Analysis of the bovine plasma proteome by matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry

In this study, the bovine plasma proteome was analysed using a three step protocol: (1) plasma was treated with a combinatorial peptide ligand library (CPLL) to assimilate the differences in concentrations of different proteins in raw plasma; (2) CPLL-treated material was fractionated by three standard electrophoretic separation techniques, and (3) samples were analysed by nano-liquid chromatography (nLC) matrix-assisted laser desorption/ionisation (MALDI) time-of-flight tandem (TOF/TOF) mass spectrometry. The efficiencies of three fractionation protocols for plasma proteome analysis were compared.After size fractionation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), resolution of proteins was better and yields of identified proteins were higher than after charge-based fractionation by preparative gel-free isoelectric focussing. For proteins with isoelectric points >6 and molecular weights ?63 kDa, the best results were obtained with a ‘shotgun’ approach, in which the CPLL-treated plasma was digested and the peptides, rather than the proteins, were fractionated by gel-free isoelectric focussing. However, the three fractionation techniques were largely complementary, since only about one-third of the proteome was identified by each approach.     

VEGF and MMP‐9: biomarkers for canine lymphoma

Vascular endothelial growth factor (VEGF) and metalloproteinase (MMP) 2 and 9 are useful biomarkers in human lymphoma. During cancerogenesis, transforming growth factor beta (TGF‐β) stimulates VEGF and MMPs production. VEGF and TGF‐β plasma levels were tested by ELISA, MMP‐2 and MMP‐9 by gelatine zymography in 37 dogs with lymphoma, 13 of which were also monitored during chemotherapy. Ten healthy dogs served as control. Lymphoma dogs showed higher act‐MMP‐9 (P < 0.01) and VEGF (P < 0.05), and lower TGF‐β than controls, and a positive correlation between act‐MMP‐9 and VEGF (P < 0.001). Act‐MMP‐9 and VEGF were significantly higher in T‐cell lymphomas, and in stage V compared with stages III–IV disease, regardless of immunophenotype. VEGF was higher in high‐grade compared with low‐grade T‐cell lymphomas. No correlation was found between cytokines levels at presentation and outcome. During chemotherapy, act‐MMP‐9 and VEGF decreased in B‐cell lymphomas (P < 0.01), suggesting a possible predictive role in this group of dogs.     

超高压液相色谱串联四级杆飞行时间质谱法同时筛查牛肉中6种镇静剂

应用超高压液相色谱串联四级杆飞行时间质谱建立了同时筛查牛肉中6种镇静剂的新方法.样品经乙腈提取后,氨基柱净化,氮吹复溶后上机测定.空白样品在2、5和10 μg/kg三个不同添加浓度下测得6种镇静剂的回收率在60％～90％,6种镇静剂的检出限为0.5～1 μg/kg,且在2 ～50 ng/mL范围内线性关系良好,相关系数r2≥0.98.在2、5和10 μg/kg三个添加浓度下获得的6种镇静剂的精确分子量,其质量偏差绝对值均低于7×10-6,适用于牛肉中6种镇静剂的检测.     

超高压液相色谱-四级杆飞行时间质谱法筛查牛奶中55种药物

应用超高压液相色谱-四级杆飞行时间质谱法（UPLC-QTOF）建立了筛查牛奶中55种药物的方法。以乙腈提取样品中的药物,提取液经过HLB固相萃取柱净化浓缩后检测。在不同添加浓度下获得了精确分子离子质量,质量偏差的绝对值低于9.9×10-6。55种药物的检出限为2～10μg/kg,在5～200 ng/mL范围内线性关系良好,相关系数r≥0.98。在牛奶中添加20～100μg/kg的药物,回收率在22.9%～114.3%之间。本方法操作简单、高效、杂质干扰少,适用于牛奶中55种药物的检测需要。     

Proteomic profiling using SELDI‐TOF mass spectrometry: a diagnostic tool for canine B‐cell lymphoma

