Proteomic analysis of human gastric juice: a shotgun approach

Gastric juice is the most proximal fluid surrounding the stomach tissue. The analysis of gastric juice protein contents will thus be able to accurately reflect the pathophysiology of the stomach. This biological fluid is also a potential reservoir of secreted biomarkers in higher concentration as compared to the serum. Unlike the rest of the gastrointestinal fluids, there were very few studies reported on gastric juice proteome. To date, the proteins that routinely populate this biofluid are largely unknown. This is partly due to the technical difficulties in processing a sample that contains a collection of other gastrointestinal fluids, especially saliva. In this study, we attempt to profile the protein components of the gastric fluids from chronic gastritis patients using a direct shotgun proteomics approach. These data represent the first report of the proteome of human gastric juice with gastritis background.     

Proteomic identification of interactions between histones and plasma proteins: Implications for cytoprotection

Extracellular histones released from cells during acute inflammation contribute to organ failure and death in a mouse model of sepsis, and histones are known to exert in vitro cytotoxicity in the absence of serum. Since addition of histones to serum and plasma is known to induce protein aggregation, we reasoned that plasma proteins may afford protection from cytotoxicity. We found that MODE‐K mouse small intestinal epithelial cells were protected from histone‐induced toxicity in the presence of 10% FCS. Therefore, the main aim of this study was to identify histone‐interacting plasma proteins that might be involved in cytoprotection. The precipitate formed following addition of calf thymus histones to human EDTA plasma was characterised by shotgun proteomics, identifying a total of 36 protein subunits, including complement components, coagulation factors, protease inhibitors and apolipoproteins. The highly sulphated glycosaminoglycan heparin inhibited histone‐induced plasma protein aggregation. Moreover, histones bound to heparin agarose were capable of pulling down plasma proteins from solution, indicating their effective cross‐linking properties. It was particularly notable that inter‐α‐trypsin inhibitor was prominent among the histone‐precipitated proteins, since it contains a chondroitin sulphate glycan chain, and suggests a potential role for this protein in histone sequestration during acute inflammation in vivo.     

The loss of phenol sulfotransferase 1 in hepatocellular carcinogenesis

Biomarkers for the detection of early hepatocellular carcinoma (HCC) are urgently needed. To identify biomarkers of HCC, we performed a comparative proteomics analysis, based on 2‐DE of HCC tissues and surrounding non‐tumor tissues. Six xenobiotic enzymes were significantly down‐regulated in the HCC tissue. Among these, phenol sulfotransferase (SULT1A1) was confirmed by Western blot analysis in 105 HCC patients. SULT1A1 showed a significant decrease in 98.1% of the HCC tissues, with 88.6% sensitivity and 66.7% specificity for the detection of HCC. Immunohistochemistry for SULT1A1 was performed and compared with glypican‐3, which is a well‐known marker of HCC. The results showed down‐regulation of SULT1A1 and up‐regulation of glypican‐3 in 52.6 and 71.9% of the HCCs, and the use of both markers improved the sensitivity up to 78.9%. Moreover, SULT1A1 was useful in differentiating early HCC from benign dysplastic nodules. Clinically, the down‐regulation of SULT1A1 was closely associated with an advanced International Union Against Cancer stage and high levels of serum α‐fetoprotein. In conclusion, the results of this study demonstrate that the loss of SULT1A1 appears to be a characteristic molecular signature of HCC. SULT1A1 might be a useful biomarker for the detection of early HCC and help predict the clinical outcome of patients with HCC.     

Peptidomic profiling of human cerebrospinal fluid identifies YPRPIHPA as a novel substrate for prolylcarboxypeptidase

Prolylcarboxypeptidase (PRCP) is a serine protease that catalyzes the cleavage of C‐terminal amino acids linked to proline in peptides. It is ubiquitously expressed and is involved in regulating blood pressure, proliferation, inflammation, angiogenesis, and weight maintenance. To identify the candidate proximal target engagement markers for PRCP inhibition in the central nervous system, we profiled the peptidome of human cerebrospinal fluid to look for PRCP substrates using a MS‐based in vitro substrate profiling assay. These experiments identified a single peptide, with the sequence YPRPIHPA, as a novel substrate for PRCP in human cerebrospinal fluid. The peptide YPRPIHPA is from the extracellular portion of human endothelin B receptor‐like protein 2.     

