Proteomic analysis of plasma from cows affected with milk fever using two-dimensional differential in-gel electrophoresis and mass spectrometry

Research in Veterinary Science 93 (2012) 857–861

Contents lists available at SciVerse ScienceDirect

Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc

Proteomic analysis of plasma from cows affected with milk fever using two-dimensional differential in-gel electrophoresis and mass spectrometry C. Xia a, H.Y. Zhang a,⇑, L. Wu a, C. Xu a, J.S. Zheng a, Y.J. Yan b, L.J. Yang a, S. Shu a a b

College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163319, PR China Department of Genomics and Proteomics, Beijing Institute of Radiation Medicine, Beijing 102206, PR China

a r t i c l e

i n f o

Article history: Received 23 April 2011 Accepted 31 October 2011

Keywords: Two-dimensional differential in-gel electrophoresis Plasma proteome Milk fever Dairy cows

a b s t r a c t Milk fever is an important metabolic disorder of dairy cows after calving, and is characterized by hypocalcemia, tetany, lateral recumbency, and eventual coma. To date, there have been many reports about the pathogenesis and pathophysiology of milk fever, but the plasma protein profile in milk fever has not been reported. The aim of our study was to investigate novel pathophysiological changes in the plasma proteome of cows affected with milk fever. Plasma samples were collected from eight Holstein cows with milk fever (T), and eight control Holstein cows without milk fever (C), at an intensive Holstein dairy farm in Heilongjiang province, China. Samples were analyzed by fluorescence two-dimensional (2D) differential in-gel electrophoresis (DIGE), followed by in-gel digestion, and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) for peptide mass fingerprinting of selected protein spots. Eight of the 23 differential protein spots in the plasma of T and C cows were isolated and identified by 2D-DIGE and MALDI-TOF-MS. The protein spots represented five unique proteins, and had significant alterations in spot volume as determined by DeCyder differential in-gel analysis (DIA) software. The upregulated proteins were identified as serpin peptidase inhibitor (angiotensin), which regulates blood pressure and maintains fluid and electrolyte homeostasis, and endopin 2B which is involved in neural regulation. The downregulated proteins were serum albumin, which acts as a transport protein, fibrinogen beta chain which is involved in blood coagulation, and IgG heavy-chain C-region (IgG-CH) which participates in the immune response. In conclusion, we were able to use proteomic technologies to identify several novel plasma proteins in cows affected with milk fever. These findings may reveal new pathophysiological changes that occur in cows with milk fever. Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.

Milk fever (MF) is a complex metabolic disorder, usually of dairy cows, that occurs around parturition. Its clinical syndromes include inappetence, tetany, recumbency, and even coma. The majority of affected cows die if untreated (Horst et al., 1997). The biochemical characteristic of this condition is severe hypocalcemia (usually <1.5 mmol/L), which most likely explains the clinical signs associated with milk fever (Goff, 2008). Investigation of this disease has chiefly focused on blood calcium, ionized calcium, or total calcium contents in the transition period because of the close connection between blood calcium and milk fever. Blood phosphorus status has also been researched because of the prominent interaction between calcium and phosphorus at the onset of lactation (Larsen et al., 2001). Several other parameters, such as magnesium, alkaline phosphatase, hydroxyproline, osteocalcin, parathormone, calcitionin, and 1,25-dihydroxy-vitamin D, which are associated with the regulation of calcium metabolism, have ⇑ Corresponding author. Fax: +86 6819090. E-mail address: [email protected] (H.Y. Zhang).