Introduction: Surface enhanced laser desorption ionization time of flight (SELDI‐TOF) mass spectrometry is a powerful new tool for biomarker discovery and diagnostic test development. In this process, proteins in biological samples are selectively bound to chip surfaces that have various chemistries and then subjected to mass spectrometry. Selectively bound proteins are separated by mass and the relative quantities of each protein compared among samples. Unlike conventional diagnostic test formats that commonly use single biomarkers, SELDI‐TOF technology allows multiple biomarkers to be used in a single assay to improve sensitivity and specificity. This technology has been used to improve diagnostic tests for human prostate and ovarian cancers. The objective of this study is to investigate the utility of this technology for the proteomic profiling of canine B‐cell lymphoma. Developing a diagnostic serum screening test for B‐cell lymphoma in dogs would be valuable as early diagnosis and treatment may confer a more favorable outcome. Methods: Serum samples were collected from 26 dogs diagnosed with B‐cell lymphoma prior to treatment and from 26 apparently healthy dogs that were similar in age, breed, and sex to the test group. Serum proteins were selectively bound to weak cationic, strong anionic, and nickel chelating chip surface chemistries under optimized buffer conditions, and subjected to mass spectrometry. The protein profiles were then compared using Biomarker Wizard and classification trees developed using Biomarker Patterns software from Ciphergen Biosystems, Inc (Fremont, CA). Results: To date several putative biomarkers have been identified. These putative biomarkers have been used to build classification tree models that predict disease in test samples. Preliminary results using cross‐validation of the test and control sample sets indicate that trees built with two or more biomarkers have greater than 90% sensitivity and specificity. Conclusions: Proteomic profiling using SELDI‐TOF technology in canine serum is feasible. Further testing is planned to confirm these initial results.     

Preliminary assessment of serum clusterin as a potential biomarker for canine lymphoma

Clusterin (CLU), also known as apolipoprotein J, is a widely expressed, heterodimeric, glycoprotein, important in tumourigenesis, apoptosis and immunoregulation. In humans, CLU expression has been associated with anaplastic large cell and Hodgkin's lymphoma. In this study, serum CLU levels in dogs with multicentric lymphoma (MLSA) were compared with healthy control dogs, using both western blot and enzyme‐linked immunosorbent assay (ELISA). Western blot confirmed the presence of CLU in dog sera at the predicted molecular weight and the relative levels detected correlated with the levels detected by ELISA. CLU level analysis by ELISA found treatment naïve dogs with MLSA had a significantly (P < .001) lower serum CLU level compared with healthy controls. However, there was no significant difference between MLSA dogs prior to treatment and in complete remission. The wide variation in serum CLU levels may limit its potential as a single candidate biomarker for MLSA, although any prognostic predictive value of serum CLU concentrations has yet to be assessed.     

Lipid profiles of canine spermatozoa as revealed via matrix‐assisted laser desorption/ionization mass spectrometry

In this study, we investigated the ability of matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) to characterize the lipid contents of canine spermatozoa. For that, samples of pure semen were analysed. Indeed, quite comprehensive lipid coverage was observed, and the most abundant phospholipid ions detected were from four phosphatidylcholines, that is those of m/z 760.6; 782.6; 808.6; and 830.6 and one of m/z 725.6 from a sphingomyelin. In conclusion, MALDI‐MS was found to offer an easy, fast, accurate, and sensitive analytical method for lipid profiling in canine spermatozoa and could be used as a tool to select sires by assessing the relationship between sperm lipid profiles and variables such as age and breeding history as well as to study the effects of cryopreservation on lipid contents.     

Changes in serum biomarkers of oxidative stress after treatment for canine leishmaniosis in sick dogs

Canine leishmaniosis (CanL) is a zoonotic disease being endemic in several parts of the world. In this study we investigated the behavior of a panel of biomarkers of oxidative stress in 12 sick dogs naturally infected by CanL before and at days 30 and 180 of a successful therapy with a standard treatment. The assays total oxidant status (TOS), trolox equivalent antioxidant capacity (TEAC), ferric reducing ability of plasma (FRAP), cupric reducing antioxidant capacity (CUPRAC), serum thiol and paraoxonase 1 (PON1) were included in the panel. In addition, correlations between biomarkers of oxidative stress and inflammation (C-reactive protein (CRP) and ferritin) and urinary protein:creatinine ratio (UPC) were calculated. Serum CUPRAC, thiol and PON1 significantly increased after treatment and were negatively correlated with CRP, ferritin and UPC. This study demonstrates that biomarkers of oxidative stress, not previously studied in leishmaniosis such as CUPRAC and thiol, can change after a successful treatment for CanL showing a potential for use in monitoring the treatment of this disease.     

