Possible Role of Extracellular Vesicles in Hepatotoxicity of Acetaminophen.

We examined proteomic profiles of rat liver extracellular vesicles (EVs) shed following treatment with a sub-toxic dose (500 mg/kg) of the pain reliever drug, acetaminophen (APAP). EVs representing the entire complement of hepatic cells were isolated after perfusion of the intact liver and analyzed with LC-MS/MS. The investigation was focused on revealing the function and cellular origin of identified EVs proteins shed by different parenchymal and non-parenchymal liver cells and their possible role in an early response of this organ to a toxic environment. Comparison of EV proteomic profiles from control and APAP-treated animals revealed significant differences. Alpha-1-macroglobulin and members of the cytochrome P450 superfamily were highly abundant proteins in EVs shed by the normal liver. In contrast, proteins like aminopeptidase N, metalloreductase STEAP4, different surface antigens like CD14 and CD45, and most members of the annexin family were detected only in EVs that were shed by livers of APAP-treated animals. In EVs from treated livers, there was almost a complete disappearance of members of the cytochrome P450 superfamily and a major decrease in other enzymes involved in the detoxification of xenobiotics. Additionally, there were proteins that predominated in non-parenchymal liver cells and in the extracellular matrix, like fibronectin, receptor-type tyrosine-protein phosphatase C, and endothelial type gp91. These differences indicate that even treatment with a sub-toxic concentration of APAP initiates dramatic perturbation in the function of this vital organ.

Changes in the proteome of extracellular vesicles shed by rat liver after subtoxic exposure to acetaminophen.

To identify changes in extracellular vesicles (EVs) secreted by the liver following drug-induced liver injury (DILI), rats were treated with a subtoxic dose (500 mg/kg) of the analgesic drug, acetaminophen (APAP). EVs were collected by liver perfusion of sham and APAP-treated rats. Changes in EVs morphology were examined by transmission electron microscopic analysis of negatively stained vesicles. Results from morphometric analysis of EVs revealed striking differences in their size and distribution. Proteome composition of EVs collected by liver perfusion was determined by mass spectrometry using methods of sample preparation that enabled better detection of both highly hydrophobic proteins and proteins with complex post-translational modifications. The collection of EVs after liver perfusion is an approach that enables the isolation of EVs shed not only by isolated hepatocytes, but also by the entire complement of hepatic cells. EVs derived after DILI had a lower content of alpha-1-macroglobulin, ferritin, and members of cytochrome 450 family. Fibronectin, aminopeptidase N, metalloreductase STEAP4, integrin beta, and members of the annexin family were detected only in APAP-treated samples of EVs. These results show that the present approach can provide valuable insights into the response of the liver following drug-induced liver injury.

Soft agar-based selection of spontaneously transformed rat prostate epithelial cells with highly tumorigenic characteristics.

The critical molecular and cellular mechanisms involved in the development and progression of prostate cancer remain elusive. In this report, we demonstrate that normal rat prostate epithelial cells (PEC) undergo spontaneous transformation at high passage (p > 85) evidenced by the acquisition of anchorage independent growth when plated on soft agar and tumorigenicity when injected into immunodeficient mice. In addition, we also report the discovery of a minor subpopulation of spontaneously transformed PEC derived from high passage PEC with the ability to migrate through a layer of 1% agar and form expanding colonies on the underlying plastic substratum. Comparison of these soft agar invasive (SAI) cells with low (p < 35), mid (p36-84) and high passage (p > 85) PEC identified marked differences in cell morphology, proliferation and motility. The SAI subpopulation was more tumorigenic than the high passage anchorage independent cultures from which they were isolated, as manifested by a decreased latency period and an increase in the size of tumors arising in immunodeficient mice. In contrast, low and mid passage cells were unable to grow on soft agar and failed to form tumors when injected into immunodeficient mice. Screening with antibody-based signaling arrays identified several differences in the altered expression levels of signaling proteins between SAI-derived cells and low or high passage PEC, including the up-regulation of EGFR and MAPK-related signaling pathways in SAI-selected cells. In summary, these studies suggest that the SAI assay selects for a novel, highly tumorigenic subpopulation of transformed cells that may represent an early step in the progression of slow growing prostatic carcinomas into more rapidly growing and aggressive tumors.

The cholangiocyte marker, BD. 1, forms a stable complex with CLIP170 and shares an identity with eIF3a, a multifunctional subunit of the eIF3 initiation complex.