also been considered in an attempt to further understand calcium homeostasis (Liesegang et al., 1998; Radostits et al., 2000). Over the past few years, new proteomic approaches have been used to study the plasma proteome in many diseases including human and mouse diabetes (Meier et al., 2005), acute coronary syndrome (Petra et al., 2004), cancer (Edward et al., 2007), and cattle ketosis and fatty liver disease (Xu and Wang, 2008; Björn et al., 2009). In comparative proteomics, accurate quantitation and good reproducibility is the basis of reliable experimental data. A recent proteomic method, two-dimensional (2D) differential ingel electrophoresis (DIGE), has been increasingly used to find new biomarkers in human diseases, because it is able to overcome the shortcomings of conventional 2DE, analyze multiple samples on the same gel, and it contains an internal standard of mixed samples on all gels for inter-gel alignment and relative quantification of spots (Laura et al., 2009). Some efforts have been made to study the mechanism of milk fever in physiology, biochemistry, and pathology (Horst et al., 1997). However, to date, there have been no reports detailing the plasma

0034-5288/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.10.025

858

C. Xia et al. / Research in Veterinary Science 93 (2012) 857–861

proteome of cows with milk fever. Thus, the goal of this study was to determine if there are novel pathophysiological changes in the plasma proteome of cows affected with milk fever. All animals used in our experiments were treated according to the International Guiding Principles for Biomedical Research Involving Animals. Eight Holstein cows with milk fever (T, 5.63 ± 0.53 years; 3.38 ± 0.74 parities; plasma Ca 1.37 ± 0.12 mmol/L) that had apparent clinical signs and hypocalcemia (Radostits et al., 2000) and eight control Holstein cows without milk fever (C, 5.5 ± 0.52 years; 3.25 ± 0.71 parities; plasma Ca 2.51 ± 0.06 mmol/L), were selected within 24 h after calving from a commercial dairy farm located at Daqing in Heilongjiang province, China. Blood samples (10 mL) from each cow were collected from the caudal vein into tubes with heparin at the time of illness after calving. The samples were immediately centrifuged at 3000g for 10 min, then frozen in liquid nitrogen, and stored at 80 °C until subsequent analyses. Plasma Ca2+ were detected by colorimetry with a semi-autoanalyzer (Eppendorf ECOM-F 6124, Germany), using commercial kits (Chang Chun Hui Li Bioengineering Ltd. Company). To prepare the samples, proteins were directly extracted from the plasma in lysis buffer (8 mol/L urea, 40 g/L 3-[(3-cholamidopropyl)dimethyl ammonio]-1-propane sulfonate (CHAPS)). For 2D-DIGE, interfering components of the plasma samples were removed using the Aurum Serum Mini Kit and Pd-10 Kit (GE Healthcare, Chalfont St Giles, UK). Proteins were diluted in 8 mol/L urea, 40 g/L CHAPS, 130 mM DTT, and 2% pharmalytes, and protein concentration was determined using the Bradford method (Rajini et al., 2009). For conventional 2DE, sample proteins (150 lg) were diluted in rehydration solution [8 mol/L urea, 20 g/L CHAPS, 65 mmol/L DTT, and 0.5% IPG buffer]. For IPG strips (pH 3–10), proteins were applied by cup-loading strips that were previously hydrated in rehydration solution (Petra et al., 2004). Proteins were then separated in the second dimension using 12.5% Tris–glycine gels in a Protean II system (Bio-Rad Laboratories Inc., Hercules, California). 2DE gels were silver stained using a protocol compatible with MS (Shevchenko et al., 1996). For 2D-DIGE separation, proteins were labeled according to the manufacturer’s instructions (GE Healthcare). Briefly, 50 lg of the C and T protein extracts were minimally labeled with 400 pmol Cy3 or Cy5 fluorescent cyanine dye for 30 min on ice in the dark. An internal standard, containing equal amounts of each protein extract, was labeled with Cy2 fluorescent dye, and used in all experiments to ensure that every protein that was expressed in all eight C and T samples, was present in the internal standard protein mixture according to the cross-label rule (Chiara et al., 2005). Since the same internal standard was run in all gels, it was normalized and matched across the gels, effectively decreasing gel-to-gel variations. The labeling reaction was with 0.5 lL of 10 mmol/L lysine for 30 min on ice in the dark. The C, T, and internal standard protein samples were adequately mixed and run on a single gel (150 lg total protein). The proteins were separated in the first dimension with 24 cm immobilized pH gradient strips (pH 3–10, NL) using the IPGphor IEF II System (GE Healthcare) following a voltage increase in four steps: hydration at 50 V for 12 h, desalination at a rapid gradient to 1000 V in 4 h, linear gradient to 70,000 V in 6 h, and steady state at 500 V for 5 h. Prior to SDS–PAGE, the strips were equilibrated and separated on 12.5% Tris–glycine gels using an Ettan Dalt II device (Amersham Biosciences, Uppsala, Sweden). The gels were scanned and visualized with a Typhoon 9410 Varialbe Mode Imager (Amersham Biosciences) using appropriate wavelengths and filters for Cy2: 488 nm, Cy3: 530 nm, and Cy5: 633 nm. Gel image analysis was performed with DeCyder 6.5 software (Amersham Biosciences). To visualize protein spots, 2D-DIGE gels were silver stained. The silver-stained spots were excised manually and then digested auto-

