BACKGROUND
mastitis, an inflammation of the mammary gland, remains the most prevalent disease and the largest economic loss in dairy cattle.
the economic impact of mastitis on the u.s dairy industry in  <dig> was estimated at $ <dig>  billion and increased up to $ <dig> billion in the year of  <dig>  <cit> .
a large number of microorganisms, most of which are bacteria, have been reported to cause bovine mastitis.
worldwide, however, the most common udder pathogens isolated from the clinical cases are staphylococci aureus , streptococcus dysgalactiae and escherichia coli.
the s. aureus is considered as a contagious mastitis-causing pathogen and remains an important mastitis pathogen in most countries  <cit> .
the frequency of mastitis in the dairy cow population could potentially be decreased by breeding for cows with better ability to resist udder disease.
therefore, identifying specific genes involved in the susceptibility or resistance to mastitis could lead to new approaches for mastitis control through genetic selection  <cit> .
mastitis involves a complex set of interactions between an invading pathogen and immune systems of the host.
proteomics and the associated bioinformatics are considered as complimentary tools for the study the dynamic interactions between the immune system and pathogens  <cit> .
most proteomic studies on mastitis conducted to date have been performed using two-dimensional electrophoresis  and liquid chromatography  coupled with tandem mass spectrometry  methods  and using milk, serum or somatic cells.
for instance, differential expression analysis of the whey from both mastitic and non-mastitic milk revealed a series of proteins including acute phase proteins , lactotransferrin and immunoglobulins that present a marked alternation during infection  <cit> .
several studies have reported proteomics profiles in milk and serum of cows infected with different pathogens  <cit> .
for instance, a total of  <dig> milk proteins were identified and more than  <dig> milk proteins associated with host defense were identified in s. aureus infected and normal milk using the isobaric tag for relative and absolute quantification  method  <cit> .
differentially expressed milk proteins at  <dig> and 14 days post-intramammary infection with different s. aureus strains have also been identified by 2de  <cit> .
forty-seven peptide biomarkers of milk for the diagnosis of mastitis were discovered using capillary electrophoresis and mass spectroscopy  <cit> .
the transcriptional response of the mammary gland to mastitis infected different pathogens has been reported in several microarray studies  <cit> , while information at the proteome level in mammary gland tissue is still limited particularly in animals with natural infections.
persistence of pathogen infections through late stages can worsen damage of the mammary gland and result in milk lost, or leave necrotic tissue and important injures  <cit> .
therefore, the investigation of changes in the protein expression upon the onset, progression and late onset of mastitis is crucial for providing a full picture of the events triggered by this disease.
the exact quantification of differentially expressed proteins has proven difficult with gel-based approaches.
nevertheless, to date, there are no proteomic studies aimed at investigating the susceptibility to s. aureus-associated mastitis in dairy cows.
here we present an exploration of the mastitis-induced changes in the proteome of mammary gland tissue using the itraq system.
this approach allows the simultaneous identification and quantitative comparison of peptides by measuring peak intensities of reporter ions in tandem mass spectrometry  spectra  <cit> .
differentially expressed proteins were used for bioinformatics analyses via the gene ontology , dynamic impact approach   <cit>  and the ingenuity pathway analysis .
RESULTS
protein spectrum and go analysis of mammary gland tissue of dairy cows
in the present study, itraq technology in combination with lc-esi-ms/ms was applied to investigate differentially expressed proteins in the healthy and mastitis-infected mammary glands of dairy cows.
a total of  <dig> proteins were identified from  <dig> peptides according to the standard of protein identification .
of which,  <dig> % proteins were identified with one peptide and  <dig> % proteins with  <dig> to  <dig> peptides.
col1a <dig>  collagen alpha- <dig> chain possesses the most of peptides.
there are  <dig> peptides covering the  <dig> % protein sequence with above 95% confidence.
several proteins like lactoferrin, myh <dig>  alpha-s1-casein, col3a <dig>  serum albumin, col6a <dig> and col1a <dig> were identified with over  <dig> peptides.
figure  <dig> shows the relative expression of two proteins  in the healthy and mastitis-infected mammary gland tissues which was calculated as a ratio by comparing the intensity of the itraq- <dig> and itraq- <dig> after normalization.figure  <dig>
ms/ms spectrum for the relative quantity of two representative proteins  col1a <dig> ;  itih <dig> .
the two lines on the top of the peak figure correspond to the peptides sequence ; amino acid with bold letters present detected sequence.
amino acid + digital present the decorated aa, while the number shows the quality deviation occurred, such as +  <dig> denotes the itraq modification is introduced by the itraq reagent.
horizontal axis is the value of m/z and the vertical axis is the normalized intensity.
b <dig>  b <dig>  b <dig> … y <dig>  y <dig>  y3… correspond to the peak of the detected b and y ions.
the peaks corresponding to itraq– <dig> and itraq- <dig> mark these  <dig> pool samples.
the intensity of two peaks was used for the quantitative of protein.
the peak figure is done by scaffold software at http://www.proteomesoftware.com/.
expressed proteins in mammary gland tissues of cows were classified by the david online program .