Evaluation of serum chondroitin sulfate and hyaluronan: biomarkers for osteoarthritis in canine hip dysplasia

Hip dysplasia (HD) is one of the most important bone and joint diseases in dogs. Making the radiographic diagnosis is sometime possible when the disease has markedly progressed. Chondroitin sulfate (CS) and hyaluronan (HA) are the most important cartilage biomolecules that are elevated in the serum taken from dogs with osteoarthritis. The serum CS and HA can be detected by an ELISA technique, with using monoclonal antibodies against CS epitope 3B3 and WF6 and the HA chain as the primary antibodies. The aim of this study was to compare the levels of serum CS (both epitopes) and HA in non-HD and HD dogs. All 123 dogs were categorized into 2 groups. The non-HD group was composed of 98 healthy dogs, while the HD group was comprised of 25 HD dogs. Blood samples were collected for analyzing the serum CS and HA levels with using the ELISA technique. The results showed that the average serum level of the CS epitope WF6 in the HD group (2,594 ± 3,036.10 ng/ml) was significantly higher than that in the non-HD group (465 ± 208.97 ng/ml) (p < 0.01) while the epitope 3B3 in the HD group (105 ± 100.05 ng/ml) was significantly lower than that in the non-HD group (136 ± 142.03 ng/ml) (p < 0.05). The amount of serum HA in the HD group (134.74 ± 59.71 ng/ml) was lower than that in the non HD group (245.45 ± 97.84 ng/ml) (p < 0.05). The results indicate that the serum CS and HA levels might be used as biomarkers for osteoarthritis in HD dogs.     

Chemotherapy followed by half-body radiation therapy for canine lymphoma

A protocol of induction chemotherapy followed by half-body radiation therapy for treatment of lymphoma was used in 94 dogs. Seventy-three (78%) dogs achieved complete remission. Substage (P = .011) and phenotype (P = .015) were identified as predictors of complete remission rate. Of these, 52 dogs received half-body irradiation. Cranial and caudal halves received a total dose of 8.0 Gy, given in 2 fractions of 4.0 Gy on consecutive days with cobalt-60 photons and a 3-week interval between halves. Median 1st remission for these dogs was 311 days. Anemia was identified as the only predictor for length of 1st remission (P = .024). Toxicoses after half-body irradiation generally were mild and infrequent and included myelosuppression and gastrointestinal signs. Thirty-one dogs relapsed and 20 resumed treatment with induction followed by maintenance chemotherapy. Seventeen (85%) dogs achieved a 2nd complete remission. Median overall remission for all 52 dogs was 486 days. Results of this study suggest that half-body radiation therapy after induction chemotherapy is well tolerated and might increase remission duration compared with conventional protocols that use chemotherapy alone, but this increase might not be long enough to be clinically relevant or to justify application of the method described herein.     

Proteomics discovery of protein biomarkers linked to yak meat tenderness as determined by label-free mass spectrometry

Tenderness is one of the most important qualities in meat. A proteomic approach is a suitable way to ensure meat tenderness. Thirty-six tenderloin samples from yak were classified as exhibiting high (n = 12) or low (n = 12) tenderness and were evaluated using label-free proteomics for the identification of the proteins and pathways most influential in tenderness variability. Between the two groups, proteomic changes were mainly caused by 33 differentially expressed proteins as displayed in reference patterns in heat maps. The expression of ENO2, SUCLG2, ETFDH, PGM1, TNNT3, TNNT1, HSDL2, GPI, ALAD, and COL1A1 proteins was very different between yak meats with high and low tenderness, and therefore, they are candidate biomarkers of yak meat tenderness. Furthermore, bioinformatics analyses revealed that the identified proteins are related to pentose phosphate, glycolysis, the citrate cycle, fatty acid metabolism, and the calcium signaling pathway.     