matically with modified trypsin (sequencing grade; Promega, Madison, WI, USA) at a final concentration of 10 mg/L in 25 mmol/L ammonium bicarbonate according to the protocol of Shevchenko et al. (1996). Peptide extraction was carried out with 2.5 g/L trifluoroacetic acid (99.5% purity; Sigma Chemical Inc., St. Louis, MO, US). For MALDI-MS/MS and database searching, an aliquot of the digestion solution was mixed with an aliquot of a-cyano -4-hydroxycinnamic acid in 50% aqueous acetonitrile and 0.1% trifluoroacetic acid. This mixture was deposited onto a 600 lm AnchorChip MALDI probe and was allowed to dry at room temperature. MALDI-MS/MS data were obtained using an ABI 4800 MALDITOF–TOF mass spectrometer (Applied Biosystems Inc., Foster City, California, USA) (Xu and Wang, 2008). Detailed analysis of peptide mass mapping data was performed using flexAnalysis software (Bruker-Daltons Inc., Billerica, USA). MALDI-MS and MS/MS data were combined through the MS BioTools program, and the NCBI database was searched using Mascot software (Matrix Science, London, UK). For statistical analyses, differences based on clinical signs and plasma Ca2+ concentrations between C and T cows were evaluated by the unpaired t-test; results were expressed as mean ± SD. Then, relative protein quantification across C and T samples was performed using DeCyder 6.5 software, which co-detected and quantified the spots on a given gel in terms of the ratios of the Cy3 and Cy5 sample volumes to the standard Cy2 volume, and matched the spots and standardized the ratios across the gels, accounting for the observed differences in the Cy2 sample volumes on the gels. This software provides a mode of differential in gel-analysis (DIA) and biological variation analysis (BVA) for determining if a protein is differentially expressed between two groups based on the fold change calculated as the ratio of the average standardized abundance corresponding to the two groups of samples, which was set at a greater than 1.5-fold threshold and P < 0.05 (Student t-test) by a DeCyder 6.5 software. In this study, we show for the first time the different proteins that are expressed in the plasma of cows with milk fever (Fig. 1A) using 2D-DIGE. The 2D-DIGE images were analyzed using DeCyder 6.5 software. Based on a threshold of a greater than 1.5-fold change and P < 0.05, 8 of 23 spots were detected as having significantly differential abundance. Five protein spots were upregulated (e.g. spot number 1076) and three protein spots were downregulated (e.g. spot number 555) in the T group (Fig. 1B). By analysis of MS/MS and a search of the bovine NCBI dataset, we identified eight peptide MS/MS spectra which are shown in Table 1. One of the main findings in our study was the identification of two significantly upregulated proteins in the plasma from cows with milk fever; namely, endopin 2B and serpin peptidase inhibitor (Table 1). Endopin 2B (E-2B) is a component of secretory vesicles where neuropeptides are produced. Bovine isoforms of endopins, which comprise endopin 1, endopin 2A, and endopin 2B, possess homology to ACT (Hwang et al., 2005). Tissue distribution studies showed expression of endopin 2B-1 and endopin 2B-2 in the bovine brain, pituitary, adrenal medulla, and other neuroendocrine tissues (Hwang et al., 2009). Endopin 2 inhibits the cysteine protease, papain, and the endogenous secretory vesicle cathepsin L which participates in the production of the biologically active enkephalin peptide neurotransmitter (Hook and Hwang, 2002). Our research suggests that E-2B regulates neuroendocrine function. Serpin peptidase inhibitors (SPIs) are a family of protease inhibitors, which have homologous structures and sequences (Rawlings and Barrett, 1993). They participate in a series of physiological and pathological processes including digestion, degradation, fertilization, embryogenesis, fibrinolysis, hormone activation, complement activation, cellular and humoral immunity, and maintenance of homeostasis (O’Brien and McVey, 1993; Silverman et al., 2001; Krem and DiCera, 2002).