these proteins were clustered into  <dig> clusters including cytoskeletal protein binding, signal peptides, etc.
gene functional annotation analysis indicated that thirty proteins such as, cd <dig>  serum amyloid  <dig>  casein alpha-s <dig>  cathelicidin  <dig>  cathelicidin  <dig>  cathelicidin antimicrobial peptide, complement component  <dig> , complement component  <dig> , complement factor b were involved in defense, immune and inflammatory reactions .
six hundred genes participated in  <dig> pathways like ecm-receptor interaction , complement and coagulation cascades , and focal adhesion  kegg pathways .table  <dig>
proteins involved in the defense, immune and inflammatory reactions by david analysis
identification and pathway analysis of the differential proteins between the healthy and mastitic cows’ mammary gland tissues
basing on the standard of the differentially expressed proteins,  <dig> proteins were significantly differentially up-regulated  while  <dig> proteins were significantly down-regulated  in mastitis-infected cow group’s proteins labeled itraq- <dig> when compared with that of the healthy group’s proteins labeled itraq − <dig> .table  <dig>
differentially expressed proteins between the healthy and mastitis-infected cows’ mammary gland tissues
up-regulated proteins in the mastitis group
down-regulated proteins in the mastitis group
in term of go database, the differentially expressed proteins were divided into cc, mf and bp categories.
the top  <dig> go terms for cc with the minimal p values were extracellular region, extracellular region part, extracellular space, extracellular matrix and extracellular matrix part .
the top  <dig> go terms for mf were growth factor binding, cell surface binding, metal ion binding, motor activity and cation binding .
the top  <dig> go terms with a p-value < <dig>  for mf were response to stress, response to stimulus, skeletal system development, response to inorganic substance, gas transport, peptide cross-linking, skin development, protein heterooligomerization, extracellular structure organization, response to steroid hormone stimulus, response to chemical stimulus and defense response .
for example,  <dig> proteins  participated in the defense, immune and inflammatory responses .
another important protein was c <dig> with a p-value  <dig> , involved also in the immune response .
of  <dig> proteins, the expression of itih <dig> protein was increased by  <dig> -fold in the mastitis group when compared with the control group.
itih <dig> protein was enriched in the membrane, cell, intracellular and intracellular part terms for cc ; it was also enriched in the nonpeptidase/peptidase/enzyme inhibitor/regulator activity terms for mf ; it is related with the response to stress/stimulus, defense response, acute or inflammatory response, amine/carbohydrate/macromolecule/nitrogen compound metabolic processes terms for bp .
moreover, according to the bp classification, the differential protein col1a <dig> with the most of peptides were involved in  <dig> go terms, such as response to stimulus, extracellular structure organization, cellular component assembly, etc.
.
col1a <dig> enriched in the  <dig> go terms for cc, for example, extracellular region part, fibrillar collagen and intracellular part .
basing on the mf classification, col1a <dig> were also related with  <dig> go terms including growth factor binding, structural molecule activity, proteins binding and binding .
pathway enrichment analysis showed that  <dig> differential proteins participated in  <dig> pathways.
among them,  <dig> proteins  were enriched in the ecm-receptor interaction pathway that serves an important role in tissue and organ morphogenesis and in the maintenance of cell and tissue structure and function,  <dig> proteins  participated in the complement coagulation cascades pathway.
the itih <dig> and flna proteins took part in the mapk signaling pathway.
two proteins fgg and hp upregulated by  <dig> - and  <dig> - fold in mastitis-infected cow’s tissues were involved in s. aureus infection pathway .
dia analysis  revealed that complement and coagulation cascade was the kegg pathway with the highest impact value in which fibrinogen complex , platelet activation  and protein binding, bridging  were the significantly impacted go terms.
focal adhesion, amoebiasis, protein digestion and absorption, and ecm-receptor interaction pathways were also significantly impacted in the dia analysis.
these pathways had in common col1a <dig>  col1a <dig> and fn <dig> proteins, with activation of skin morphogenesis , cartilage development involved in endochondral bone morphogenesis , collagen fibril organization , platelet activation  and protein binding, bridging  go terms .
some go terms related to a specific protein but not to any pathway with a high impact and inhibition were casein, alpha/beta  for alpha-s2b-casein , cartilage development involved in endochondral bone morphogenesis  and endochondral ossification  for hspg <dig> and csn1s <dig> proteins, platelet activation  for alb, and oxidoreductase activity, acting on the aldehyde or oxo group of donors, nad or nadp as acceptor  for aldh1a <dig> .
validation of two candidate proteins col1a <dig> and itih4
among the identified proteins, two proteins col1a <dig> and itih <dig> were considered as interesting protein for further studies.
firstly, the relative expressions of col1a <dig>  and itih <dig>  in the healthy and mastitis-infected cows’ mammary gland tissues were investigated by the western blotting.
the results showed that the expression of two proteins significantly increased in the mastitis-infected group when compared with the healthy control group .
these two proteins were also detected in the mammary gland tissue of two groups by the immunohistochemical staining method .
