Supplementary Data A

Transcription

1 Supplementary Data A Proteomic data and MS/MS fragmentation spectra from 2-DE and GeLC-MS/MS analysis of the bovine teat canal lining and the outer teat skin epithelium

2 Table A1: 2-DE proteins spots identified by nanolc-esi-q\tof MS\MS. Spot # Identified protein (species) NCBI accession code Predicted MW (kda) Predicted pi No. peptides matched Sequence coverage (%) 1 heat shock cognate 71 kda protein gi serum albumin precursor gi Chain A, Crystal Structure Of Bovine Serum Albumin gi serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 4-like gi PREDICTED: keratin, type II cytoskeletal 1 gi keratin, type I cytoskeletal 10 gi keratin, type I cytoskeletal 10 gi keratin, type I cytoskeletal 10 gi keratin, type I cytoskeletal 10 gi PREDICTED: keratin, type II cytoskeletal 1 gi PREDICTED: keratin, type II cytoskeletal 3 gi PREDICTED: keratin, type II cytoskeletal 3 gi PREDICTED: keratin, type II cytoskeletal 1 gi PREDICTED: keratin, type II cytoskeletal 3 gi PREDICTED: keratin, type II cytoskeletal 1 gi PREDICTED: keratin, type II cytoskeletal 1 gi PREDICTED: keratin, type II cytoskeletal 1 gi PREDICTED: keratin, type II cytoskeletal 3 gi PREDICTED: keratin, type II cytoskeletal 1 gi PREDICTED: keratin, type II cytoskeletal 1 gi PREDICTED: keratin, type II cytoskeletal 1 gi PREDICTED: keratin, type II cytoskeletal 3 gi Mowse score

3 18 PREDICTED: keratin, type II cytoskeletal 1 gi PREDICTED: keratin, type II cytoskeletal 1 gi PREDICTED: keratin, type II cytoskeletal 1 gi PREDICTED: SERPIN B3-like gi annexin A2 gi keratin, type I cytoskeletal 17 gi keratin, type II cytoskeletal 4 gi Chain A, The Structure Of Crystalline Profilin-Beta-Actin gi keratin, type II cytoskeletal 4 gi keratin, type I cytoskeletal 14 gi keratin, type II cytoskeletal 4 gi keratin, type II cytoskeletal 5 gi keratin, type II cytoskeletal 4 gi keratin 6A gi keratin, type II cytoskeletal 5 gi keratin, type II cytoskeletal 79 gi SERPIN B4 protein gi keratin, type II cytoskeletal 4 gi keratin, type II cytoskeletal 5 gi PREDICTED: keratin, type II cytoskeletal 1 gi keratin, type II cytoskeletal 6A gi SERPIN B4 protein gi keratin, type II cytoskeletal 5 gi keratin, type II cytoskeletal 4 gi keratin, type II cytoskeletal 79 gi keratin 6A gi PREDICTED: keratin, type II cytoskeletal 1 gi

4 31 PREDICTED: SERPIN B3-like gi PREDICTED: keratin, type II cytoskeletal 6A isoform 3 gi keratin, type II cytoskeletal 79 gi keratin, type II cytoskeletal 6A gi keratin 6A gi keratin, type II cytoskeletal 79 gi keratin, type II cytoskeletal 4 gi keratin, type II cytoskeletal 6A gi PREDICTED: keratin, type II cytoskeletal 6A isoform 3 gi keratin, type II cytoskeletal 79 gi keratin 6A gi PREDICTED: keratin, type II cytoskeletal 6A isoform 3 gi keratin, type II cytoskeletal 79 gi keratin, type II cytoskeletal 4 gi keratin, type II cytoskeletal 6A gi PREDICTED: keratin, type II cytoskeletal 6A isoform 3 gi keratin, type II cytoskeletal 79 gi keratin 6A gi PREDICTED: keratin, type II cytoskeletal 6A isoform 3 gi keratin, type II cytoskeletal 79 gi keratin, type II cytoskeletal 6A gi PREDICTED: keratin, type II cytoskeletal 6A isoform 3 gi keratin, type II cytoskeletal 79 gi EEF1A1 protein gi PREDICTED: SERPIN B3-like gi PREDICTED: SERPIN B4 gi PREDICTED: SERPIN B4-like gi

5 42 PREDICTED: SERPIN B4-like gi alpha S1 casein, partial gi caspase-14 gi protein sigma (ovis aries) gi PREDICTED: keratin, type II cytoskeletal 1 gi spot not identified 47 PREDICTED: SERPIN B3-like gi PREDICTED: SERPIN B3-like gi annexin I gi annexin A2 gi glyceraldehyde-3-phosphate dehydrogenase gi glyceraldehyde-3-phosphate dehydrogenase gi glyceraldehyde-3-phosphate dehydrogenase gi gasdermin-a gi heat shock protein beta-1 gi phosphoglycerate mutase 1 gi triosephosphate isomerase gi protein S100A9 gi protein S100A9 gi PREDICTED: non-specific cytotoxic cell receptor protein 1 homolog gi Chain X, The Cys121ser Mutant Of Beta-Lactoglobulin gi Chain X, The Cys121ser Mutant Of Beta-Lactoglobulin gi calmodulin gi caspase-14 gi PREDICTED: LOW QUALITY PROTEIN: aspartic peptidase, retroviral-like 1 gi calmodulin-like 5 gi fatty acid-binding protein, adipocyte gi