C. Xia et al. / Research in Veterinary Science 93 (2012) 857–861

859

Fig. 1. 2D-DIGE of plasma proteome from cows with milk fever. (A) Spot map corresponding to the mixed internal standard, which is common to all the gels analyzed. 24 cm immobilized pH gradient strips (pH 3–10, NL) were used for isoelectric focusing and 12.5% SDS–PAGE gels were used for the second dimension. Eight spots indicate proteins identified by MALDI-TOF/TOF that showed significant differential expression between the C and T samples and correlate with those shown in Table 1. (B) Graphical representation of two differential expression protein spots (spot number 1076 and 555 seen in A). Each cycled spot (up) of the two selected spots in the 2D gel created a corresponding 3D relative abundance image (down) by DeCyder 6.5 software and processed by quantitative analysis between C and T samples.

Since E-2B and SPI belong to a family of protease inhibitors and acute reactive proteins, they may enhance host defenses and regulate neuroendocrine function (Krem and DiCera, 2002; Hwang et al., 2005). Although there have been no reports on the effects of milk fever on E-2B and SPI in cattle, our studies suggest that the upregulation of E-2B and SPI proteins in the plasma may be related to adaptive alterations of neuroendocrine function when milk fever occurs. Another main finding was the identification of three significantly downregulated proteins in the plasma from cows with milk fever; namely, fibrinogen beta chain, IgG heavy-chain C-region (IgG-CH), and albumin (Table 1). As a precursor of fibrin, fibrinogen (FG) is a protein synthesized by the liver. FG is composed of pairs of three polypeptide chains, Aa, Bb and c. Fibrinogen beta chain (FG-b) synthesis is the limiting factor in fibrinogen production (Laffan, 2001). In addition, as a predictor of cardiovascular disease, elevated levels of plasma FG closely correlate with coronary artery diseases, such as myocardial infarction, ischemic heart disease,

and venous thrombosis (Koening, 2003). Furthermore, calcium (Ca2+) plays a pivotal role in the physiology and biochemistry of organisms and the cell, where it acts as a second messenger, in neurotransmitter release from neurons, in contraction of all muscle cell types, in fertilization, in the blood-clotting cascade, and in maintaining proper bone formation (Murray et al., 2010). Therefore, the downregulation of plasma FG-b indicated that milk fever has a negative affect on blood coagulation and signal transduction, which may be closely related to hypocalcemia. We found two interesting phenomena in our studies; namely the downregulation of plasma IgG heavy-chain C-region (IgG-CH) and albumin in cows affected with milk fever. Antibodies, which are also known as immunoglobulins (Igs), are gamma globulin proteins that are found in the blood or other bodily fluids of vertebrates (Litman et al., 1993). IgG is the most important antibody of the primary immune response. IgG contributes to immunity in three ways: preventing pathogens from entering or damaging cells by binding to them, stimulating removal of

