phase proteins such as alpha1-antitrypsin. In serum from sheep affected by RTB, we also found an increase in EP. This protein belongs to a large family of serine proteinase (elastase, trypsin, plasmin, chymotrypsin) inhibitors that are essential regulators of a wide variety of biological processes [48
]. In particular, EP can strongly inhibit elastase. This protease plays a crucial role in tissue destruction and inflammation in numerous diseases, including chronic obstructive pulmonary disease, cystic fibrosis, adult respiratory distress syndrome, and ischemic-reperfusion injury. EP may play a major role in inflammation and during response to infections in which elastase is particularly involved. In bovine serum, EP content is high, suggesting its involvement in the acute phase response, with other protease inhibitors [49
]. The increase in RTB sheep serum of α1BGP, a plasma protein related to the immunoglobulin supergene family, is not clear because its biological role is currently unknown. However, it might be involved in the reaction to infection, and it is considered an acute phase protein. It increases in Mycobacterium bovis
infected cows [50
], but decreases in pigs with viral infections [6
]. Even if EP and α1BGP are minor acute phase proteins, their trends suggest further studies are needed to clarify their roles in respiratory/infections disease in sheep and to assess their potential use as putative biomarkers.