6 68 PREDICTED: galectin-7 gi fatty acid-binding protein, epidermal gi hemoglobin subunit beta gi Chain A, A Novel Allosteric Mechanism In Haemoglobin. gi protein S100A7 gi PREDICTED: protein S100A7-like gi protein S100A7 gi PREDICTED: protein S100A7-like gi protein S100A7 gi protein S100A7 gi PREDICTED: protein S100A7-like gi PREDICTED: protein S100A7-like gi protein S100A7 gi PREDICTED: protein S100A7-like gi PREDICTED: protein S100A7-like gi protein S100A7 gi S100 calcium binding protein A11 (calgizzarin) gi protein S100A8 gi protein S100A8 gi protein S100A8 gi protein S100A12 gi protein S100A12 gi Chain F, Crystal Structure Of Human Stam1 Vhs Domain In Complex With Ubiquitin gi PREDICTED: galectin-7 gi

7 Table A2: Quantitative changes in 43 differentially expressed teat canal lining and teat skin proteins by 2-DE (p<0.05). Group A - TCL Group B - Teat skin SSP Identified protein Average Spot intensity CV (%) Average Spot intensity CV (%) Fold change p-value (n=3) 105 calmodulin-like protein sigma S100A keratin, type I cytoskeletal S100A S100A caspase S100A7-like keratin, type I cytoskeletal S100A S100A7-like S100A7-like S100A keratin, type II cytoskeletal keratin, type II cytoskeletal keratin, type II cytoskeletal E keratin, type II cytoskeletal keratin, type II cytoskeletal galectin keratin, type II cytoskeletal keratin, type II cytoskeletal

8 5613 keratin, type II cytoskeletal keratin, type II cytoskeletal S100A S100A S100A SERPIN B4-like keratin, type II cytoskeletal 6A keratin, type II cytoskeletal 6A keratin, type II cytoskeletal 6A SERPIN B4-like keratin, type II cytoskeletal 6A keratin, type II cytoskeletal 6A keratin, type II cytoskeletal keratin, type II cytoskeletal keratin, type II cytoskeletal hemoglobin subunit beta hemoglobin subunit alpha keratin, type II cytoskeletal 6A keratin, type II cytoskeletal 6A keratin, type II cytoskeletal keratin, type II cytoskeletal keratin, type II cytoskeletal Spot intensity is an average optical density (OD) of the same spot from three replicate samples belonging to the same experimental group. Coefficient of variation (CV) shows spot variation across these three samples. Fold change is calculated from spot intensity with teat skin as the denominator.

9 Figure A1: PDQuest histograms of 2-DE spot intensities for differentially expressed teat canal lining and teat skin protein spots. The standard spot (SSP) number is displayed beneath each histogram. The number at the upper right hand corner of the histogram is the normalised quantity of the maximum bar in the graph. The other bars are drawn proportionally to the highest bar. Intensity bars from individual teat canal lining and teat skin spots are labelled in green and red, respectively.

10 Table A3: Summary of total MS/MS spectra from each tryptic digest fraction for the teat canal lining group from 6 cows # Fraction # Average SD Total % CV 8.1% SD, standard deviation; CV, coefficient of variation Table A4: Summary of total MS/MS spectra from each tryptic digest fraction for the teat skin group from 6 cows # Fraction # Average SD Total % CV 8.4% SD, standard deviation; CV, coefficient of variation

11 Table A5: Proteins with 2 or more peptides identified by GeLC-MS/MS from TCL and teat skin replicate samples at 5 % FDR Total unique peptide count Teat canal lining Teat skin Identified proteins (species) Accession MW [kda] pi 005 keratin, type II cytoskeletal 1 gi keratin, type I cytoskeletal 10 gi keratin, type II cytoskeletal 6A gi keratin, type II cytoskeletal 6A isoform 3 gi keratin, type II cytoskeletal 4 gi keratin, type II cytoskeletal 5 gi keratin, type I cytoskeletal 14 gi keratin, type II cytoskeletal 79 gi keratin, type I cytoskeletal 17 gi keratin, type II cytoskeletal 3 gi profilaggrin-like gi SERPIN B3-like gi SERPIN B3-like gi SERPIN B4-like protein gi SERPIN B3-like gi filaggrin gi SERPIN B3-like gi bovine serum albumin gi SERPIN B3 gi protein sigma (ovis aries) gi caspase-14 gi junction plakoglobin gi S100A12 gi

12 S100A9 gi keratin, type II cytoskeletal 2 oral gi annexin A1 gi beta-lactoglobulin gi protein zeta gi hemoglobin subunit beta gi hornerin gi alpha-s1-casein precursor gi desmoplakin gi keratin, type II cytoskeletal 2 epidermal gi ubiquitin gi fatty acid-binding protein, epidermal gi pancreatic adenocarcinoma upregulated factor-like gi glyceraldehyde-3-phosphate dehydrogenase gi nucleoside diphosphate kinase B gi S100A8 gi alpha-s2-casein precursor gi S100A7 gi beta-casein precursor gi S100A7-like gi malate dehydrogenase, cytoplasmic gi triosephosphate isomerase gi actin, beta gi hemoglobin subunit alpha gi gasdermin 1 gi annexin A2 gi heat shock cognate 71 kda protein gi IGL@ protein gi L-lactate dehydrogenase A chain gi