We have previously described the generation of a monoclonal antibody recognizing a novel cholangiocyte marker, designated BD.1, that is expressed by fetal and adult rat cholangiocytes but not hepatocytes or the hepatic progenitor cells known as oval cells. In the present report, we have undertaken a comprehensive examination of BD.1 expressed by long-term cultures of bile duct epithelial cells (BDEC) and prostate epithelial cells (PEC). We show that with continued passage, the levels of BD.1 expressed by BDEC and PEC drop significantly, a decrease that is temporally associated with transition from a diploid to an aneuploid karyotype. Cell cycle analysis revealed cell cycle dependent expression of BD.1 characterized by decreased BD.1 levels within the first 10 h after release from serum starvation followed by reacquisition as cells entered S phase. MAb BD.1 recognized a 170 kDa protein in Western blots and showed strong reactivity with a 170 kDa band in blots prepared from phosphoproteins isolated by metal affinity chromatography. Analysis by mass spectrometry of tryptic peptides generated from BD.1 purified by continuous elution electrophoresis identified the plus end microtubule-binding protein, CLIP170, in the fraction reactive with MAb BD.1. Double immunofluorescence with MAb BD.1 and a MAb specific for CLIP170 showed that both were reactive with intrahepatic bile ducts. However, overexpression or siRNA knockdown of CLIP170 in 293T cells did not significantly alter BD.1 levels, indicating that CLIP170 and BD.1 were distinct, co-migrating proteins. Immunoprecipitation analysis with MAb BD.1 and anti-CLIP170 antibodies showed that under microtubule depolymerizing conditions the two proteins could be co-precipitated with both antibodies, leading us to conclude they were capable of forming stable complexes. Two different protocols were devised to enrich for the CLIP170 binding protein recognized by MAb BD.1. Analysis of tryptic peptides by LC-ESI-MS/MS identified BD.1 as eIF3a, the largest subunit of the elongation initiation factor 3 (eIF3) complex. This identity was confirmed by the simultaneous knockdown of both BD.1 and eIF3a by eIF3a-specific siRNAs and by the strong reactivity of MAb BD.1 with the 170 kDa protein immunoprecipitated with the anti-eIF3a antibody, 5H10. Based on these findings, we concluded that the BD.1 antigen was identical to eIF3a, a multifunctional subunit of the eIf3 complex shown here to associate with microtubules through its interactions with CLIP170.

The inhibitory effect of rapamycin on the oval cell response and development of preneoplastic foci in the rat.

Oval cell activation occurs under conditions of severe liver injury when normal hepatocyte proliferation is blocked. Recent studies have shown that a subset of hepatocellular carcinomas expresses oval cell markers, suggesting that these cells are targets of hepatocarcinogens. However, the signaling pathways that control oval cell activation and proliferation are not well characterized. Based on the role of the nutrient signaling kinase complex, mTORC1, in liver development, we investigated the role of this pathway in oval cell activation. Oval cell proliferation was induced in male Fisher rats by a modification of the traditional choline deficient plus ethionine model (CDE) or by 2-acetylaminoflourene treatment followed by 2/3 partial hepatectomy with or without initiation by diethylnitrosamine. To assess the role of mTOR in the oval cell response and development of preneoplastic foci, the effect of the mTORC1 inhibitor, rapamycin, was studied in all models. Rapamycin induced a significant suppression of the oval cell response in both models, an effect that coincided with a decrease in oval cell proliferation. Rapamycin administration did not affect the abundance of neutrophils or natural killer cells in CDE-treated liver or the expression of key cytokines. Gene expression studies revealed the fetal hepatocyte marker MKP-4 to be expressed in oval cells. In an experimental model of hepatic carcinogenesis, rapamycin decreased the size of preneoplastic foci and the rate of cell proliferation within the foci. mTORC1 signaling plays a key role in the oval cell response and in the development of preneoplastic foci. This pathway may be a target for the chemoprevention of hepatocellular carcinoma.

A new stem cell biology: the continuum and microvesicles.

