LC-MS/MS Identification of Species-Specific Muscle Peptides in Processed Animal Proteins

An innovative analytical strategy has been applied to identify signature peptides able to distinguish among processed animal proteins (PAPs) derived from bovine, pig, fish, and milk products. Proteomics was first used to elucidate the proteome of each source. Starting from the identified proteins and using a funnel based approach, a set of abundant and well characterized peptides with suitable physical-chemical properties (signature peptides) and specific for each source was selected. An on-target LC-ESI-MS/MS method (MRM mode) was set up using standard peptides and was then applied to selectively identify the PAP source and also to distinguish proteins from bovine carcass and milk proteins. We believe that the method described meets the request of the European Commission which has developed a strategy for gradually lifting the “total ban” toward “species to species ban”, therefore requiring official methods for species-specific discrimination in feed

Gene enrichment analysis of DEGs specific of the clinical and preclinical stage of the disease.

<p>The most relevant GO terms (y axis) associated to clinical (<b>A</b>) and preclinical (<b>B</b>) phase are listed according to decreasing statistical significance from top to bottom. The threshold for statistical significance is marked by the green lines. The enrichment analysis was performed using DAVID bioinformatics tool 6.7 (NIAID/NIH, USA).</p

Comparison of test performance under the manufacturer's dilution protocol <i>versus</i> the 50% w/v protocol.

<p>The vertical axis reports the different rapid tests challenged. The horizontal axis reflects the odds ratio magnitude.</p

Heat maps representing the DEGs found in clinical and preclinical cattle with atypical BSE.

<p>Two heat maps were generated using the <i>heatmap</i>.<i>2</i> function from the <i>gplots</i> library in <i>R</i> statistical environment. DEGs were hierarchically clustered with complete linkage using the Euclidean metric. The heat maps represent the most significant DEGs (p value ≤.0.05 and fold change ≥ 2) in clinical (A) and preclinical (B) animals compared to the control group. Animals are reported in the x-axis while the differentially expressed probes are in the y-axis.</p

Identification of common DEGs in blood of preclinical and clinical atypical BSE-infected cattle.

<p>(A) Venn diagram showing the number of differentially expressed probe sets in blood of clinical and preclinical cattle. The intersection in grey represents 35 differentially expressed probe sets corresponding to 32 differentially expressed genes (DEGs) that are in common between the two stages of the disease. (B) Expression pattern of the common 32 DEGs. The histograms represent the fold change relative to the control group. PvsCtrl = preclinical versus control, CvsCtrl = clinical versus control.</p

List of DEGs characterized by an up-down/down-up pattern of expression.^a

<p>List of DEGs characterized by an up-down/down-up pattern of expression.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153425#t003fn001" target="_blank">^a</a></p

Test performance compared to the <i>IDEXX® HerdCheck BSE-scrapie short protocol</i>.

<p>The vertical axis reports the different rapid tests challenged. The horizontal axis reflects the odds ratio magnitude using <i>IDEXX® HerdCheck</i> BSE-scrapie short protocol as reference test. Panels A, B, C, D (left column) report the results obtained under the w/v protocol; panels E, F, G, H (right column) display the results under the manufacturers' instructions. BSE forms studied: panels A, E: strong C-type; panels B, F: weak C-type; panels C, G: H-type; panels D, H: L-type. All the weak C type water dilutions series tested negative with Bio-Rad® TeSeE™ SAP (notably, the optical densities were for all the three replicates of the 1∶2 dilution just under the cut-off value), thereby, the odd ratio could not be calculated (Subfigure B).</p

Detection limits obtained by the different rapid tests for the different BSE forms. The number of positives out of three replicates is also reported.

<p>Detection limits obtained by the different rapid tests for the different BSE forms. The number of positives out of three replicates is also reported.</p

Functional classification of differentially expressed genes in blood of infected cattle versus control group^a.

<p>Functional classification of differentially expressed genes in blood of infected cattle versus control group<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153425#t002fn001" target="_blank">^a</a>.</p

DEGs in common between CvsP, PvsCtrl and CvsCtrl comparisons.

<p>(A) Venn diagram revealed the presence of 22 DEGs in common between CvsP and PvsCtrl comparisons. One DEG (SEL1L3, Sel-1 Suppressor Of Lin-12-Like 3) was common in the three comparisons. (B-C) The normalized expression values of the 22 common DEGs for CvsP and PvsCtrl comparisons are represented by the histograms. As indicated by the schematic figures on the right, these 22 DEGs showed an oscillatory pattern of expression: (B) 9/22 were upregulated in PvsCtrl and then downregulated in CvsP comparisons, while (C) 13/22 were downregulated in PvsCtrl and upregulated in CvsP comparison, respectively. (D) SEL1L3, the only gene found in common among the three comparisons (CvsP, CvsCtrl and PvsCtrl), showed a progressive downregulation during the preclinical and the clinical phase of the infection. P = preclinical, C = clinical and Ctrl = control, <i>vs</i> = <i>versus</i>. DEGs = differentially expressed genes.</p