13 tropomyosin 4-like gi casein para kappaa gi repetin gi S100A2 gi transitional endoplasmic reticulum ATPase isoform 3 (canis lupus familiaris) gi SERPIN B12 gi heat shock 27kDa protein 1 gi insulin-degrading enzyme isoform X1 (IDE) gi proteasome subunit beta type-2 isoform 2 gi ganglioside GM2 activator precursor gi retroviral-like aspartic protease 1 gi annexin A8 gi purine nucleoside phosphorylase, chain A gi chloride intracellular channel protein 1 gi collagen, type VI, alpha 3-like isoform 4 gi F-box only protein 50 gi tubulin alpha-1b chain (mus musculus) gi alpha-actinin 1 gi calmodulin-like 5 gi IgM heavy chain constant region gi thioredoxin gi elongation factor 2 gi fatty acid-binding protein, adipocyte gi galectin-7 gi peptidylprolyl isomerase A gi cystatin-a gi histone H2A type 2-C gi histone H2B gi vimentin gi

14 peroxiredoxin-1 gi apolipoprotein A-I precursor gi elongation factor 1 alpha gi profilin gi protein DJ-1 gi S100A11 (calgizzarin) gi S100A14 gi protein beta/alpha (homo sapiens) gi protein epsilon (homo sapiens) gi S ribosomal protein S7 (homo sapiens) gi S ribosomal protein L11 isoform 2 (homo sapiens) gi S ribosomal protein L12 isoform X1 gi ADP-ribosylation factor 3 gi AHNAK nucleoprotein 2 gi aldose reductase gi enolase (alpha) gi angiogenin gi annexin A4 gi annexin V=CaBP33 isoform [cattle, brain, peptide, 320 aa] gi band 6 polypeptide B6P, partial gi basigin precursor gi beta-enolase gi cofilin-1 (sus scrofa) gi collagen alpha-2(vi) chain isoform X1 gi cornulin-like gi cullin 1-like gi cystatin-b gi desmocollin gi

15 desmoglein gi desmosomal cadherin gi epiplakin gi F-actin-binding protein gi fatty acid synthase isoform X1 gi gamma-glutamylcyclotransferase gi gelsolin isoform b gi glutathione S-transferase Mu 1 isoform X1 gi glutathione S-transferase P gi heat shock protein HSP 90-alpha gi heat shock protein HSP 90-beta gi histone H2A type 1 gi histone H3 family 3A gi histone H4 gi keratin 15 gi keratin 77-like gi keratin, type I cytoskeletal 16 gi keratin, type I cytoskeletal 19 gi keratin, type I cytoskeletal 27 gi keratin, type II cytoskeletal 73 gi lactoferrin gi L-lactate dehydrogenase B chain gi mediator of RNA polymerase II transcription subunit 1 gi mitochondrial malate dehydrogenase 2, NAD gi myosin regulatory light chain 12B-like isoform 2 gi macrophage migration-inhibitory factor [cattle, Peptide, 114 aa] gi peroxiredoxin-2 gi phosphatidylethanolamine binding protein gi

16 plakophilin-1 gi plasmalemmal porin gi putative RNA-binding protein 3 gi ras-related protein Rab-10 (mus musculus) gi ras-related protein Rab-11A isoform X1 gi ras-related protein Rab-1A gi ras-related protein Rab-1B gi ribosomal protein L12 gi secernin-3 gi SERPIN B13 isoform 1 gi suprabasin isoform X1 gi titin gi transgelin-2 gi tyrosine 3/tryptophan 5 -monooxygenase activation protein gi ZBTB42 protein-like gi zinc finger protein 638 gi zymogen granule protein 16 homolog B gi Cells highlighted in grey correspond to biological replicates (n = 6) where no peptides were identified in either the TCL or teat skin group.

17 (A) (B) Figure A2: Representative MS/MS spectra identifying the two diagnostic peptides for S100A7. (A) Fragmentation pattern for precursor ion at m/z ; sequence: DNFPNFLGACEK. (B) Fragmentation pattern for precursor ion at m/z ; sequence: GRDYLSNIFEK. Peaks corresponding to b (red) and y (blue) ions are marked. Diagnostic amino acids detected are labelled with an asterisk.

18 (A) K/ ENFPNFLSACEK /R (B) R/ QYLSDIFEK /K Figure A3: Representative MS/MS spectra identifying the two diagnostic peptides for S100A7-like protein. (A) Fragmentation pattern for precursor ion at m/z ; sequence: ENFPNFLSACEK. (B) Fragmentation pattern for precursor ion at m/z ; sequence: QYLSDIFEK. Peaks corresponding to b (red) and y (blue) ions are marked. Diagnostic amino acids detected are labelled with an asterisk.

19 Table A6: Differential expression of keratin proteins in the teat canal lining and teat skin proteomes. Average peptide count (n=6) Identified keratin protein Teat canal lining Teat skin Fold change p-value keratin, type II cytoskeletal x 10-5 keratin, type II cytoskeletal 2 epidermal x keratin, type II cytoskeletal 2 oral x 10-2 keratin, type II cytoskeletal x 10-4 keratin, type II cytoskeletal x 10-7 keratin, type II cytoskeletal x 10-4 keratin, type II cytoskeletal 6A x 10-4 keratin, type II cytoskeletal 6A isoform * 0.01 keratin, type I cytoskeletal x 10-3 keratin, type I cytoskeletal x 10-4 keratin * keratin, type I cytoskeletal * 0.41 keratin, type I cytoskeletal * 0.02 keratin, type I cytoskeletal * keratin, type I cytoskeletal * keratin, type II cytoskeletal * keratin 77-like 0.5* keratin, type II cytoskeletal * 0.02 * Proteins absent in either of the two samples were given a value of 0.5 so that a fold change could be calculated. Fold change was calculated from the average peptide count with TCL as the denominator. p-values were derived from the six teat canal lining and six teat skin samples and were calculated as a two-tailed test with even distribution.