The hierarchical models of stem cell biology have been based on work first demonstrating pluripotental spleen-colony-forming units, then showing progenitors with many differentiation fates assayed in in vitro culture; there followed the definition and separation of "stem cells" using monoclonal antibodies to surface epitopes and fluorescent-activated cell characterization and sorting (FACS). These studies led to an elegant model of stem cell biology in which primitive dormant G0 stem cells with tremendous proliferative and differentiative potential gave rise to progressively more restricted and differentiated classes of stem/progenitor cells, and finally differentiated marrow hematopoietic cells. The holy grail of hematopoietic stem cell biology became the purification of the stem cell and the clonal definition of this cell. Most recently, the long-term repopulating hematopoietic stem cell (LT-HSC) has been believed to be a lineage negative sca-1+C-kit+ Flk3- and CD150+ cell. However, a series of studies over the past 10 years has indicated that murine marrow stem cells continuously change phenotype with cell cycle passage. We present here studies using tritiated thymidine suicide and pyronin-Hoechst FACS separations indicating that the murine hematopoietic stem cell is a cycling cell. This would indicate that the hematopoietic stem cell must be continuously changing in phenotype and, thus, could not be purified. The extant data indicate that murine marrow stem cells are continually transiting cell cycle and that the purification has discarded these cycling cells. Further in vivo BrdU studies indicate that the "quiescent" LT-HSC in G0 rapidly transits cycle. Further complexity of the marrow stem cell system is indicated by studies on cell-derived microvesicles showing that they enter marrow cells and transcriptionally alter their cell fate and phenotype. Thus, the stem cell model is a model of continuing changing potential tied to cell cycle and microvesicle exposure. The challenge of the future is to define the stem cell population, not purify the stem cell. We are at the beginning of elucidation of quantum stemomics.

Mammalian plasma membrane proteins as potential biomarkers and drug targets.

Defining the plasma membrane proteome is crucial to understand the role of plasma membrane in fundamental biological processes. Change in membrane proteins is one of the first events that take place under pathological conditions, making plasma membrane proteins a likely source of potential disease biomarkers with prognostic or diagnostic potential. Membrane proteins are also potential targets for monoclonal antibodies and other drugs that block receptors or inhibit enzymes essential to the disease progress. Despite several advanced methods recently developed for the analysis of hydrophobic proteins and proteins with posttranslational modifications, integral membrane proteins are still under-represented in plasma membrane proteome. Recent advances in proteomic investigation of plasma membrane proteins, defining their roles as diagnostic and prognostic disease biomarkers and as target molecules in disease treatment, are presented.

Accumulation of neoplastic traits prior to spontaneous in vitro transformation of rat cholangiocytes determines susceptibility to activated ErbB-2/Neu.

Cholangiocarcinoma, a severe form of biliary cancer, has a high mortality rate resulting partially from the advanced stage of disease at earliest diagnosis. A better understanding of the progressive molecular and cellular changes occurring during spontaneous cholangiocarcinogenesis is needed to identify potential biomarkers for diagnosis/prognosis or targets for novel therapeutics. Here, we show that with continued passage (p) in vitro, rat bile duct epithelial cells (BDEC) accumulated neoplastic characteristics that by mid-passage (p31-85) included alterations in morphology, increased growth rate, growth factor independence, decreased cell adhesion, loss of cholangiocyte markers expressed at low passage (p<30), and onset of aneuploidy. At high passage (p>85), BDEC cultures showed increasing numbers of cells expressing activated, tyrosine phosphorylated ErbB-2/Neu, a receptor tyrosine kinase previously reported to be at elevated levels in cholangiocarcinomas. Enrichment for high passage ErbB-2/Neu-positive cells yielded several anchorage-independent sub-lines with elevated levels of activated ErbB-2/Neu and increased expression of cyclooxygenase-2 (COX-2). When injected into immunodeficient beige/nude/xid mice, these sub-lines formed poorly differentiated cystic tumors strongly positive for rat cholangiocyte markers, a finding consistent with a previous report showing the susceptibility of high passage, non-tumorigenic BDEC to transformation by activated ErbB-2/Neu. Mid passage BDEC, in contrast, were resistant to the transforming activity of activated ErbB-2/Neu and remained anchorage dependent in vitro and non-tumorigenic in vivo following stable transfection. Based on these findings, we concluded that during progression to high passage, cultured BDEC undergo preneoplastic changes that enhance their susceptibility to transformation by ErbB-2/Neu. The ability to generate cells at different points in the process of spontaneous neoplastic transformation offers a valuable model system for identifying molecular features that determine whether over-expression of activated ErbB-2/Neu is necessary and sufficient to induce neoplastic conversion.

SELDI-TOF as a method for biomarker discovery in the urine of aristolochic-acid-treated mice.

