Proteomics profiling of swine serum following lipopolysaccharide stimulation.

RATIONALE: There are no approved animal drugs for management of inflammation in swine due to lack of validated animal models. To assess efficacy, it was essential to develop proteomics approaches to identify suitable biomarkers of inflammation as presented in this study. METHODS: Serum samples were collected from a group of four pigs prior to (baseline) and 24 and 48 h following lipopolysaccharide (LPS) stimulation to reveal proteomic changes during inflammation. Two other pigs served as untreated controls. Proteins were separated by either one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional (2D) gel electrophoresis (2DE) prior to analysis by nano-flow liquid chromatography (nLC) coupled to tandem mass spectrometry (MS/MS). RESULTS: We identified 165 proteins using SDS-PAGE, of which 47 proteins were also detected by 2DE prior to nLC/MS/MS. More than half (72%) of all characterized proteins were modulated as a result of LPS stimulation, many of which are known to be involved with innate and adaptive immunity. Pig serum samples obtained 24 h after LPS initiation of inflammation showed protein modulations of serum albumin, serotransferrin, light and heavy immunoglobulin chains (IGs), and major acute phase proteins including haptoglobin (HPT), serum amyloid A2 (SAA2), C-reactive protein (CRP), ß-2-glycoprotein 1 (B-2GP1), alpha-2-HS-glycoprotein (A2HS), α-1-antitrypsin (A1AT), and α-1-acid glycoprotein (A1AG). SAA2 was distinguished from the other SAA isoforms by the unique peptide sequence of SAA2. CONCLUSIONS: The results provided proteomics analysis of swine serum due to LPS stimulation and indicated the importance of SAA2, which appears to be unique and may be regarded as a potential clinical diagnostic biomarker of inflammation.

Detection of casein phosphopeptides in goat milk.

The aims of this study were to profile casein phosphopeptides in goat milk, to accurately determine the site of phosphorylation, and to evaluate whether or not any of the casein phosphorylation patterns were specific to a given physiological condition. Goat milk, collected before and after experimental induction of endotoxin mastitis, was separated by SDS-PAGE. Casein bands were digested with trypsin and the resulting peptides were analyzed by nLC-MS/MS. Eight out of nine predicted tryptic phosphopeptides corresponding to 18 different phosphorylation sites were detected in αS1-, αS2-, and ß-casein. Characterization of the phosphorylation sites illustrated the capability of tandem MS to accurately localize phosphorylated residues among a number of other putative sites. Despite an apparent lower abundance, almost all of the phosphopeptides were also detected in milk samples obtained from the goats following experimental induction of endotoxin mastitis. However, a tetra-phosphopeptide in αS2-casein was only observed in the milk samples obtained from healthy animals. The absence of this multiphosphopeptide in the mastitic goat milk samples could indicate changes in phosphorylation as a result of disease and potentially be used as a marker for milk quality. This study represents the first comprehensive analysis of casein phosphoproteome and reveals a much higher level of phosphorylation than previously demonstrated in goat milk.