20 Table A7: Proteins identified in all 12 GeLC samples from teat canal lining and teat skin Average peptide count Identified proteins Accession number Teat canal lining Teat skin Fold change Keratins keratin, type II cytoskeletal 1 gi keratin, type I cytoskeletal 10 gi keratin, type II cytoskeletal 2 epidermal gi keratin, type II cytoskeletal 2 oral gi keratin, type II cytoskeletal 3 gi keratin, type II cytoskeletal 4 gi keratin, type II cytoskeletal 5 gi keratin, type II cytoskeletal 6A gi keratin, type I cytoskeletal 14 gi Cytoskeleton desmoplakin gi hornerin gi junction plakoglobin gi filaggrin gi profilaggrin-like gi tubulin alpha-1b chain (mus musculus) gi Major milk proteins alpha-s1-casein precursor gi beta-casein precursor gi beta-lactoglobulin gi S100 calcium-binding proteins S100A7 gi S100A7-like gi S100A8 gi S100A9 gi S100A12 gi Cellular proteins actin, beta gi annexin A1 gi annexin A2 gi ubiquitin gi fatty acid-binding protein, epidermal gi heat shock 27kDa protein 1 gi L-lactate dehydrogenase A chain gi malate dehydrogenase, cytoplasmic gi thioredoxin gi transitional endoplasmic reticulum ATPase gi isoform 3 (canis lupus familiaris) Serum proteins bovine serum albumin gi hemoglobin subunit alpha gi hemoglobin subunit beta gi Apoptosis galectin-7 gi caspase-14 gi gasdermin 1 gi Fold change is calculated from average peptide counts with teat skin counts used as the denominator.

21 Supplementary Data B Experiments to determine monoclonal and polyclonal antibody specificity

22 B1: Immunohistochemical staining of super mammary lymph node. Figure B2: Immune cell staining of super mammary lymph node tissue. Cryosections of super mammary lymph node tissues were probed with anti-mhc class II monoclonal antibody (a), anti-cd205 monoclonal antibody (b), and anti-cd3 monoclonal antibody (c). The bound anti-mhc class II antibody was detected using Alexa Fluor-594-IgG2a while the anti-cd205 and anti-cd3 antibodies were detected with Alexa Fluor-594-IgG (red signal). Cryosections probed with anti-cd8 monoclonal antibody (d), and anti-cd14 monoclonal antibody (e) were detected with Alexa Fluor-488-IgM labelled secondary antibody (green signal). Nuclei were counterstained with DAPI (blue signal). Section thickness: 5 µm. Scale bar = 100 µm.

23 Supplementary Data C Proteomic data and MS/MS fragmentation spectra from 2-DE and GeLC-MS/MS analysis of late-lactating teat canal lining and 14 d involution teat canal lining

24 Figure C1: PDQuest histograms of 2-DE spot intensities for differentially expressed latelactating and 14 d involution teat canal lining protein spots. The standard spot (SSP) number is displayed beneath each histogram. The number at the upper right hand corner of the histogram is the normalised quantity of the maximum bar in the graph. The other bars are drawn proportionally to the highest bar. Intensity bars from individual latelactating and 14 d involution teat canal lining spots are labelled in red and green, respectively.

25 Table C1: Summary of LC-MS/MS results for the four spiked proteins in E. coli lysate a. Gel slice SpC Peptide count Sequence coverage Conalbumin fmol Ave SD SEM fmol Ave SD SEM fmol Ave SD SEM SpC Peptide count Sequence coverage Ovalbumin fmol Ave SD SEM fmol Ave SD SEM fmol Ave SD SEM SpC Peptide count Sequence coverage Carbonic fmol Ave SD SEM fmol Ave SD SEM fmol Ave SD SEM Anhydrase SpC Peptide count Sequence coverage Lysozyme fmol Ave SD SEM fmol Ave SD SEM fmol Ave SD SEM SpC, spectral count; Ave, average; SD, standard deviation; SEM, standard error of the mean

26 (A) (B) (C) Figure C2: Correlation between SpC, peptide count and sequence coverage with protein abundance level. In each experiment four non- E. coli proteins were added to E. coli lysate at different concentrations representing different protein abundance levels. For all added proteins, a linear correlation was observed between SpC and relative protein abundance (A). Regression coefficients decreased between peptide count and relative protein abundance (B) and sequence coverage and relative protein abundance (C). The results are an average of four technical replicates. Error bars represent the standard error of the mean.