however, it should be considered that the apparent difference of col1a <dig> and itih <dig> observed by western blotting and ihc methods were smaller than that seen by itraq , although consistent with the observations made by itraq, respectively, might be due to differences in specificity of the commercially available antibodies used in this work, which are not specific for bovine proteins.
in the present study, for example, several collagen alpha family proteins  present the similar differentially expressed tendency from  <dig> -fold to  <dig> -fold .
therefore, the quantitative conclusions should be drawn only by means of quantitative proteomic approaches or alternative quantitative assays.figure  <dig>
expression and localization of bovine col1a <dig> and itih <dig> proteins.
a, b: the localization and expression of col1a <dig> in the healthy cow  and mastitis cow’s  mammary gland tissues by immunohistochemical staining.
brown indicates the positive col1a <dig> protein expression; blue presents the negative.
d, e: the localization and expression of itih <dig> in the hc and mc group’s mammary gland tissues by immunohistochemical staining.
brown indicates the positive itih <dig> protein; blue presents the negative.
c, f: the results of western blotting reveal the significant increase of col1a <dig> and itih <dig> proteins in mc group compared to hc group .
discussion
in this study, we performed a comprehensive evaluation of proteomic profile in the mammary glands of cows infected naturally by s. aureus, providing new data on the in vivo events occurring in the lactating mammary epithelium during persistent infection.
subsequently, go enrichment and pathway analyses of proteins in combination with western blotting and immunohistochemistry detection for proteins of interest were focused on the consistent results involved in the immune, inflammatory and defense response for mastitis infection.
generally, host defense against microbial pathogens requires appropriate coordination of multiple signaling pathways.
these pathways are triggered by innate immune recognition of microbial molecules, and initiate an inflammatory cascade that involved recruitment of leukocytes to the site of infection, activation of antimicrobial effector mechanisms and induction of an adaptive immune response  <cit> .
in the present study, we found the global expressed and differentially expressed proteins participated in respective  <dig> and  <dig> pathways including mapk, ecm-receptor interaction, complement and coagulation cascades, and focal adhesion kegg pathways .
mapk play key roles in activating host innate immune responses and are frequent targets of pathogenic effectors in animal systems  <cit> .
the complement and coagulation cascades are connected to the immune system  <cit> .
cells of the immune system are activated by soluble chemokine signals and must migrate through endothelial cell or solid tissue barriers to reach sites of inflammation or infection during the progress of performing host-defense functions.
adhesive interactions of immune cells with endothelium, extracellular matrix components, and cells of solid organs are the critical control point of the immune response.
both the soluble chemokine and cell adhesion receptor-mediated migration signals must converge on common intracellular targets to engage the cell migration machinery  <cit> .
specifically, in this study, several differentially expressed proteins, such as fibrinogen proteins , complement proteins  expressed in mammary gland participated in many pathways encompassing the mapk, complement and coagulation cascades, focal adhesion, etc.
the complement system plays a fundamental role in innate immunity in addition to enhancing adaptive immune responses and is therefore a primary line of defense against infection  <cit> .
the beginning of the pathogenesis of mastitis is determined by the binding of s. aureus to platelets  <cit> .
this binding is likely to be mediated in part by soluble bridging molecules  like fibrinogen   <cit> , that link the organism to the platelet surface .
platelets play a key role in innate defenses against staphylococci by releasing platelet microbicidal proteins that kill many s. aureus isolates.
platelet binding may be an important mechanism for host against mastitic infections, because platelets attached to damaged cell surfaces may serve as binding center for organisms circulating in the blood  <cit> .
moreover, clinical and subclinical mastitis is characterized by abnormalities in the coagulation process due to excessive fibrin deposition in microvasculature leading to clot formation  <cit> .
c <dig> protein was numerically down-regulated in the mastitic cow’s mammary gland with the p value nearing significance.
a2m protein belongs to the complement and coagulation cascades pathway, which has been reported that it is involved in the mastitis susceptibility in our previous publication  <cit> .
the combined use of itraq labelling and lc esi-ms/ms is a powerful tool for the proteomics identification of the dairy cow’s mammary gland tissues.
in this study, only  <dig> proteins were detected in the cow’s mammary gland.
whereas, a transcriptome profiling study reveals more than  <dig>  differential expressed genes  in the streptococcus uberis-induced mastitis mammary gland when compared with noninfected control quarters  <cit> .
obviously, the number of proteins is by far, less than the amount of genes at the mrnas level.
the divergence might be caused by these factors, such as distinct response to different bacteria  <cit>  and strain types  <cit> , different infection ways , different infection stages , translation regulation of mrnas to proteins, or limitation of detection method.
on the other hand, the contrasting data between proteomics and transcriptomics analysis, suggests that changes in protein profiles between the healthy and mastitis-infected cows’ mammary gland tissues provide information that is complementary to transcriptional profiling studies, revealing potentially crucial aspects of the mastitis infection response.
especially, when the mammary gland was at the state of fibrosis for the serious infection, expression of some proteins associated with metabolism would be shut down.
three s <dig> calcium-binding proteins s <dig> calcium-binding protein a <dig> , s100a <dig> and s100a <dig>  are involved in the immune, defense and inflammatory responses, and have proinflammatory functions and direct antimicrobial effects  <cit> .
