The serum proteome of Equus caballus

Abstract

We constructed a reference two-dimensional protein map for horse (Equus caballus) serum. The serum proteins were separated by two-dimensional electrophoresis (2-DE); 29 different gene products were identified. Proteins represented by 25 spots/spot groups were identified by tandem nanoelectrospray mass spectrometry (MS), four by matrix-assisted laser desorption ionization time-of-flight (TOF) MS and one was sequenced by TOF-TOF technology. The identities of four proteins were deduced by similarity to the human plasma protein database. In selected cases, i.e. the immunoglobulins, immunoblotting with specific antibodies provided additional information about the respective proteins. Albumin was detected as the full-length protein and as fragments of various sizes. Spots representing products of different mass and charge were also detected for alpha1-antitrypsin, haptoglobin and transthyretin. Thus, despite the fact that the Equus caballus genome is incompletely characterized, we were able to identify almost all moderate to high abundance proteins stained in the serum 2-DE pattern.

Protein stains for proteomic applications:Which, when, why?

“…Changes range from less than 100% to several thousand percent. Figure 8 compiles histograms of some spots or spot chains, most of them already identified [21]. They show different staining behavior, as illustrated, which may sometimes be explained by the amino acid composition of the molecules.…”

“…After fixing and washing steps, gels were kept in staining solution for 4 days, to achieve optimal staining. Similar staining recipes, sometimes slightly shortened, have also been used for establishing protein maps [20][21][22] or for monitoring protein level changes [23].…”

“…Proteins with low affinity to CBB may be detected. Other Zn 21 binding biopolymers, like peptides, glycolipids, oligonucleotides, multimolecular complexes, may be visualized as well. The association of zinc to negatively charged or polarizable groups may occur through His, Cys, Glu and Asp in proteins, phosphate and amino groups in nucleic acids and polyhydroxyls (from the sugar moieties) and carboxylate (from sialic acid) and phosphate (from lipid A) in lipopoly-/lipooligosaccharides [84].…”

“…A reverse staining using imidazole and zinc salts for protein detection in electrophoresis gels was introduced a few years later [83]. It exploits the ability of biopolymers (in particular, proteins and protein-SDS complexes) to bind Zn 21 and that of imidazole to react with unbound Zn 21 to produce insoluble zinc imidazolate (seen as deep whitestained background); bound Zn 21 (on proteins) does not stain. Proteins with low affinity to CBB may be detected.…”

“…Classical 2-DE was performed in accordance to existing protocols ( [21] and http://www.expasy.org/ch2d/), followed by different staining/detection methods and scanning under appropriate conditions (Table 5). Protein amounts were adapted to the staining technique to be applied, being in the range of 0.17-5 mL original serum (Table 5).…”