27 Table C2: Summary of LC-MS/MS results for the E. coli proteins in each of the five dilution series lysates. The results are from the four technical replicates. Abbreviations used for E. coli proteins are as follows: PFL, pyruvate formate-lyase [2PFL_A]; DnaK, chaperone protein DnaK [ZP_ ]; TEF Tu, translation elongation factor Tu [EKW71632]; PPH, phosphopyruvate hydratase [ZP_ ]; Oxidoreductase, [ZP_ ]; OMP A, outer membrane protein A [ZP_ ]; Global, global DNA-binding transcriptional dual regulator H-NS [WP_ ]; Stress F, universal stress protein F [WP_ ]. Overall coefficient of variation (CV) was 14.9 %. Gel slice SpC (PFL) SpC (DnaK) Conalbumin fmol Ave. SD SEM %CV fmol Ave. SD SEM %CV % % % % % % % % % % SpC (TEF Tu) SpC (PPH) Ovalbumin fmol Ave. SD SEM %CV fmol Ave. SD SEM %CV % % % % % % % % % % SpC (Oxireductase) SpC (OMP A) Carbonic fmol Ave. SD SEM %CV fmol Ave. SD SEM %CV Anhydrase % % % % % % % % % % SpC (Global) SpC (Stress F) Lysozyme fmol Ave. SD SEM %CV fmol Ave. SD SEM %CV % % % % % % % % % % SpC, spectral count; Ave, average; SD, standard deviation; SEM, standard error of the mean; CV, coefficient of variation

28 (A) (B) (C) (D) Figure C3: Correlation between E. coli protein SpC with added protein abundance levels. In each experiment two of the most abundant E. coli proteins were identified and their SpC plotted relative to the concentration of the added non-e. coli proteins. For all E. coli proteins, a nonlinear correlation was observed between SpC and relative added protein abundance for conalbumin (A), ovalbumin (B), carbonic anhydrase (C), and lysozyme (D). The results are an average of four technical replicates. Error bars represent the standard error of the mean. Abbreviations used for E. coli proteins are as follows: PFL, pyruvate formate-lyase [2PFL_A]; DnaK, chaperone protein DnaK [ZP_ ]; TEF Tu, translation elongation factor Tu [EKW71632]; PPH, phosphopyruvate hydratase [ZP_ ]; Oxidoreductase, [ZP_ ]; OMP A, outer membrane protein A [ZP_ ]; Global, global DNA-binding transcriptional dual regulator H-NS [WP_ ]; Stress F, universal stress protein F [WP_ ].

29 Table C3: Summary of total MS/MS spectra from each tryptic digest fraction for the 14 d involution teat canal lining group from 7 cows Fraction # Average SD Total % CV 8.1% SD, standard deviation; CV, coefficient of variation

30 Table C4: 86 proteins with 2 or more peptides identified by GeLC-MS/MS from 14 d TCL replicate samples at 5 % FDR Identified proteins Accession MW [kda] pi Total unique peptide count 14 d Involution TCL keratin, type II cytoskeletal 1 gi keratin, type I cytoskeletal 10 gi keratin, type II cytoskeletal 6A gi keratin, type II cytoskeletal 4 gi keratin, type II cytoskeletal 79 gi keratin, type II cytoskeletal 3 gi keratin, type II cytoskeletal 6C gi keratin, type I cytoskeletal 14 gi keratin, type II cytoskeletal 5 gi profilaggrin-like gi keratin, type I cytoskeletal 17 gi filaggrin-like gi filaggrin-like, partial gi serpin B3-like gi serpin B3-like gi serpin B4-like protein gi serpin B3-like gi lactoferrin gi keratin, type II cytoskeletal 2, oral gi serum albumin precursor gi

31 keratin, type II cytoskeletal 2, epidermal gi annexin A1 gi protein S100A9 gi protein S100A12 gi annexin A2 gi glyceraldehyde-3-phosphate dehydrogenase gi caspase-14 gi beta-lactoglobulin gi hemoglobin subunit beta gi hornerin gi cytokeratin 8 (370 AA) gi alpha S1 casein, partial gi protein S100A7 gi hemoglobin alpha chain gi protein S100A8 gi protein S100A7-like gi ubiquitin gi imunoglobulin light chain, lambda gi fatty acid-binding protein, epidermal gi desmoplakin gi hornerin-like gi beta-casein gi gasdermin 1 gi malate dehydrogenase, cytoplasmic gi IgM heavy chain constant region gi F-box only protein 50 gi protein S100A2 gi

32 cystatin-a gi cathelicidin-1 precursor gi nucleoside diphosphate kinase B gi transitional endoplasmic reticulum ATPase 3 gi serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 12-like gi lactate dehydrogenase-a gi S100 calcium binding protein A11 gi beta actin gi L-lactate dehydrogenase B chain gi serotransferrin precursor gi pancreatic adenocarcinoma upregulated factor-like gi proteasome subunit beta type-2 isoform 2 gi mammalian 20s proteasome gi protein DJ-1 gi lactoperoxidase precursor gi alpha-s2-casein precursor gi casein para kappaa gi IgG1 heavy chain constant region, partial gi thioredoxin gi junction plakoglobin isoform X1 gi apolipoprotein A-I precursor gi histone H2B gi serpin B3-like gi

33 serpin B3 gi hypothetical protein BOS_17317 gi keratin, type I cytoskeletal 16 gi keratin, type II cytoskeletal 78 gi keratin, type I cytoskeletal 24 gi proteasome subunit alpha type-7 isoform X3 gi protein S100A14 gi polyubiquitin-c gi cathepsin O preproprotein-like gi heat shock cognate 71 kda protein gi protein sigma gi zymogen granule protein 16 homolog B gi galectin-7 gi tropomyosin alpha-3 chain isoform 8 gi heat shock 27kDa protein 1 gi cyclophilin I gi