860

C. Xia et al. / Research in Veterinary Science 93 (2012) 857–861

Table 1 List of identified proteinsa in plasma of cows with milk fever. Fold changec

Protein named

Accession no.e

Theo. Mrf

Theo. pIg

Sequence coverageh (%)

172

2.22

Serum albumin

gi|30794280

69,278

5.82

16

9

96

191

1.87

25

15

225

460 197

1.30 1.23

27 27

18 10

431 172

357

1.65

24

8

184

555

1.93

1076

1.88

1127

1.23

Spot no.b

a b c d e f g h i j k

46,175

5.57

Peptides counti

Mascot score MSj

Serpin peptidase inhibitor

gi|31340900

Fibrinogen b chain Endopin 2B

gi|1346006

53,306

8.45

24

15

89

gi|38683423

46,972

5.82

16

7

124

Ig G heavy chains of C region

gi|89611

36,020

8.04

25

9

229

Mascot score MS/ MSk

Mascot peptides identified by MS/MSg

Biological function

24 28 27 26 44 41 51 71 25 48 75 20

YLYEIAR LGEYGFQNALIVR YLYEIAR+M RHPEYAVSVLLR LGEYGFQNALIVR YLYEIAR+2C RIHELYLPK SNYELNDILSQLGIR LTPETLTR RIHELYLPK SNYELNDILSQLGIR SILENLR+C

Transportation

23 36 37 32 29

LTPETLTR LAVSHVIHK FSISSHYQLK GLSASIVR SYFLYSK

Homeostasis

Metabolism; blood blotting Neural regulation Defense response

Spots for which the volume ratio was >1.5 based on DeCyder 6.5 software analysis were identified by MALDI-TOF/TOF MS. Spots number in Fig. 1A. Spots in the same line were identified as same protein. Spot volume ratio (milk fever versus control). Spots in the same line were identified as same protein. Protein ID accessed from NCBI database. Theoretical molecular weight in kDa. Theoretical pI. Protein sequence coverage in percentage. Number of matched peptides. Protein candidate score as provided by Mascot. Ion score of peptide analyzed by MS/MS.

pathogens by macrophages, and triggering destruction of pathogens by stimulating other immune responses such as the complement pathway (Pier et al., 2004). Milk fever increases susceptibility to infection due to failure of the cervical os and teat end to completely close after calving and milking, thus allowing bacteria to have access to the endometrium and mammary gland (James, 1999). Goff (2006) demonstrated that the ability of the immune cells to respond to bacterial infection is directly reduced by low blood calcium. In our studies, the downregulation of IgG-CH demonstrated that milk fever contributes to an increase in infectious diseases which may be related to decreased synthesis of IgG caused by hypocalcemia at calving. Bovine serum albumin (BSA) is a single polypeptide chain consisting of approximately 583 amino acid residues and no carbohydrates (Frank, 1975). It is the most abundant protein in bovine blood, and is produced in the liver. BSA has many important biological functions, including maintaining oncotic pressure, transporting many materials such as hormones, fat-soluble vitamins, free fatty acids, unconjugated bilirubin, drugs, bind calcium ions (Ca2+), buffer pH (Peters, 1975). However, low levels of serum albumin are often associated with liver disease, kidney disease, enteropathy, hemodilution in pregnancy, increased vascular permeability, and acute disease states (Frank, 1975). So, it still needs to be confirmed afterward as to problem of the down-regulating expression of serum IgG-CH and BSA in cows affected with milk fever. FG-b, IgG-CH, and BSA belong to the family of negative reactive proteins. A reduction in their synthesis could be helpful in generating a greater amount of amino acids that could be used in the synthesis of acute phase proteins that enhance the host defense system (Peters, 1975). However, this may also contribute to an increase in infectious diseases due to low immunity caused by milk fever after calving. Our final finding was the identification of different modified forms of BSA and the SPI inhibitor. The 2D-DIGE method was able