it has been reported that there is  <dig>  fold to  <dig>  fold up-regulation of s100a <dig> expression for three cows at the infection dose of 1 × 105
s. aureus when compared with the intra-animal controls  <cit> .
furthermore, significantly increased s100a <dig> protein expression was detected in the milk whey from the infected udders in all three cows at 16 h post-infection  <cit> .
in the present study, proteins s100a <dig>  s100a <dig> and s100a <dig>  were up-regulated by  <dig> ,  <dig>  and  <dig>  folds in the mastitis-infected mammary glands, which is consistent with the previous report, further demonstrating their roles in the immune response to pathogen infection.
type i collagen, which is encoded by two subunits of col1a <dig> and one subunit of col1a <dig> wound together to form a triple-helix, of which the coordinated transcription rates ensure a 2: <dig> ratio.
it is the most abundant protein in vertebrates and is the main structural protein of skin, bone, teeth, and tendon  <cit> .
moreover, it plays a major role in tissue and organ development, cell migration, proliferation, and differentiation, wound healing, tissue remodeling, and homeostasis and therefore it participates in the maintenance of organ morphology and function.
its excessive deposition results in the fibrosis of tissues  <cit> .
bovine mammary glands are composed of glandular tissue and connective tissue.
it is easy to understand the most abundant expression of col1a <dig>  our finding of type i collagen protein expression is consistent with the above result.
during infection of the mammary glands, the tissue damage can initially be caused by bacteria and their products.
certain bacteria like s. aureus produce toxins that destroy cell membranes and damage milk-producing tissue, whereas other bacteria are able to invade and multiply within the bovine mammary epithelial cells.
then, more immune cells migrate into the mammary gland and harm the blood-milk barrier.
damage of the mammary epithelium gets worsen and it finally results in fibrosis  <cit> .
therefore, regulating the bovine col1a <dig> expression for reducing tissue damage may be a cost-effective way to reduce the losses caused by mastitis.
focal adhesion, amoebiasis, ecm receptor interacting and protein digestion and absorption pathways were highly impacted due to the activation of several proteins that might have a role in the conformational changes in the parenquimal epithelial cells due to s. aureus infection .
heparan sulfate proteoglycan  <dig>  or perlecan blocks endothelial cell adhesion to fibronectin and type i collagen in the submucosal connective tissue from the parenquimal tissue of the infected mammary gland.
fibronectin plays a major role in cell adhesion, growth, migration and differentiation, and it is important for processes such as wound healing  <cit> .
altered fibronectin expression has been associated with fibrosis  <cit> .
milk obtained from cows with clinical mastitis possessed high counts of somatic cells and very high levels of protease activity which hydrolyzes casein  <cit> .
this process was also detected by dia analysis by a high impacted inhibition of caseins .
the acute-phase response occurs in animals and elevates app as a consequence of infection, inflammation, or trauma.
app including serum amyloid a, haptoglobin, alpha-1-acid glycoprotein, alpha-1-proteinase inhibitor  and itih <dig> have been shown to increase in different inflammatory processes, in both in vivo and in vitro models, and involved several inflammatory diseases, such as crohn’s disease and ulcerative colitis .
itih <dig> protein is a 120kd glycoprotein, which is prone to be cleaved to produce fragments of different length, such as 100 kda and 35 kda fragment, which have been identified as the biomarkers for down syndrome  <cit> , amyotrophic lateral sclerosis  <cit> , and cancer  <cit> .
a report showed that all heifers responded to the mastitis with increased itih <dig> concentrations  <cit> .
severely infected heifers showed maximum itih <dig> concentrations at 72 h after bacterial injection.
the concentration of the protein in serum was 6- to 12- fold higher than before the bacterial challenge.
in the four animals with milder clinical mastitis, the protein exhibited only a three- to four-fold increase during the infection process and had reached a plateau by 48 h after the challenge.
in both cases, the itih <dig> concentrations decreased to normal values about 2 weeks after the injection  <cit> .
in this study, however, at the late stage of infection, the itih <dig> protein was first found to be significantly increased in the mammary gland tissues as compared to the control case.
in this study, itraq method is unable to differentiate cleaved fragments from whole protein.
the result of western blotting implies that 100 kda fragment of bovine itih <dig> plays an important role in the defense and immune responses to infection of mastitis.
CONCLUSIONS
proteomics analysis highlighted the effects of proteins like collagen, fibrinogen, fibronectin, casein alpha and heparan sulfate proteoglycan  <dig> among others in mammary gland tissues infected naturally by s. aureus.
this study also established a protein database characteristic of the mammary response during late on-set of s. aureus naturally occurring mastitis.
the itraq technology and lc-ms/ms data generated, can be used as a reference for further research in this field.
ours is the first to examine the late response to natural mastitis providing a ‘closer look’ into the mechanisms associated with host tissue damage.
the differentially expressed proteins we discovered in this study, such as col1a <dig> and itih <dig>  may serve as potential genes for the susceptibility to mastitis in dairy cows.