34 Table C5: Normalised total SpCs of 25 proteins showing two-fold change in protein abundance between late-lactating and 14 d involution TCL (p < 0.05) Late-lactating TCL 14 d involution TCL Identified proteins LL Ave d14 Ave p-value Fc lactoferrin vimentin cofilin glyceraldehyde-3-phosphate dehydrogenase fatty acid binding protein, epithelial repetin transgelin purine nucleoside phosphorylase histone H2A type 1-B/E tubulin alpha-1b chain histone H2B type 1-D collagen, type VI, alpha 3-like isoform nucleoside diphosphate kinase B L-lactate dehydrogenase A chain peroxiredoxin

35 Table B5 continued: protein sigma Junction plakoglobin insulin-degrading enzyme precursor protein zeta peptidylprolyl isomerase A aspartic peptidase, retroviral-like galectin heat shock protein beta heat shock cognate 71 kda protein beta-actin Fc, fold count

36 Supplementary Data D Microbiological methods and proteomic GeLC-MS/MS analysis of the bovine teat canal lining 24 h post bacterial challenge

37 D1 Preparation of cultures for teat canal inoculation The extent of turbidity, or optical density (OD), was used to measure the cell biomass indirectly as the bacteria grew in liquid culture. Two cultures were made for each of the three microbes. One culture for measuring OD and the other culture for glycerol stocks. All three bacterial cultures exhibited standard, in vitro, temporal bacterial growth curves with characteristic lag, exponential and stationary phases (Figure D1). Both the E.coli and S. aureus cultures were stopped and harvested for glycerol-stock storage at late-log phase when their absorbance reading reached an OD of 0.8. S. uberis not only grew slower than the other two cultures, but its stationary phase was also lower (OD of 0.4). Based on this growth curve, the S. uberis culture was stopped and 0.5 ml aliquots harvested for glycerol-stock storage at late-log phase when its absorbance reading reached an OD of Glycerol stocks of bacteria were stored at -80 C until required. In preparation for teat infusion, bacteria were recovered from frozen glycerol stock solutions, washed in 1 ml of one-quarter strength ringer s solution containing 0.1 % (w/v) proteose-peptone for 1 h at 37 C. One hundred microlitres of this suspension was diluted into 9.9 ml of sterile skim milk. After 16 h growth at 37 C, ten-fold serial dilutions were performed to estimate the bacterial concentration in the skim milk suspension (See Methods Figure 2.2). The suspensions were then adjusted by dilution to give final inoculum concentrations of 2.5 x 10 8 cfu/ml (~500,000 cfu in 2 µl of skim milk), 1.25 x 10 6 cfu/ml (~2500 cfu in 2 µl of skim milk), and 1.25 x 10 6 cfu/ml (~2500 cfu in 2 µl of skim milk), for E. coli, S. aureus, and S, uberis, respectively. Minimal cell death (< 3 %) was observed when the stock skim milk suspensions were stored overnight at 4 C (data not shown). Stock skim milk suspensions were therefore made two days preceding the on-farm inoculations, allowing for a 16 h growth phase and then a 24 h storage phase. This storage phase enabled an accurate bacterial count for each inoculum to be determined prior to administration (Table D1). Dilutions of the inoculums were also replated after the on-farm inoculations. The results from this analysis showed negligible change (< 5.0 %) from the preceding dilution series (data not shown).

38 Figure D1: Growth curves for E.coli, S. aureus and S. uberis in brain heart infusion broth.

39 Table D1: Bacterial concentrations for teat canal inoculations. Day 1 Colony counts prior to teat canal inoculations Bacteria Number of colonies Average Dilution cfu a /ml SD b E. coli TMTC c TMTC TMTC 1 x 10-3 TMTC TMTC TMTC 1 x x x x x 10-6 S. uberis TMTC TMTC TMTC 1 x x x x x x 10-5 S. aureus TMTC TMTC TMTC 1 x x x x x x 10-5 Day 2 Colony counts prior to teat canal inoculations Bacteria Number of colonies Average Dilution cfu/ml SD E. coli TMTC TMTC TMTC 1 x x x x x x 10-7 S. uberis TMTC TMTC TMTC 1 x x x x x x 10-5 S. aureus TMTC TMTC TMTC 1 x x x x x x 10-5 a) cfu, colony forming units; b) SD, standard deviation; c) TMTC, Too many to count

40 Table D2: Significantly altered 2-DE proteins spots from Control verses E. coli infection groups SSP Con-1 Con-2 Con-3 E. coli-1 E. coli-2 E. coli-3 (Ave) Con SD (Ave) E. coli SD Fc p-value (n = 3) Identified protein caspase lactoglobulin keratin actin heat shock protein beta spot not identified serpin B3-like serpin B3-like serum albumin S100A serpin B3-like keratin 4/ keratin keratin 6A ubiquitin hemoglobin subunit beta spot not identified

41 Table D3: Significantly altered 2-DE proteins spots from Control verses S. aureus infection groups SSP Con-1 Con-2 Con-3 S.aureus-1 S.aureus-2 S.aureus-3 (Ave) Con SD (Ave) S. aureus SD Fc p-value Identified protein caspase spot not identified heat shock cognate 71 kda protein keratin 4/ keratin serum albumin spot not identified serpin B3-like keratin ubiquitin beta hemoglobin spot not identified spot not identified keratin 6A non-specific cytotoxic cell receptor protein 1 homolog