Aristolochic acids (AAs) present in Aristolochia plants are substances responsible for Chinese herbs nephropathy. Recently, strong indications have also been presented, which dietary poisoning with AA is responsible for endemic (Balkan) nephropathy (EN), an enigmatic renal disease that affects rural population living in some countries in Southeastern Europe. A mouse model was applied to follow the effects of two forms of AA, AAI and AAII. SDS-PAGE and SELDI-TOF mass spectrometry with normal phase chips were used to evaluate changes in the urine of treated animals. These two methods are demonstrated to be comparable. The use of SELDI-TOF MS for rapid analysis of a large number of samples and the combination of this method with nano-LC-ESI MS/MS for protein identification were demonstrated. Biomarker discovery after analysis of large cohort of EN patients will be the final aim of these investigations.

Proteomic analysis for process development and control of therapeutic protein separation from human plasma.

The use of proteomics technology during the development of a new process for plasma protein separation was demonstrated. In a two-step process, the two most abundant proteins, HSA and IgG, were removed in a first step of anion-exchange chromatography using a gel with very high capacity. Subsequently, two fractions containing medium and low abundance proteins were re-chromatographed on a smaller column with the same type of gel. Collected fractions were separated by SDS-PAGE and 2-D electrophoresis, and excised proteins were digested with trypsin and identified by LC-ESI-MS/MS. This proteomic analysis proved to be a useful method for detection of low abundance therapeutic proteins and potential harmful contaminants during process development. Based on this method, low abundance therapeutic proteins, such as vitamin-K-dependent clotting factors and inhibitors, could be identified as present in target fractions after chromatographic separation. In addition, the tracking of potentially dangerous impurities and designing proper steps for their removal are important outcomes when developing, refining or controlling a new fractionation schema. For the purpose of in-process control, in-solution digestion of complete fractions followed by protein identification with LC-ESI-MS/MS was demonstrated as a rapid and simple alternative to the entire analysis including 1-D or 2-D electrophoretic steps.

Proteomic characterization of plasma-derived clotting factor VIII-von Willebrand factor concentrates.

Proteomic methods were used to identify the levels of impurities in three commercial plasma-derived clotting factor VIII-von Willebrand factor (FVIII/VWF) concentrates. In all three concentrates, significant amounts of other plasma proteins were found. In Octanate and Haemoctin, two concentrates developed in the 1990s, the major impurities identified were inter-alpha inhibitor proteins, fibrinogen and fibronectin. These two concentrates were also found to contain additional components such as clotting factor II (prothrombin) that are known activators of FVIII. In Wilate, a recently developed FVIII/VWF concentrate, the amount of these impurities was significantly reduced. Batch-to-batch variations and differences between three investigated products were detected using iTRAQ, an isotope labeling technique for comparative MS, demonstrating the potential value of this technique for quality control analysis. The importance of thorough proteomic investigations of therapeutic FVIII/VWF preparations from human plasma is also discussed.

Membrane proteins as diagnostic biomarkers and targets for new therapies.

Membrane proteins, especially plasma membrane proteins, form one of the most interesting classes of proteins among disease biomarker candidates. Because of their localization on the surface of cells and organelles, membrane proteins also represent potential drug targets. In this review, developments in the characterization of membrane proteins and their role in the treatment of disease, in particular cancer treatment, are presented.

Proteomics as a tool for optimization of human plasma protein separation.

The application of proteomics technology in purification of proteins from human plasma and for characterization of plasma-derived therapeutics has been recently discussed. However, until now, the impact of this technology on the plasma protein fractionation and analysis of the final product has not been realized. In the present work, we demonstrate the use of proteomic techniques the monitoring of the first step of the plasma fractionation by use of anion-exchange chromatography. This chromatographic method is frequently used in the purification scheme for isolation of vitamin K dependent clotting factors II, VII, IX and X, and clotting inhibitors protein C and protein S, as well as inter-alpha inhibitor proteins (IaIp). After the removal of immunoglobulin G and non-binding proteins in the flow-through fraction, albumin and weakly bound proteins were eluted with low concentration of sodium chloride. The proteins that strongly bind to the anion-exchange column were eluted by higher salt concentrations. The fractions of interest were analyzed, and proteins were identified by LC-ESI-MS/MS. By use of this method, not only candidates for therapeutic concentrates, but also some potentially harmful components were identified. This strategy was very helpful for further process optimization, fast identification of target proteins with relatively low abundance, and for the design of subsequent steps in their removal or purification.

