Fig. 1.
Schematic workflow of cervical-mucus proteomic biomarker discovery in swamp buffalo. LC-MS/MS – liquid chromatography with tandem mass spectrometry; FDR – false detection rate; PCA/PLS-DA – principal component analysis and partial least-squares discriminant analysis; GO – Gene Ontology database; PRM – parallel reaction monitoring
Fig. 2.
Venn diagram of retained features after QC (good swamp-buffalo cervical mucus (GCM) vs poor cervical mucus (PCM))
Fig. 3.
Principal component analysis of swamp-buffalo cervical mucus proteins. (A) Score plot of PC1 vs PC2 showing group separation with 95% confidence ellipses. (B) Biplot illustrating the contribution of selected proteins to group separation. GCM – good cervical mucus; PCM – poor cervical mucus; CLU– clusterin; SPP1 – osteopontin; LTF – lactotransferrin; SLPI – secretory leukocyte protease inhibitor; LCN2 – lipocalin-2; MPO – myeloperoxidase; PTX3 – pentraxin 3; CRP – C-reactive protein; S100A12 – S100 calcium-binding protein A12
Fig. 4.
Volcano plot of differential proteins in good swamp-buffalo cervical mucus and in poor cervical mucus. FC – fold change; MPO – myeloperoxidase; ITGA6 – integrin α6; LTF – lactotransferrin; CLU – clusterin; SLPI – secretory leukocyte protease inhibitor
Fig. 5.
Partial least-squares discriminant analysis variable-importance (VIP) scores for the top 15 proteins in swamp-buffalo cervical mucus. GCM – good cervical mucus; PCM – poor cervical mucus; MPO – myeloperoxidase; LTF – lactotransferrin; LCN2 – lipocalin-2; ITGA6 – integrin α6; CLU – clusterin; ACTA2 – actin α2, smooth muscle; S100A12 – S100 calcium-binding protein A12; SPP1 – osteopontin; PTX3 – pentraxin 3; CRP – C-reactive protein; PRDX6 – peroxiredoxin 6; SLPI – secretory leukocyte protease inhibitor; LGALS3 – galectin-3; SOD1 – superoxide dismutase 1; HSPB6 – heat-shock protein B6
Table 1.
Proteins expressed differentially between good cervical mucus (GCM) and poor cervical mucus (PCM) from swamp buffalo
| Protein | FC | Log2 (FC) | P-value | Direction |
|---|---|---|---|---|
| MPO | 1.44 × 10−2 | -6.117 | 1.31 × 10−13 | PCM |
| LTF | 4.17 × 101 | 5.381 | 2.09 × 10−13 | GCM |
| ITGA6 | 3.53 × 102 | 8.462 | 2.33 × 10−12 | GCM |
| LCN2 | 4.63 | 2.211 | 3.00 × 10−12 | GCM |
| CLU | 8.94 × 102 | 9.804 | 7.05 × 10−12 | GCM |
| S100A12 | 1.35 × 10−2 | -6.212 | 1.19 × 10−11 | PCM |
| ACTA2 | 5.83 | 2.544 | 1.81 × 10−11 | GCM |
| SPP1 | 1.53 × 101 | 3.931 | 1.88 × 10−11 | GCM |
| PTX3 | 1.35 × 10−2 | -6.211 | 4.19 × 10−11 | PCM |
| CRP | 1.34 × 10−2 | -6.217 | 8.29 × 10−11 | PCM |
| PRDX6 | 2.61 × 104 | 14.673 | 9.64 × 10−11 | GCM |
| ITGB1 | 1.54 × 101 | 3.942 | 2.08 × 10−10 | GCM |
| SLPI | 4.26 × 101 | 5.414 | 2.89 × 10−11 | GCM |
| LGALS3 | 9.35 × 104 | 16.512 | 4.22 × 10−10 | GCM |
| SOD1 | 2.70 × 104 | 14.721 | 5.82 × 10−10 | GCM |
| HSPB6 | 7.10 × 102 | 9.472 | 6.99 × 10−10 | GCM |
| ANXA5 | 2.63 × 104 | 14.685 | 7.67 × 10−11 | GCM |