42 Table D4: Significantly altered 2-DE proteins spots from Control verses S. uberis infection groups (Ave) (Ave) SSP Con-1 Con-2 Con-3 S.uberis-1 S.uberis-2 S.uberis-3 SD SD Fc p-value Identified protein Con S. uberis spot not identified spot not identified spot not identified spot not identified caspase spot not identified lactoglobulin keratin S100A serum albumin spot not identified keratin 4/ keratin keratin spot not identified ubiquitin S100A spot not identified spot not identified annexin A non-specific cytotoxic cell receptor protein 1 homolog keratin 1

43 Table D5: Summary of total MS/MS spectra from each teat canal lining tryptic digest fraction from the bacterial challenge study group of 4 cows # Treatment Con EC SA SU Con EC SA SU Quarter FL BL FR BR FL BR FR BL Fraction Fraction Fraction Fraction Fraction Fraction Fraction Fraction Total # Treatment Con EC SA SU Con EC SA SU Quarter FR BR FL BL FR BR BL FL Fraction Fraction Fraction Fraction Fraction Fraction Fraction Fraction Total Fraction 1 Fraction 2 Fraction 3 Fraction 4 Fraction 5 Fraction 6 Fraction 7 Fraction 8 Average SD % CV SD, standard deviation; CV, coefficient of variation

44 Table D6: Normalised raw SpC data for experimentally induced bacterial challenge study # Protein Acc. No. Normalised SpCs Control E. coli S. aureus S. uberis Ratio Protein Name Mwt gi keratin, type II cytoskeletal 1 63 kda gi keratin, type I cytoskeletal kda gi keratin, type II cytoskeletal 6A 61 kda gi cytokeratin type II, component IV isoform 2 59 kda gi keratin, type II cytoskeletal 3 65 kda gi keratin, type II cytoskeletal 4 58 kda gi keratin, type II cytoskeletal 5 63 kda gi keratin, type II cytoskeletal kda gi profilaggrin-like 56 kda gi keratin, type II cytoskeletal 2 (epidermal) 64 kda gi keratin, type I cytoskeletal kda gi filaggrin-like 180 kda gi S100-A9 16 kda gi S100-A12 11 kda gi serpin B3-like 44 kda gi keratin, type I cytoskeletal kda gi alpha S1 casein, partial 24 kda gi serpin B3-like 44 kda gi serpin B3-like 42 kda gi serpin B4 44 kda gi hornerin 175 kda gi S100-A8 10 kda gi hemoglobin beta-a chain 16 kda gi caspase kda gi junction plakoglobin 82 kda gi beta-lactoglobulin 18 kda E. coli /Con S. aureus /Con S. uberis /Con

45 27 gi desmoplakin 332 kda gi serum albumin precursor 69 kda gi S100-A7 12 kda gi S100-A7 isoform X2 12 kda gi nucleoside diphosphate kinase B 17 kda gi hemoglobin subunit alpha 15 kda gi hornerin-like 164 kda gi glyceraldehyde-3-phosphate dehydrogenase 36 kda gi fatty acid binding protein, epidermal 15 kda gi protein sigma 28 kda gi kappa casein 18 kda gi gasdermin-a 50 kda gi beta casein 25 kda gi galectin-7-like 15 kda gi alpha-s2-casein precursor 26 kda gi immunoglobulin mu heavy chain constant region 50 kda gi IGL@ protein 25 kda gi fatty acid-binding protein, adipocyte 15 kda gi polyubiquitin-b 34 kda gi histone H2A type 1-like 34 kda gi protein zeta/delta 28 kda gi retroviral-like aspartic protease 1 34 kda gi cystatin-a 11 kda gi S100-A14 11 kda gi histone H2B type 1-D 14 kda gi heat shock protein beta-1 22 kda gi S100-A2 11 kda gi 74 annexin I 39 kda gi beta-actin 42 kda gi malate dehydrogenase, cytoplasmic 36 kda gi proteasome subunit beta type-2 23 kda

46 58 gi F-box only protein kda gi ganglioside GM2 activator precursor 21 kda gi L-lactate dehydrogenase A chain 37 kda gi protein DJ-1 20 kda gi annexin A2 39 kda gi keratin, type II cytoskeletal kda gi pancreatic adenocarcinoma upregulated factor-like 17 kda gi alpha-actinin kda gi peroxiredoxin 1 22 kda gi peptidylprolyl isomerase A 17 kda gi thioredoxin 12 kda gi repetin 78 kda gi serpin B12 46 kda gi insulin-degrading enzyme precursor 118 kda gi desmoglein-1 precursor 112 kda gi transitional endoplasmic reticulum ATPase 89 kda gi triosephosphate isomerase 27 kda gi heat shock cognate 71 kda protein 71 kda gi PSMA7 protein 27 kda gi tropomyosin alpha-3 chain isoform 4 29 kda gi L-lactate dehydrogenase B chain 37 kda gi profilin 15 kda gi beta2-microglobulin 12 kda gi AHNAK nucleoprotein kda gi serotransferrin precursor 78 kda gi ezrin 69 kda gi myosin light polypeptide 6 isoform 1 17 kda gi cornulin-like 87 kda gi fibrinogen gamma-b chain isoform X1 49 kda