Comparative proteomics of rat liver and Morris hepatoma 7777 plasma membranes.

Plasma membranes from normal rat liver and hepatocellular carcinoma Morris hepatoma 7777 were selectively solubilized by use of different reagents. After selective solubilization, proteins were identified by nano-HPLC-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS). Using simple software, the patterns of proteins identified in membrane solubilizates from liver and hepatoma were compared. Proteins identified in Morris hepatoma 7777 and not in the corresponding membrane solubilizate from liver, mostly members of the annexin and heat shock protein families, are discussed as potential candidate markers for hepatocellular carcinomas.

Primitive stem cells residing in the skeletal muscle of adult pigs are mobilized into the peripheral blood after trauma.

This study was designed to determine if trauma causes the release of adult-derived blastomere-like stem cells (BLSCs) from skeletal muscle into the circulating blood of adult pigs. Experimental procedures followed the guidelines of Fort Valley State University's Institutional Animal Care and Utilization Committee. Pigs were traumatized by splenectomy followed by pancreatectomy. Blood samples and skeletal muscle biopsies were taken before and after trauma. Adult-derived BLSCs were isolated from skeletal muscle and blood samples following established procedures. Nontraumatized skeletal muscle contained approximately 277 million BLSCs per gram of muscle. After trauma, skeletal muscle contained approximately 2 million BLSCs per gram of muscle. Blood taken before trauma contained approximately 22 million BLSCs per milliliter, whereas approximately 512 million BLSCs per milliliter were present within the blood after trauma. Blood values were statistically significant with a P < 0.05. This report is the first demonstration that trauma causes the release of adult-derived BLSCs from skeletal muscle into blood. Further studies are required to elucidate the roles that adult-derived BLSCs play in the response to injury and in the healing process. Surgeons must take a role in this evolving field.

Carcinoembryonic antigen-related cell adhesion molecule 1a-4L suppression of rat hepatocellular carcinomas.

Carcinoembryonic antigen (CEA)-related cell adhesion molecule 1 (CEACAM1) is a member of the CEA family of immunoglobulin-like adhesion molecules with two major splice variants, CEACAM1(a)-4L and CEACAM1(b)-4S, differing in the length of their COOH-terminal cytoplasmic tail. Both forms are down-regulated in prostate and liver carcinomas relative to normal tissues. We have previously shown in a nude mouse xenograft model that restoration of CEACAM1(a)-4L expression in human prostate carcinoma cells (PC-3) suppresses tumorigenicity, an effect observed with carcinomas from several other tissues but never established for hepatocellular carcinomas. In this report, we have examined the effect of CEACAM1(a)-4L on tumorigenicity of 1682A, a rat hepatocellular carcinoma that grows on the omentum when injected into the peritoneal cavity. Results show that restoration of CEACAM1(a)-4L expression at levels 13- and 0.45-fold compared with negative controls or normal hepatocytes, respectively, completely suppressed the formation of 1682A tumor nodules on the omentum at 3 weeks after injection. In contrast, 1682A cells infected with CEACAM1(b)-4S or an empty retroviral vector formed multiple clusters of tumor nodules. Although tumor nodules of 1682A cells positive and negative for CEACAM1(a)-4L did not display significant differences in histologic organization, aggregates formed in vitro by 1682A-L were smaller in size and displayed enlarged intercellular spaces relative to their 1682A-V counterparts. Restoration of CEACAM1(a)-4L expression did not elevate levels of apoptosis but seemed to cause an increase in the length of G1. This is the first demonstration of CEACAM1(a)-4L-induced tumor suppression in liver carcinomas using a quantifiable i.p. syngeneic transplantation model.

Use of short monolithic columns for isolation of low abundance membrane proteins.

Convective interaction media (CIM) monoliths provide a stationary phase with a high binding capacity for large molecules and are capable of high flow rates at a very low pressure drop. Used as anion- and cation-exchangers or with affinity ligands such as antibodies, these columns have the potential for processing large volumes of complex biological mixtures within a short time. In the present report, monoclonal antibodies against several rat liver plasma membrane proteins were bound and cross-linked to protein A or protein G CIM affinity columns with a bed volume of only 60 microL. Antigens recognized by bound antibodies and co-eluting (interacting) proteins were rapidly isolated in a single step from either total plasma membrane extracts or subfractions isolated using anion-exchange CIM disk-shaped columns. The isolated antigens and co-eluting proteins were subsequently identified by immunoblot or by LC-MS/MS.