| bTrappin | 2.15 | 1.107 | 9.96 × 10−10 | GCM |
| TTR | 6.99 × 102 | 9.449 | 1.00 × 10−9 | GCM |
| VTN | 4.63 | 2.211 | 1.13 × 10−9 | GCM |
| HSPB1 | 3.43 × 101 | 5.101 | 1.14 × 10−9 | GCM |
| FN1 | 3.80 × 102 | 8.569 | 1.31 × 10−9 | GCM |
| COL3A1 | 8.03 × 104 | 16.293 | 1.38 × 10−9 | GCM |
| THBS2 | 9.60 × 104 | 16.551 | 1.48 × 10−9 | GCM |
| GPX1 | 2.74 × 104 | 14.744 | 1.61 × 10−9 | GCM |
| ACTA1 | 9.54 × 104 | 16.541 | 1.87 × 10−9 | GCM |
1 FC – fold change; MPO – myeloperoxidase; LTF – lactotransferrin; ITGA6 – integrin α6; LCN2 – lipocalin-2; CLU – clusterin; S100A12 – S100 calcium-binding protein A12; ACTA2 – actin α2, smooth muscle; SPP1 – osteopontin; PTX3 – pentraxin 3; CRP – C-reactive protein; PRDX6 – peroxiredoxin 6; ITGB1 – integrin β1: SLPI – secretory leukocyte protease inhibitor; LGALS3 – galectin-3; SOD1 – superoxide dismutase 1; HSPB6 – heat-shock protein B6; ANXA5 – annexin A5; bTrappin – trappin-family whey acidic protein-domain protease inhibitor; TTR – transthyretin; VTN – vitronectin; HSPB1 – heat-shock protein B1; FN1 – fibronectin 1; COL3A1 – collagen type 3 α1 chain; THBS2 – thrombospondin 2; GPX1 – glutathione peroxidase 1; ACTA1 – actin α1, skeletal muscle
Fig. 6.
Functional enrichment of direction-specific protein sets in swamp-buffalo cervical mucus. GO:MF – Molecular Function in the Gene Ontology database; GO:BP – Biological Process in the Gene Ontology database; GO:CC – Cellular Component in the Gene Ontology database; KEGG – Kyoto Encyclopedia of Genes and Genomes; REAC – Reactome pathway database; WP – WikiPathways; MIRNA – microRNA-related pathways; CORUM – Comprehensive Resource of Mammalian Protein Complexes
Table 2.
Functional annotation of selected proteins and implications for swamp-buffalo cervical mucus quality
| Source | Term ID | Term name | Adjusted P-value | Protein |
|---|---|---|---|---|
| GO:MF | GO:0001849 | complement component C1q complex binding | 3.528 × 10-3 | PTX3; CRP |
| GO:BP | GO:0009620 | response to fungus | 1.310 × 10-3 | MPO; PTX3 |
| GO:BP | GO:0044793 | negative regulation by host of viral process | 2.456 × 10-2 | PTX3 |
| GO:CC | GO:0005576 | extracellular region | 7.656 × 10-7 | MPO; LTF; LCN2; SPP1; PTX3; CRP; SLPI; LGALS3; HSPB6 |
1 GO:MF – Molecular Function in the Gene Ontology database; GO:BP – Biological Process in the Gene Ontology database; GO:CC – Cellular Component in the Gene Ontology database; PTX3 – pentraxin 3; CRP – C-reactive protein; MPO – myeloperoxidase; LTF – lactotransferrin; LCN2 – lipocalin-2; SPP1 – osteopontin; SLPI – secretory leukocyte protease inhibitor; LGALS3 – galectin-3; HSPB6 – heat-shock protein B6
Fig. 7.
ROCs for candidate biomarkers in swamp-buffalo cervical mucus. GCM – good cervical mucus; PCM – poor cervical mucus; MPO – myeloperoxidase; LCN2 – lipocalin-2; PTX3 – pentraxin 3; CRP – C-reactive protein; HSPB6 – heat-shock protein B6; SLPI – secretory leukocyte protease inhibitor