 mastitis, an inflammation of the mammary gland, remains the most prevalent disease and the largest economic loss in dairy cattle. the economic impact of mastitis on the u.s dairy industry in was estimated at $ billion and increased up to $ billion in the year of. a large number of microorganisms, most of which are bacteria, have been reported to cause bovine mastitis. worldwide, however, the most common udder pathogens isolated from the clinical cases are staphylococci aureus, streptococcus dysgalactiae and escherichia coli. the s. aureus is considered as a contagious mastitis-causing pathogen and remains an important mastitis pathogen in most countries. the frequency of mastitis in the dairy cow population could potentially be decreased by breeding for cows with better ability to resist udder disease. therefore, identifying specific genes involved in the susceptibility or resistance to mastitis could lead to new approaches for mastitis control through genetic selection. mastitis involves a complex set of interactions between an invading pathogen and immune systems of the host. proteomics and the associated bioinformatics are considered as complimentary tools for the study the dynamic interactions between the immune system and pathogens. most proteomic studies on mastitis conducted to date have been performed using two-dimensional electrophoresis and liquid chromatography coupled with tandem mass spectrometry methods and using milk, serum or somatic cells. for instance, differential expression analysis of the whey from both mastitic and non-mastitic milk revealed a series of proteins including acute phase proteins, lactotransferrin and immunoglobulins that present a marked alternation during infection. several studies have reported proteomics profiles in milk and serum of cows infected with different pathogens. for instance, a total of milk proteins were identified and more than milk proteins associated with host defense were identified in s. aureus infected and normal milk using the isobaric tag for relative and absolute quantification method. differentially expressed milk proteins at and 14 days post-intramammary infection with different s. aureus strains have also been identified by 2de. forty-seven peptide biomarkers of milk for the diagnosis of mastitis were discovered using capillary electrophoresis and mass spectroscopy. the transcriptional response of the mammary gland to mastitis infected different pathogens has been reported in several microarray studies, while information at the proteome level in mammary gland tissue is still limited particularly in animals with natural infections. persistence of pathogen infections through late stages can worsen damage of the mammary gland and result in milk lost, or leave necrotic tissue and important injures. therefore, the investigation of changes in the protein expression upon the onset, progression and late onset of mastitis is crucial for providing a full picture of the events triggered by this disease. the exact quantification of differentially expressed proteins has proven difficult with gel-based approaches. nevertheless, to date, there are no proteomic studies aimed at investigating the susceptibility to s. aureus-associated mastitis in dairy cows. here we present an exploration of the mastitis-induced changes in the proteome of mammary gland tissue using the itraq system. this approach allows the simultaneous identification and quantitative comparison of peptides by measuring peak intensities of reporter ions in tandem mass spectrometry spectra. differentially expressed proteins were used for bioinformatics analyses via the gene ontology, dynamic impact approach and the ingenuity pathway analysis.  protein spectrum and go analysis of mammary gland tissue of dairy cows in the present study, itraq technology in combination with lc-esi-ms/ms was applied to investigate differentially expressed proteins in the healthy and mastitis-infected mammary glands of dairy cows. a total of proteins were identified from peptides according to the standard of protein identification. of which, % proteins were identified with one peptide and % proteins with to peptides. col1a collagen alpha- chain possesses the most of peptides. there are peptides covering the % protein sequence with above 95% confidence. several proteins like lactoferrin, myh alpha-s1-casein, col3a serum albumin, col6a and col1a were identified with over peptides. figure  shows the relative expression of two proteins in the healthy and mastitis-infected mammary gland tissues which was calculated as a ratio by comparing the intensity of the itraq- and itraq- after normalization.figure ms/ms spectrum for the relative quantity of two representative proteins col1a; itih. the two lines on the top of the peak figure correspond to the peptides sequence; amino acid with bold letters present detected sequence. amino acid + digital present the decorated aa, while the number shows the quality deviation occurred, such as +  denotes the itraq modification is introduced by the itraq reagent. horizontal axis is the value of m/z and the vertical axis is the normalized intensity. b b b … y y y3… correspond to the peak of the detected b and y ions. the peaks corresponding to itraq– and itraq- mark these pool samples. the intensity of two peaks was used for the quantitative of protein. the peak figure is done by scaffold software at http://www.proteomesoftware.com/. expressed proteins in mammary gland tissues of cows were classified by the david online program. these proteins were clustered into clusters including cytoskeletal protein binding, signal peptides, etc. gene functional annotation analysis indicated that thirty proteins such as, cd serum amyloid casein alpha-s cathelicidin cathelicidin cathelicidin antimicrobial peptide, complement component, complement component, complement factor b were involved in defense, immune and inflammatory reactions. six hundred genes participated in pathways like ecm-receptor interaction, complement and coagulation cascades, and focal adhesion kegg pathways.table proteins involved in the defense, immune and inflammatory reactions by david analysis identification and pathway analysis of the differential proteins between the healthy and mastitic cows’ mammary gland tissues basing on the standard of the differentially expressed proteins, proteins were significantly differentially up-regulated while proteins were significantly down-regulated in mastitis-infected cow group’s proteins labeled itraq- when compared with