Identification of members of the annexin family in the detergent-insoluble fraction of rat Morris hepatoma plasma membranes.

For proteomic analysis, plasma membranes of rat hepatocellular carcinoma Morris hepatoma 7777 were selectively solubilized according to the previously developed method [D. Josic, K. Zeilinger, Methods Enzymol. 271 (1996) 113-134]. If the Triton X100 insoluble pellet is subsequently extracted, several proteins can be solubilized. These proteins can be classified in two groups according to their molecular size. The proteins with apparent molecular weights in SDS-PAGE between 70 and 75 kDa belong to the first group. Smaller proteins, with apparent molecular weights between 30 and 45 kDa, are members of the second group. The main protein of higher molecular weight was also found in the Triton X100 insoluble extract from normal rat liver plasma membranes. This protein was identified as Annexin A6. The proteins from the second group are practically absent in the Triton X100 insoluble extract from rat liver. These proteins are present in relatively high concentrations in plasma membranes of Morris hepatoma 7777. Both groups of detergent-insoluble proteins from Morris hepatoma 7777 were further analyzed with SELDI-TOF and LC electrospray ionization mass spectrometry. From the first group, Annexin A6, together with two other integral plasma membrane proteins, was identified. In the second group of proteins with apparent molecular weights between 30 and 45kDa, further members of the annexin family, Annexins A1, A2, A4, A5 and A7 were identified. The possible role of these low molecular size annexins as potential cancer biomarkers is discussed.

V(D)J recombination of chromosomally integrated, wild-type deletional and inversional substrates occur at similar frequencies with no preference for orientation.

Efficient and correct recombination of V(D)J substrates results in the generation of antibodies. The RSS substrates are oriented in two directions with respect to each other: deletional and inversional. Deletional recombination results in the formation of the coding joint and excision of the intervening sequences. Inversional recombination retains all the genomic sequences and forms both a coding joint and a signal joint. A bias for deletional recombination has been characterized with specific loci in vivo and recapitulated in experiments using extrachromosomal substrates. We constructed retroviral substrates of RSS in the deletional and inversional orientation. We introduced the substrates into wild-type and scid pre-B cells and measured the frequency of functional recombination in addition to open/shut recombination. We also mutated the RSSs to determine whether mutated sequences influenced orientation bias. We show that pre-B cells recombine the wild-type substrates at a 1.6 ratio of deletion:inversion. Nonamer mutated substrates recombined with a deletional bias whereas heptamer mutated substrates recombined with an inversional bias. A spacer length mutation and drastic mutations in the RSS abolish all recombination. These results suggest that there is no orientation bias with wild-type RSSs but that orientation bias occurs when RSSs are mutated.

Affinity purification and enzymatic cleavage of inter-alpha inhibitor proteins using antibody and elastase immobilized on CIM monolithic disks.

Epoxy-activated monolithic CIM disks seem to be excellent supports for immobilization of protein ligands. The potential use of enzymes, immobilized on monolithic disks for rapid preparative cleavage proteins in solution was investigated. Digestion of complex plasma proteins was demonstrated by using inter-alpha inhibitors with elastase, immobilized on epoxy-activated CIM disks. Recently, a monoclonal antibody against human inter-alpha inhibitor proteins (MAb 69.31) was developed. MAb 69.31 blocks the inhibitory activity of inter-alpha inhibitor proteins to serine proteases. These results suggest that the epitope defined by this antibody is located within or proximal to the active site of the inhibitor molecule. This antibody, immobilized on monolithic disk, was used for very rapid isolation of inter-alpha proteins. The isolated complex protein was used for enzymatic digestion and isolation of cleavage products, especially from inter-alpha inhibitor light chain to elucidate precisely the target sequence for MAb 69.31 by N-terminal amino acid sequencing. Bovine pancreatic elastase immobilized on monolithic disk cleaves inter-alpha inhibitor protein complex into small fragments which are still reactive with MAb 69.31. One of these proteolytic fragments was isolated and partially sequenced. It could be shown that this sequence is located at the beginning of two proteinase inhibitor domains of the inter-alpha inhibitor light chain (bikunin). Elastase immobilized on monolithic disk offers a simple and rapid method for preparative isolation of protease cleavage fragments. The immobilized enzyme is stable and still active after repeated runs. A partial or complete digestion can be achieved by varying the flow rate.