Academic and molecular matrices: a study of the transformations of connective tissue research at the University of Manchester (1947-1996)

This paper explores the different identities adopted by connective tissue research at the University of Manchester during the second half of the 20th century. By looking at the long-term redefinition of a research programme, it sheds new light on the interactions between different and conflicting levels in the study of biomedicine, such as the local and the global, or the medical and the biological. It also addresses the gap in the literature between the first biomedical complexes after World War II and the emergence of biotechnology. Connective tissue research in Manchester emerged as a field focused on new treatments for rheumatic diseases. During the 1950s and 60s, it absorbed a number of laboratory techniques from biology, namely cell culture and electron microscopy. The transformations in scientific policy during the late 70s and the migration of Manchester researchers to the US led them to adopt recombinant DNA methods, which were borrowed from human genetics. This resulted in the emergence of cell matrix biology, a new field which had one of its reference centres in Manchester. The Manchester story shows the potential of detailed and chronologically wide local studies of patterns of work to understand the mechanisms by which new biomedical tools and institutions interact with long-standing problems and existing affiliations.     

A novel procedure for protein extraction from formalin-fixed paraffin-embedded tissues

Most of the archived pathological specimens in hospitals are kept as formalin-fixed paraffin-embedded tissues (FFPE) for long-term preservation. Up to now, these samples are only used for immunohistochemistry in a clinical routine as it is difficult to recover intact protein from these FFPE tissues. Here, we report a novel, short time-consuming and cost-effective method to extract full-length, non-degraded proteins from FFPE tissues. This procedure is combined with an effective and non-toxic deparaffinisation process and an extraction method based on antigen-retrieval, high concentration of SDS and high temperature. We have obtained enough intact protein to be detected by Western blotting analysis. This technique will allow utilising these stored FFPE tissues in several applications for protein analysis helping to advance the translational studies in cancer and other diseases.     

Proteomic changes in articular cartilage of human endemic osteoarthritis in China

Kashin-Beck disease (KBD) is a chronic endemic osteochondropathy with unclear pathogenesis. It is a degenerative disease similar to osteoarthritis, but with different manifestations of cartilage damage. The aim of this investigation was to show the protein changes in KBD cartilage and to identify the candidate proteins in order to understand the pathogenesis of the disease. Proteins were extracted from the media of primary cell cultures of KBD and normal chondrocytes, and separated by two-dimensional fluorescence difference gel electrophoresis (2-D DIGE). MALDI-TOF/TOF analysis revealed statistically significant differences in 27 proteins from KBD chondrocyte cultures, which consisted of 17 up-regulated and ten down-regulated proteins. The results were further validated by Western blot analysis. The proteins identified are mainly involved in cellular redox homeostasis and stress response (MnSOD, Hsp27, Peroxiredoxin-1, and Cofilin-1), glycolysis (PGK-1, PGM-1, α-enolase), and cell motility and cytoskeletal organization (Actin, Calponin-2, and Keratin). These KBD-associated proteins indicate that cytoskeletal remodeling, glycometabolism, and oxidative stress are abnormal in KBD articular cartilage.     

Proteomic analysis of the coagulation reaction in plasma and whole blood using PROTOMAP

Proteases are critical in many physiological processes and the human genome encodes for 566 predicted proteolytic enzymes. Therefore, there is great interest in identifying and characterizing physiologic protease-substrate relationships. The coagulation cascade is a well-described network of serine proteases. However, new interactions of the coagulation cascade with other biological pathways have been discovered only recently. Therefore, we hypothesized that a non-biased protease degradomics analysis of the physiologic coagulation reaction would identify new interactions between the coagulation cascade and other pathways. We used the recently described PROTOMAP technique to profile the complete coagulation degradome. This analysis detected virtually all of the proteins of the coagulation cascade and identified a majority of the expected proteolytic events, suggesting significant coverage of the coagulation degradome. Multiple potential new proteolytic cleavages were detected, including two of transmembrane proteins that may be shed from the surface of blood cells. In addition, this analysis was able to identify several new potentially secreted proteins. A significant majority of the newly identified events were of proteins involved in innate immunity (complement and inflammation). This highlights potential new areas of crosstalk between these linked systems. Future studies will elucidate the details and functional consequences of these proteolytic events during coagulation.     

A comparative MudPIT analysis identifies different expression profiles in heart compartments

Cardiomyopathies indistinctly affect atrial and ventricular cardiac compartments with alterations of their mechanical and/or electrical activity. To understand the main mechanisms involved in these pathological alterations, a detailed knowledge of the physiology of the healthy heart is critical. In the present work, we utilize multidimensional protein identification technology to characterize the murine left ventricle (LV), right ventricle (RV), and atria (A) proteomes, identifying thousands of distinct proteins. Moreover, using multidimensional algorithm protein map tool, relative abundances of proteins among the heart chambers were investigated. In sum, we found 16 and 55 proteins were more abundant in LV compared to RV and A, respectively; 47 and 60 proteins were more abundant in RV than LV and A, respectively; and, 81 and 74 proteins were more abundant in A than LV and RV, respectively. This detailed characterization of myocardial compartment proteome represents an important advancement in the knowledge of heart physiology, and may contribute to the identification of key features underlying the onset of cardiomyopathy.