that of the healthy group’s proteins labeled itraq −.table differentially expressed proteins between the healthy and mastitis-infected cows’ mammary gland tissues up-regulated proteins in the mastitis group down-regulated proteins in the mastitis group in term of go database, the differentially expressed proteins were divided into cc, mf and bp categories. the top go terms for cc with the minimal p values were extracellular region, extracellular region part, extracellular space, extracellular matrix and extracellular matrix part. the top go terms for mf were growth factor binding, cell surface binding, metal ion binding, motor activity and cation binding. the top go terms with a p-value < for mf were response to stress, response to stimulus, skeletal system development, response to inorganic substance, gas transport, peptide cross-linking, skin development, protein heterooligomerization, extracellular structure organization, response to steroid hormone stimulus, response to chemical stimulus and defense response. for example, proteins participated in the defense, immune and inflammatory responses. another important protein was c with a p-value, involved also in the immune response. of proteins, the expression of itih protein was increased by -fold in the mastitis group when compared with the control group. itih protein was enriched in the membrane, cell, intracellular and intracellular part terms for cc; it was also enriched in the nonpeptidase/peptidase/enzyme inhibitor/regulator activity terms for mf; it is related with the response to stress/stimulus, defense response, acute or inflammatory response, amine/carbohydrate/macromolecule/nitrogen compound metabolic processes terms for bp. moreover, according to the bp classification, the differential protein col1a with the most of peptides were involved in go terms, such as response to stimulus, extracellular structure organization, cellular component assembly, etc.. col1a enriched in the go terms for cc, for example, extracellular region part, fibrillar collagen and intracellular part. basing on the mf classification, col1a were also related with go terms including growth factor binding, structural molecule activity, proteins binding and binding. pathway enrichment analysis showed that differential proteins participated in pathways. among them, proteins were enriched in the ecm-receptor interaction pathway that serves an important role in tissue and organ morphogenesis and in the maintenance of cell and tissue structure and function, proteins participated in the complement coagulation cascades pathway. the itih and flna proteins took part in the mapk signaling pathway. two proteins fgg and hp upregulated by - and - fold in mastitis-infected cow’s tissues were involved in s. aureus infection pathway. dia analysis revealed that complement and coagulation cascade was the kegg pathway with the highest impact value in which fibrinogen complex, platelet activation and protein binding, bridging were the significantly impacted go terms. focal adhesion, amoebiasis, protein digestion and absorption, and ecm-receptor interaction pathways were also significantly impacted in the dia analysis. these pathways had in common col1a col1a and fn proteins, with activation of skin morphogenesis, cartilage development involved in endochondral bone morphogenesis, collagen fibril organization, platelet activation and protein binding, bridging go terms. some go terms related to a specific protein but not to any pathway with a high impact and inhibition were casein, alpha/beta for alpha-s2b-casein, cartilage development involved in endochondral bone morphogenesis and endochondral ossification for hspg and csn1s proteins, platelet activation for alb, and oxidoreductase activity, acting on the aldehyde or oxo group of donors, nad or nadp as acceptor for aldh1a. validation of two candidate proteins col1a and itih4 among the identified proteins, two proteins col1a and itih were considered as interesting protein for further studies. firstly, the relative expressions of col1a and itih in the healthy and mastitis-infected cows’ mammary gland tissues were investigated by the western blotting. the results showed that the expression of two proteins significantly increased in the mastitis-infected group when compared with the healthy control group. these two proteins were also detected in the mammary gland tissue of two groups by the immunohistochemical staining method. however, it should be considered that the apparent difference of col1a and itih observed by western blotting and ihc methods were smaller than that seen by itraq, although consistent with the observations made by itraq, respectively, might be due to differences in specificity of the commercially available antibodies used in this work, which are not specific for bovine proteins. in the present study, for example, several collagen alpha family proteins present the similar differentially expressed tendency from -fold to -fold. therefore, the quantitative conclusions should be drawn only by means of quantitative proteomic approaches or alternative quantitative assays.figure expression and localization of bovine col1a and itih proteins. a, b: the localization and expression of col1a in the healthy cow and mastitis cow’s mammary gland tissues by immunohistochemical staining. brown indicates the positive col1a protein expression; blue presents the negative. d, e: the localization and expression of itih in the hc and mc group’s mammary gland tissues by immunohistochemical staining. brown indicates the positive itih protein; blue presents the negative. c, f: the results of western blotting reveal the significant increase of col1a and itih proteins in mc group compared to hc group. discussion in this study, we performed a comprehensive evaluation of proteomic profile in the mammary glands of cows infected naturally by s. aureus, providing new data on the in vivo events occurring in the lactating mammary epithelium during persistent infection. subsequently, go enrichment and pathway analyses of proteins in combination with western blotting and immunohistochemistry detection for proteins of interest were focused on the consistent results involved in the immune, inflammatory and defense response for mastitis infection. generally, host defense against microbial pathogens requires appropriate coordination of multiple signaling pathways. these pathways are triggered by innate immune recognition of microbial molecules, and initiate an inflammatory cascade that involved recruitment