47 87 gi peptidoglycan recognition protein 1 21 kda gi ras-related protein Rab kda gi ATP-dependent RNA helicase DDX3X 73 kda gi creatine kinase M chain 43 kda gi prelamin-a/c isoform X1 74 kda gi S ribosomal protein S20 isoform 2 13 kda gi putative RNA-binding protein 3 10 kda gi protein POF1B 57 kda gi cathelicidin-1 precursor 18 kda gi myoglobin 17 kda gi lactoferrin 78 kda gi elongation factor 2 95 kda gi calmodulin-like isoform 2 17 kda gi xanthine dehydrogenase 147 kda gi histone H kda gi glutathione S-transferase P 24 kda gi apolipoprotein A-I precursor 30 kda gi S ribosomal protein L11 20 kda gi keratin, type I cytoskeletal kda gi heat shock protein HSP 90- alpha isoform X1 89 kda gi tripeptidyl-peptidase kda gi ribosomal protein L12 16 kda gi pyruvate kinase isozymes M1/M2 58 kda gi S ribosomal protein S7 22 kda gi cofilin-1 19 kda gi AHNAK nucleoprotein 587 kda gi plastin-3 72 kda gi eukaryotic translation elongation factor 1 alpha 1-like 50 kda gi transthyretin precursor 16 kda gi transgelin-2 22 kda gi tubulin alpha-1b chain 50 kda

48 118 gi ATP-citrate synthase isoform X1 123 kda gi proteasome subunit beta type-3 23 kda gi phosphatidylethanolaminebinding protein 1 21 kda gi short-chain dehydrogenase/ reductase family 9C member 7 35 kda gi epiplakin 376 kda gi annexin A5 36 kda gi myosin kda gi filamin A, alpha 276 kda gi actin, alpha 1, skeletal muscle 42 kda gi clathrin heavy chain kda gi enolase, alpha 47 kda gi vimentin 54 kda gi malate dehydrogenase, mitochondrial 30 kda gi purine nucleoside phosphorylase 32 kda gi collagen, type VI, alpha 3-like isoform kda gi annexin A8 37 kda gi collagen alpha-2(vi) chain isoform X1 109 kda gi kallikrein-10 precursor 31 kda gi myosin kda gi annexin IV 36 kda gi proteasome subunit beta type-4 precursor 29 kda gi peroxiredoxin-2 22 kda gi proteasome subunit alpha type-3 isoform X1 28 kda

49 Table D7: Normalised total, EmPAI and NSAF ratios for significantly regulated teat canal lining proteins Normalised total ratio Average normalised SpC E. coli S. aureus S. uberis p-value verses control # Protein name Control E.coli S. aureus S. uberis /Control /Control /Control E.coli S. aureus S. uberis 28 serum albumin precursor glyceraldehyde-3-phosphate dehydrogenase heat shock protein beta pancreatic adenocarcinoma upregulated factor-like serotransferrin precursor lactoferrin apolipoprotein A-I precursor EmPAI ratio Average EmPAI value E. coli S. aureus S. uberis p-value verses control # Protein name Control E.coli S. aureus S. uberis /Con /Con /Con E.coli S. aureus S. uberis 28 serum albumin precursor glyceraldehyde-3-phosphate dehydrogenase heat shock protein beta pancreatic adenocarcinoma upregulated factor-like serotransferrin precursor * 41.3* 17.0* lactoferrin apolipoprotein A-I precursor NSAF ratio Average NSAF value E. coli S. aureus S. uberis p-value verses control # Protein name Control E.coli S. aureus S. uberis /Con /Con /Con E.coli S. aureus S. uberis 28 serum albumin precursor glyceraldehyde-3-phosphate dehydrogenase heat shock protein beta pancreatic adenocarcinoma upregulated factor-like serotransferrin precursor * 6.63* 2.40* lactoferrin apolipoprotein A-I precursor * For serotransferrin fold calculations, the control value was set to 0.01 and to calculate EmPAI and NSAF ratios, respectively.

50 Table D8: Quickscore summary of MHC class II signal in teat-end tissue regions from 24 h bacteria challenge cows. # Treatment Tissue region Control E. coli S. aureus S. uberis Teat canal Fürstenberg s rosette Teat sinus Teat canal Fürstenberg s rosette Teat sinus Teat canal Fürstenberg s rosette Teat sinus Teat canal Fürstenberg s rosette Teat sinus Immunofluorescent signal intensities in the cryosections were subjectively graded as weakly positive (+), mildly positive (++), moderately positive (+++) and strongly positive (++++). Table D9: Quickscore summary of CH138A signal in teat-end tissue regions from 24 h bacteria challenge cows. # Treatment Tissue region Control E. coli S. aureus S. uberis Teat canal Fürstenberg s rosette Teat sinus Teat canal Fürstenberg s rosette Teat sinus Teat canal Fürstenberg s rosette Teat sinus Teat canal Fürstenberg s rosette Teat sinus Immunofluorescent signal intensities in the cryosections were subjectively graded as weakly positive (+), mildly positive (++), moderately positive (+++) and strongly positive (++++).

51 (A) Con EC SA SU Teat canal Con EC SA SU Teat sinus Con EC SA SU Fürstenberg's rosette (B) Con EC SA SU Teat canal Con EC SA SU Teat sinus Con EC SA SU Fürstenberg's rosette Figure D2: Semi-quantitative comparison of the proportion CD205 + and CD3 + cells in 24 h challenged teat-end tissues. Positively stained CD205 expressing cells associated with the epithelial bilayer (A), and CD3 expressing cells located within the epithelial layer (B) were counted from seven nonadjacent fields per slide at 200 x magnification and then averaged. The results are cumulative data from four latelactating cows and are expressed as a proportion of the total number of nuclei in each field. Con; Control inoculation, EC; E. coli, SA; S. aureus, SU; S. uberis. Error bar: ± SEM.