Wickramasinghe et al. BMC Genomics 2012, 13:45 http://www.biomedcentral.com/1471-2164/13/45 highest expression of cathepsins. Enzymes involved in the plasminogen activation pathway had high expression in peak lactation. Therefore, milk collected in earlier stages of lactation might have the minimum protease effect on the quality of dairy products. Plasmin in milk has been shown to cause bitterness in infant formula, and cathepsins have been shown to contribute to pro- teolysis of Swiss cheese [53]. With the present gene expression analysis results one can suggest that late lac- tation milk should be preferable to peak lactation milk for infant formula production and peak lactation milk may be optimal for Swiss cheese production. More research on biochemical and immunological assays on milk proteases are necessary to draw conclusions on selecting milk from the best lactation stage for the pro- duction of different dairy products. Expression of genes in ubiquitin- proteasome pathway Ubiquitin-proteasome pathway (UPP) is a non-lysoso- mal, ATP-dependent pathway involved in degradation of proteins in the cell [54]. We have observed an enrich- ment of GO terms associated with UPP in peak and late lactation MSC. Lemay et al. [31] identified UPP as the most significantly enriched pathway during lactation and involution in mouse with the highest number of genes up regulated during early involution of the mammary gland. Because of the important regulatory functions performed by UPP, and the significant enrichment results observed in the current study and the mouse mammary gland, we analyzed the expression of 48 genes [54] belonging to UPP (Table 4). In UPP, degradation initiates by the covalent linkage of cellular proteins to multiple molecules of ubiquitin proteins (UB). Eight genes encoding UB and UB-like proteins (UBL) were expressed in bovine MSC. Among these genes, UBA52 had the highest expression at all three stages of lactation. UBC showed a significant high expression in peak lactation whilst most other UB genes showed higher expression in late lactation. Activity of three enzymes, UB-activating enzyme (E1), UB-conjugat- ing enzyme (E2) and UB-protein ligase (E3) are required for the attachment of UB to the target proteins. Most the genes encoding these three enzymes had signifi- cantly higher expression in peak and late lactation. Once the target proteins are attached to UB there will be degradation by proteolytic activity in proteasomes. Six genes encoding proteasome proteins were expressed in the milk samples, and except for ATAD3A, all the others showed increased expression in late lactation. Deubiquitination enzymes regulate the overall proteoly- sis of UPP by removal of conjugated UB by proteolysis. Six deubiquitination enzymes were expressed in MSC Table 4 Expression values of genes encoding enzymes in ubiquitin-proteasome pathway at 15, 90 and 250 days of lactation Protein UB activating enzyme (E1) UBA1 UB-conjugating enzyme (E2) 128.64 134.10 Gene symbol1 CCDC34 BRCA1 UBE4B UBE4A UBE2R2 UFD1L UBE2I UBE2C UBE2B SKP2 CUL5 ANAPC10 SMURF2 CUL4B CUL2 BTRC SMAD3 ANAPC5 ANAPC1 CUL3 CUL1 FBXW2 SMAD2 CUL4A SKP1 STUB1 RBX1 SUMO1 UBL7 UBAP1 NEDD8 UBD UBC RPS27A UBA52 0.88 0.70 1.15 49.97 18.95 22.41 8.78 32.96 28.78 50.89 98.49 106.18 32.48 163.89 259.41 112.33 1095.10 332.69 551.89 451.18 630.29 1710.22 1277.97 1903.08 Page 10 of 14 Day 2502 1.47 2.71 13.01 17.83 26.34 39.23 42.38 42.98 43.34 1.07 2.39 3.05 4.45 5.29 5.96 6.21 6.36 6.94 8.20 8.59 14.64 25.13 26.03 44.91 48.03 52.47 76.38 0.23 0.56 6.28 12.68 14.05 23.79 2.39 11.38 13.15 69.91 Day 152 57.96 0.32 0.48 5.83 8.19 10.91 27.59 14.54 6.43 16.53 0.24 0.93 1.63 1.48 3.02 1.62 2.11 3.03 3.14 1.73 3.62 4.88 9.39 5.46 15.25 14.72 42.41 33.24 0.60 0.05 2.65 4.84 4.54 7.26 0.66 8.23 4.43 18.19 Day 902 1.14 1.87 9.83 23.34 21.70 45.93 37.27 19.54 48.69 1.00 1.80 3.12 4.72 4.87 6.14 12.92 10.07 7.08 8.01 8.21 14.32 27.72 20.50 59.93 61.88 55.86 42.01 0.37 0.46 5.23 18.33 10.15 17.51 3.37 13.67 9.99 49.89 UB-protein ligase (E3) UB and UB-like proteins Deubiquitination enzymes USP2 USP9X USP21 VCPIP1 USP8 USP10 Proteasome ATAD2 ATAD3A ATAD1 PSMA1