of leukocytes to the site of infection, activation of antimicrobial effector mechanisms and induction of an adaptive immune response. in the present study, we found the global expressed and differentially expressed proteins participated in respective and pathways including mapk, ecm-receptor interaction, complement and coagulation cascades, and focal adhesion kegg pathways. mapk play key roles in activating host innate immune responses and are frequent targets of pathogenic effectors in animal systems. the complement and coagulation cascades are connected to the immune system. cells of the immune system are activated by soluble chemokine signals and must migrate through endothelial cell or solid tissue barriers to reach sites of inflammation or infection during the progress of performing host-defense functions. adhesive interactions of immune cells with endothelium, extracellular matrix components, and cells of solid organs are the critical control point of the immune response. both the soluble chemokine and cell adhesion receptor-mediated migration signals must converge on common intracellular targets to engage the cell migration machinery. specifically, in this study, several differentially expressed proteins, such as fibrinogen proteins, complement proteins expressed in mammary gland participated in many pathways encompassing the mapk, complement and coagulation cascades, focal adhesion, etc. the complement system plays a fundamental role in innate immunity in addition to enhancing adaptive immune responses and is therefore a primary line of defense against infection. the beginning of the pathogenesis of mastitis is determined by the binding of s. aureus to platelets. this binding is likely to be mediated in part by soluble bridging molecules like fibrinogen, that link the organism to the platelet surface. platelets play a key role in innate defenses against staphylococci by releasing platelet microbicidal proteins that kill many s. aureus isolates. platelet binding may be an important mechanism for host against mastitic infections, because platelets attached to damaged cell surfaces may serve as binding center for organisms circulating in the blood. moreover, clinical and subclinical mastitis is characterized by abnormalities in the coagulation process due to excessive fibrin deposition in microvasculature leading to clot formation. c protein was numerically down-regulated in the mastitic cow’s mammary gland with the p value nearing significance. a2m protein belongs to the complement and coagulation cascades pathway, which has been reported that it is involved in the mastitis susceptibility in our previous publication. the combined use of itraq labelling and lc esi-ms/ms is a powerful tool for the proteomics identification of the dairy cow’s mammary gland tissues. in this study, only proteins were detected in the cow’s mammary gland. whereas, a transcriptome profiling study reveals more than differential expressed genes in the streptococcus uberis-induced mastitis mammary gland when compared with noninfected control quarters. obviously, the number of proteins is by far, less than the amount of genes at the mrnas level. the divergence might be caused by these factors, such as distinct response to different bacteria and strain types, different infection ways, different infection stages, translation regulation of mrnas to proteins, or limitation of detection method. on the other hand, the contrasting data between proteomics and transcriptomics analysis, suggests that changes in protein profiles between the healthy and mastitis-infected cows’ mammary gland tissues provide information that is complementary to transcriptional profiling studies, revealing potentially crucial aspects of the mastitis infection response. especially, when the mammary gland was at the state of fibrosis for the serious infection, expression of some proteins associated with metabolism would be shut down. three s calcium-binding proteins s calcium-binding protein a, s100a and s100a are involved in the immune, defense and inflammatory responses, and have proinflammatory functions and direct antimicrobial effects. it has been reported that there is fold to fold up-regulation of s100a expression for three cows at the infection dose of 1 × 105  s. aureus when compared with the intra-animal controls. furthermore, significantly increased s100a protein expression was detected in the milk whey from the infected udders in all three cows at 16 h post-infection. in the present study, proteins s100a s100a and s100a were up-regulated by, and folds in the mastitis-infected mammary glands, which is consistent with the previous report, further demonstrating their roles in the immune response to pathogen infection. type i collagen, which is encoded by two subunits of col1a and one subunit of col1a wound together to form a triple-helix, of which the coordinated transcription rates ensure a 2: ratio. it is the most abundant protein in vertebrates and is the main structural protein of skin, bone, teeth, and tendon. moreover, it plays a major role in tissue and organ development, cell migration, proliferation, and differentiation, wound healing, tissue remodeling, and homeostasis and therefore it participates in the maintenance of organ morphology and function. its excessive deposition results in the fibrosis of tissues. bovine mammary glands are composed of glandular tissue and connective tissue. it is easy to understand the most abundant expression of col1a our finding of type i collagen protein expression is consistent with the above result. during infection of the mammary glands, the tissue damage can initially be caused by bacteria and their products. certain bacteria like s. aureus produce toxins that destroy cell membranes and damage milk-producing tissue, whereas other bacteria are able to invade and multiply within the bovine mammary epithelial cells. then, more immune cells migrate into the mammary gland and harm the blood-milk barrier. damage of the mammary epithelium gets worsen and it finally results in fibrosis. therefore, regulating the bovine col1a expression for reducing tissue damage may be a cost-effective way to reduce the losses caused by mastitis. focal adhesion, amoebiasis, ecm receptor interacting and protein digestion and absorption pathways were highly impacted due to the activation of several proteins that might have a role in the conformational changes in the parenquimal epithelial cells due to s. aureus infection. heparan sulfate proteoglycan or perlecan