to show that they ran at different positions on the gel (Fig. 1A) and had different expression levels (Table 1). Some reports have indicated that most variants of one protein show similar changes on a gel in normal states. However, the disease-state may cause some changes in specific modified forms of plasma proteins (Anderson and Anderson, 2002; Chiara et al., 2005). Therefore, the specific modification of BSA and SPI in the plasma may correlate with milk fever. In summary, our present proteomic approach is the first to show differences in the expression of plasma proteins in cows with or without milk fever. These proteins have different biological functions, including protein transport, maintenance of homeostasis, neural regulation, defense response, anticoagulation of blood, and immune function. Our data reveal that when milk fever occurs, there are some plasma proteome alterations which are helpful or unfavorable for normal biological functions, and may affect the development of milk fever. However, it is necessary to further confirm these studies, especially in relation to IgG and albumin, and to further study the proteins that are differentially expressed in the plasma of cows affected with milk fever, as these studies may contribute to a better understanding of the pathogenesis of milk fever. Acknowledgements We thank the Beijing Proteomics Research Center for providing facilities to carry out the work and also thank Chinese National Science Foundation (30840060 and 30972235) and Heilongjiang Province Science Foundation (C200916) for funding support. References Anderson, N.L., Anderson, N.G., 2002. The human plasma proteome: history, character, and diagnostic prospects. Molecular and Cellular Proteomics 1, 845–867.

C. Xia et al. / Research in Veterinary Science 93 (2012) 857–861 Björn, K., Albrecht, D., Kuhla, S., Metges, C.C., 2009. Proteome analysis of fatty liver in feed-deprived dairy cows reveals interaction of fuel sensing, calcium, fatty acid, and glycogen metabolism. Physiological Genomics 37, 88–98. Chiara, D.A., Arena, S., Talamo, F., Ledda, L., Renzone, G., Ferrara, L., Scaloni, A., 2005. Comparative proteomic analysis of mammalian animal tissues and body fluids: bovine proteome database. Journal of Chromatography B 815, 157–168. Edward, F., Patz Jr., Michael, J.C., Elizabeth, B.G., Irina, K., Xiang, R.G., James, E.H., 2007. Panel of serum biomarkers for the diagnosis of lung cancer. Journal of Clinical Oncology 25, 5578–5583. Frank, W.P., 1975. The Plasma Proteins: Structure, Function and Genetic Control, vol. 1, second ed. Academic Press, New York, pp. 141–147. Goff, J.P., 2006. Major advances in our understanding of nutritional influences on bovine health. Journal of Dairy Science 89, 1292–1301. Goff, J.P., 2008. The monitoring, prevention, and treatment of milk fever and subclinical hypocalcemia in dairy cows. Veterinary Journal 176, 50–57. Hook, V.Y., Hwang, S.R., 2002. Novel Secretory vesicle serpins, endopin 1 and endopin 2: endogenous protease inhibitors with distinct target protease specificities. Biological Chemistry 383, 1067–1074. Horst, R.L., Goff, J.P., Reinhardt, T.A., Buxton, D.R., 1997. Strategies for preventing milk fever in dairy cattle. Journal of Dairy Science 80, 1269–1280. Hwang, S.R., Christina, Z.G., Jill, L.W., Hook, V.Y., 2005. Demonstration of GTG as an alternative initiation codon for the serpin endopin 2B-2. Biochemical and Biophysical Research Communications 327, 837–844. Hwang, S.R., Richard, B., Thomas, T., Hook, V.Y., 2009. Endopin serpin protease inhibitors localize with neuropeptides in secretory vesicles and neuroendocrine tissues. Neuroendocrinology 89, 210–216. James, K.D., 1999. Biology of dairy cows during the transition period: the final frontier? Journal of Dairy Science 82, 2259–2273. Koening, W., 2003. Fibrin(ogen) in cardiovascular disease: an update. Thrombosis and Haemostasis 89, 601–609. Krem, M., Dicera, E., 2002. Evolution of enzyme cascades from embryonic development to blood coagulation. Trends in Biochemical Sciences 27, 67–74. Laffan, M.A., 2001. Fibrinogen polymorphisms and disease. European Heart Journal 22, 2224–2226. Larsen, T., Moller, G., Bellio, R., 2001. Evaluation of clinical and clinical chemical parameters in periparturient cows. Journal of Dairy Science 84, 1749–1758. Laura, E., McNamara, Matthew, J.D., Mathis, O.R., Richard, B., 2009. Fluorescence two-dimensional difference gel electrophoresis for biomaterial applications. Journal of the Royal Society, Interface 1, 1–11. Liesegang, A.M., Sassi, L., Risteli, J., Eicher, R., Wanner, M., Riond, J.L., 1998. Comparison of bone markers during hypocalcemia in dairy cows. Journal of Dairy Science 81, 2614–2622.