Principles of treatment for canine lymphoma

Canine lymphoma is one of the most commonly diagnosed canine neoplasms. It is helpful to classify lymphoma anatomically, because these forms each have common histories and clinical signs. Anatomic forms include multicentric, alimentary, mediastinal, and cutaneous forms. Because lymphoma is a systemic disease, systemic chemotherapy is the most appropriate modality for its treatment. Lymphoma cells are sensitive to chemotherapy, and complete remission rates are high when these patients are treated with conventional chemotherapy. Treated dogs maintain a good quality of life, and treatment can provide resolution of many presenting signs and abnormalities. The fundamental goals of chemotherapy are to induce a durable remission and to re-induce a remission after one or more relapses. Other therapies, such as surgery and radiation therapy, are appropriate in certain situations. Prognostic factors will also be summarized.     

Evaluation of the prognostic significance of BCL6 gene expression in canine high-grade B-cell lymphoma

The clinical usefulness of BCL6 gene expression was evaluated as a prognostic indicator in dogs with high-grade B-cell lymphoma. Forty-four dogs were diagnosed with centroblastic or B-cell immunoblastic type lymphoma according to the updated Kiel classification. BCL6 mRNA expression was measured by real-time PCR and its relationship with prognosis was analyzed. Progression-free and overall survival was not significantly different between the high BCL6 expression group (higher than the median) and the low BCL6 expression group (lower than the median) (P=0.99 and P=0.61, respectively). No correlation between BCL6 and prognosis was observed in this study, which is inconsistent with findings reported for human diffuse large B-cell lymphoma. BCL6 protein expression was not detected in the 11 dogs evaluated by immunohistochemistry. Furthermore, BCL6 protein expression was assessed in 13 archived paraffin-embedded high-grade canine lymphoma tissues and all were also negative. The results suggest that most canine high-grade B-cell lymphomas correspond to human diffuse large B-cell lymphoma with no immunohistochemical expression of BCL6.     

免疫共沉淀联合质谱技术筛选宿主因子CypB的互作蛋白

为探讨亲环蛋白B(cyclophilin B,CypB)促进羊传染性脓疱病毒(orf virus,ORFV)复制增殖的机制,以ORFV早期感染MDBK细胞为试验材料,利用免疫共沉淀技术筛选与CypB互作的蛋白。免疫共沉淀产物的Western blot和SDS-PAGE的分析结果显示,在大小约为25 000处可见有明显的差异条带。切下该差异蛋白条带进行质谱鉴定分析,将获得的原始质谱数据使用Mascot软件中的串级质谱数据搜索功能(MS/MS Ions Search)进行搜索鉴定,初步筛选获得6个可能与CypB相互作用的病毒相关蛋白。为进一步深入研究CypB在ORFV早期侵染过程中的作用奠定基础。     

A case of atypical canine lymphoma with oral mass and multiple osteolysis

An 8-year-old female Golden Retriever had an oral mass and lameness. Multiple osteolysis of the systemic skeleton without monoclonal gammopathy was shown on electrophoresis of serum and urine samples. Cytological and histopathological examinations of the oral mass revealed atypical polymorphic cells similar to myeloid cells, and bone marrow aspiration indicated that these abnormal cells also might have invaded the bone marrow. These cells were negative to peroxidase and non-specific esterase staining, and clonal expansion of B lymphocytes could be detected by polymerase chain reaction (PCR) assay for antigen receptor gene rearrangement. The case was diagnosed as atypical lymphoma and treated by multi-drug chemotherapy. On the 142nd day after the first admission, the case had remission and the oral mass and multiple osteolysis were improved.     

Confirmation of indandione rodenticide toxicoses by mass spectrometry/mass spectrometry.

Mass spectrometry/mass spectrometry (MS/MS) with collision-activated dissociation (CAD) was utilized to unequivocally distinguish 1,3-indandione rodenticides in 2 cases of anticoagulant toxicosis. Anecdotal evidence provided by the veterinarian in a case involving feedlot cows and physical evidence at the site of occurrence in a similar case involving lambs strongly implicated diphenadione (diphacinone; DP) in both instances. However, high performance liquid chromatography indicated chlorophacinone (CP), not DP, was present in the blood samples obtained from both cows and lambs. Intact 1,3-indandiones exhibit poor gas chromatographic properties, so procedures were developed for analysis by MS/MS using a direct exposure probe for sample introduction. The EI mass spectra of DP and CP contained a base peak at m/z 173, with molecular ions (M+) at m/z 340 and m/z 374 (Cl isotope cluster), respectively. Corresponding MS/MS CAD parent ion spectra of m/z 173 showed an ion of m/z 340 for DP and 374 (Cl cluster) for CP. CAD analysis of the blood extracts showed a parent ion scan of m/z 173 identical to that of CP, with the m/z 374 (Cl cluster). (Additional evidence was obtained by MS/MS examination of the CAD daughter ion spectrum of m/z 374.) Blood extracts from the affected animals revealed CAD daughter ion spectra for m/z 374 identical to that of reference CP. Positive confirmation of CP in both cases led to identification of the source of the toxicant and prevention of further animal exposures.     