blocks endothelial cell adhesion to fibronectin and type i collagen in the submucosal connective tissue from the parenquimal tissue of the infected mammary gland. fibronectin plays a major role in cell adhesion, growth, migration and differentiation, and it is important for processes such as wound healing. altered fibronectin expression has been associated with fibrosis. milk obtained from cows with clinical mastitis possessed high counts of somatic cells and very high levels of protease activity which hydrolyzes casein. this process was also detected by dia analysis by a high impacted inhibition of caseins. the acute-phase response occurs in animals and elevates app as a consequence of infection, inflammation, or trauma. app including serum amyloid a, haptoglobin, alpha-1-acid glycoprotein, alpha-1-proteinase inhibitor and itih have been shown to increase in different inflammatory processes, in both in vivo and in vitro models, and involved several inflammatory diseases, such as crohn’s disease and ulcerative colitis. itih protein is a 120kd glycoprotein, which is prone to be cleaved to produce fragments of different length, such as 100 kda and 35 kda fragment, which have been identified as the biomarkers for down syndrome, amyotrophic lateral sclerosis, and cancer. a report showed that all heifers responded to the mastitis with increased itih concentrations. severely infected heifers showed maximum itih concentrations at 72 h after bacterial injection. the concentration of the protein in serum was 6- to 12- fold higher than before the bacterial challenge. in the four animals with milder clinical mastitis, the protein exhibited only a three- to four-fold increase during the infection process and had reached a plateau by 48 h after the challenge. in both cases, the itih concentrations decreased to normal values about 2 weeks after the injection. in this study, however, at the late stage of infection, the itih protein was first found to be significantly increased in the mammary gland tissues as compared to the control case. in this study, itraq method is unable to differentiate cleaved fragments from whole protein. the result of western blotting implies that 100 kda fragment of bovine itih plays an important role in the defense and immune responses to infection of mastitis.  proteomics analysis highlighted the effects of proteins like collagen, fibrinogen, fibronectin, casein alpha and heparan sulfate proteoglycan among others in mammary gland tissues infected naturally by s. aureus. this study also established a protein database characteristic of the mammary response during late on-set of s. aureus naturally occurring mastitis. the itraq technology and lc-ms/ms data generated, can be used as a reference for further research in this field. ours is the first to examine the late response to natural mastitis providing a ‘closer look’ into the mechanisms associated with host tissue damage. the differentially expressed proteins we discovered in this study, such as col1a and itih may serve as potential genes for the susceptibility to mastitis in dairy cows. 
bioinformatics analyses of differentially expressed proteins were performed by means of gene ontology, metabolic pathways, transcriptional regulation networks using blast2go software, the dynamic impact approach and ingenuity pathway analysis. this paper is the first to show the protein expression in the late response to a mastitic pathogen, thus, revealing mechanisms associated with host tissue damage. the up-regulated expression of col1a and itih in the mastitic mammary gland may be associated with tissue damage and repair during late stages of infection. the bioinformatics analyses highlighted the effects of mastitis on proteins such as collagen, fibrinogen, fibronectin, casein alpha and heparan sulfate proteoglycan our findings provide additional clues for further studies of candidate genes for mastitis susceptibility. this systems approach also could help identify biomarkers for monitoring clinical and subclinical mastitis. compared with healthy mammary gland tissue, proteins were significantly up-regulated while were significantly down-regulated in response to mastitis due to natural infections with staphylococci aureus. proteomics and bioinformatics may help us better understand the biological adaptations occurring during bovine mastitis. the aim of the present study was to use isobaric tags for relative and absolute quantification to screen potential proteins associated with mastitis at late infectious stage.
 proteomics and bioinformatics may help us better understand the biological adaptations occurring during bovine mastitis. this systems approach also could help identify biomarkers for monitoring clinical and subclinical mastitis. the aim of the present study was to use isobaric tags for relative and absolute quantification to screen potential proteins associated with mastitis at late infectious stage.  healthy and mastitic cows’ mammary gland tissues were analyzed using itraq combined with two-dimensional liquid chromatography-tandem mass spectrometry. bioinformatics analyses of differentially expressed proteins were performed by means of gene ontology, metabolic pathways, transcriptional regulation networks using blast2go software, the dynamic impact approach and ingenuity pathway analysis. at a false discovery rate of 5%, a total of proteins were identified from peptides, which were matched with spectra. compared with healthy mammary gland tissue, proteins were significantly up-regulated while were significantly down-regulated in response to mastitis due to natural infections with staphylococci aureus. up-regulation of collagen, type i, alpha and inter-alpha inhibitor h in the mastitis-infected tissue was confirmed by western blotting and immunohistochemistry.  this paper is the first to show the protein expression in the late response to a mastitic pathogen, thus, revealing mechanisms associated with host tissue damage. the bioinformatics analyses highlighted the effects of mastitis on proteins such as collagen, fibrinogen, fibronectin, casein alpha and heparan sulfate proteoglycan our findings provide additional clues for further studies of candidate genes for mastitis susceptibility. the up-regulated expression of col1a and itih in the mastitic mammary gland may be associated with tissue damage and repair during late stages of infection. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords itraqproteomicscol1a1itih4dairy cowmastitisissue-copyright-statement© the author 2014 