861

Litman, G.W., Rast, J.P., Shamblott, M.J., 1993. Phylogenetic diversification of immuno-globulin genes and the antibody repertoire. Molecular Biology and Evolution 10, 60–72. Meier, M., Kaiser, T., Herrmann, A., Knueppel, S., Hillmann, M., Koester, P., Danne, T., Haller, H., Fliser, D., Mischak, H., 2005. Identification of urinary protein pattern in type 1 diabetic adolescents with early diabetic nephropathy by a novel combined proteome analysis. Journal of Diabetes and Its Complications 19, 223–232. Murray, L., Wilkinson, I.B., Edward, H., Davidson, A.F., Ahmad, R.M., 2010. Calcium and phosphate physiology. Oxford Handbook of Clinical Medicine 8, med9780199 232178-div1-15013. O’Brien, D., McVey, J., 1993. Blood coagulation, inflammation, and defense. In: Sim, E. (Ed.), The Natural Immune System, Humoral Factors. IRL Press, New York, pp. 257–280. Peters, T., 1975. In: Putman, F.W. (Ed.), The Plasma Proteins. Academic Press, pp. 133–181. Petra, J., Cáceres, M., Antonio, G.M., Antonio, L.F., Carlos, M., Antonio, N., Juan, G., Sergio, A.O., Carolina, C., Marta, B., de Raimundo, A., Juan, J.G., Jerónimo, F., Luis, A.R., 2004. Proteomic analysis of plasma from patients during an acute coronary syndrome. Journal of the American College of Cardiology 44, 1578–1583. Pier, G.B., Lyczak, J.B., Wetzler, L.M., 2004. Immunology, Infection, and Immunity. ASM Press, ISBN: 978-1-55581-246-1. Radostits, O.M., Gay, C.C., Blood, D.C., Hinchcliff, K.W., 2000. Veterinary Medicine, ninth ed. Har-court Publishers Ltd., pp. 1418–1427. Rajini, S., Barkha, P.P., Mazen, J.H., 2009. Comparison of total protein concentration in skeletal muscle as measured by the Bradford and Lowry assays. Journal of Biochemistry 145, 791–797. Rawlings, R.D., Barrett, A.J., 1993. Evolutionary families of peptidases. Journal of Biochemistry 290, 205–218. Shevchenko, A., Wilm, M., Vorm, O., Mann, M., 1996. Mass spectrometric sequencing of proteins silver-staining polyacrylamide gels. Analytical Chemistry 68, 850–858. Silverman, G.A., Bird, P.I., Carrell, R.W., Church, F.C., Coughlin, P.B., Gettins, P.G., Irving, J.A., 2001. The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature. Journal of Biological Chemistry 276, 33293–33329. Xu, C., Wang, Z., 2008. Comparative proteomic analysis of livers from ketotic cows. Veterinary Research Communications 32, 263–273.