Glycoproteomic profiling of serum peptides in canine lymphoma and transitional cell carcinoma

Differential expression of fucosylated glycoproteins has been correlated with malignancy and metastatic potential in various types of neoplasia. Utilizing glycoproteomics techniques, changes in fucosylated serum peptides associated with naturally occurring canine lymphoma and transitional cell carcinoma (TCC) have been evaluated. In both types of neoplasia, the majority of the fucosylated peptides that changed increased with the cancer. In one lymphoma case that was examined over the course of the disease, the same fucosylated peptides that increased during pre-chemotherapy decreased during post-chemotherapy, and then subsequently increased upon recurrence of the lymphoma. When comparing all the fucosylated peptides that increased in both types of cancer, there were only two peptides in common allowing discrimination between lymphoma and TCC based on their peptide profiles. These results emphasize the prospect of glycopeptide profiling in proteomics for use in discovering a panel of non-invasive, diagnostic or prognostic biomarkers of cancer.     

Identification of Arcanobacterium pluranimalium by matrix-assisted laser desorption ionization-time of flight mass spectrometry and,as novel target,by sequencing pluranimaliumlysin encoding gene pla

In the present study 13 Arcanobacterium pluranimalium strains isolated from various animal origin could successfully be identified phenotypically by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and genotypically by sequencing 16S rDNA and the pluranimaliumlysin encoding gene pla. The detection of mass spectra by MALDI-TOF MS and the novel genotypic approach using gene pla might help to identify A. pluranimalium in future and might elucidate the role this species plays in infections of animals.     

Diagnostic use of serum gamma-glutamyltransferase in canine liver disease.

gamma-Glutamyltransferase (GGT) activity in canine kidney, pancreas, and liver is similar to previously reported values for other species. The low serum GGT activity in dogs (0 to 10 IU/L) may be related to relatively less liver GGT than in some other domestic animals. While determination of serum GGT in dogs may aid in the differentiation of sources of alkaline phosphatase, GGT alone appears to offer little in the diagnosis of canine liver disease. Clinical studies, as well as experimentally induced bile duct obstruction, have shown canine GGT increases to coincide with increases in alkaline phosphatase. The occasional benefit from knowledge of serum GGT in dogs would not seem to merit determination of GGT activity in routine serum chemistry panels for this species.     

Characterization of house dust mite and scabies mite allergens by use of canine serum antibodies

OBJECTIVE: To identify the major allergenic proteins from the 3 main species of dust mites to which dogs react (Dermatophagoides farinae, D. pteronyssinus, and Euroglyphus maynei) and evaluate the potential cross-reactivity of dust mite allergens with antigens from the ectoparasitic mite Sarcoptes scabiei var canis. SAMPLE POPULATION: Sera from 83 dogs with atopic dermatitis. PROCEDURE: Sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting using serum from atopic dogs was used to identify IgE-binding proteins in extracts of the 4 mite species. RESULTS: Sera of atopic dogs contained IgE against 23, 17, 25, and 17 allergens from D. farinae, D. pteronyssinus, E. maynei, and S. scabiei, respectively. Unlike the situation for humans, the major allergens for dogs are mostly proteins that are larger than 90 kd molecular weight. Dermatophagoides farinae and E. maynei appear to be more allergenic for dogs than is D. pteronyssinus. Some dogs with serum IgE against dust mites also had IgE against antigens of S. scabiei var canis. CONCLUSIONS AND CLINICAL RELEVANCE: Multiple dust mite allergens induce an IgE response in dogs. These allergens are mostly greater than 90 kd molecular weight.