Preliminary proteome analysis of rabbit serum with hepatic failure

The administration of dimethylnitrosamine (DMN) into rabbit induced liver fibrosis/cirrhosis and finally caused a lethal hepatic failure. Blood collected from the rabbit was centrifuged and the supernatant was analyzed by two-dimensional gel electrophoresis (2-DE) for the study of proteome in serum. Compared with 2-DE gel of serum from healthy rabbit, a significant reduction in the number of protein spots having molecular weights (MWs) below 21 kDa was observed in the gels of the serum from the rabbit treated with DMN, while the secretion of albumin was kept at a high level. Separated spots in the two-dimensional gel were cut, digested with trypsin, and analyzed by MALDI-TOF mass spectrometry. Serum amyloid A-3 protein precursor (SAA3) and other serum amyloid A (SAA) protein precursors were identified by matching the peptide masses with those in database. In the SAA family of acute-phase/inflammatory response proteins, SAA3 is mainly synthesized in the liver. The SAA3 secreted level in the serum decreased with time after DMN administration as the result of hepatic dysfunctions.     

Cryodetector mass spectrometry profiling of plasma samples for HELLP diagnosis: an exploratory study

The cryodetector MS-based screening method enables the rapid and reliable separation of hemolysis, elevated liver enzymes, and low platelets syndrome (HELLP) patients from healthy women. For developing the assay, plasma protein abundances from patients suffering from HELLP were profiled before and after delivery and compared to healthy pregnant and nonpregnant control individuals, using a TOF mass spectrometer equipped with a cryodetector system. The spectra were well reproducible when acquisition conditions were kept constant and the overall appearance of the profiles was well comparable. Peak areas of ten selected ion signals (9.5, 11.8, 14.0, 28.1, 43.4, 50.5, 60.1, 66.6, 74.5, and 79.8 kDa) from each spectrum were subjected to statistical analysis. Significant differences in ion intensities between the groups could be measured even without sample fractionation. The most striking difference between most of the spectra from HELLP patients and nonHELLP samples was the presence/absence of the 11.8 kDa ion signal (p-value: 0.0000365), most likely belonging to serum amyloid A (SAA). Other significant differences in ion intensities between HELLP and control samples were observed in the peak areas of the 14.0, 28.1, 50.5, and 74.5 kDa ion signals. Using the cryodetector MS data for sorting plasma samples into either the HELLP group (n = 8) or the nonHELLP group (n = 22), a sensitivity of 87.5% and a specificity of 100% were achieved. The positive predictive value of our screening assay was 100% and a negative predictive value of 95.6% was obtained with the samples included in this study.     

Proteomic Analysis of Haptoglobin and Amyloid A Protein Levels in Patients with Vivax Malaria

Advancements in the field of proteomics have provided great opportunities for the development of diagnostic and therapeutic tools against human diseases. In this study, we analyzed haptoglobin and amyloid A protein levels of vivax malaria patients with combinations of depletion of the abundant plasma proteins, 2-dimensional gel electrophoresis (2-DE), image analysis, and mass spectrometry in the plasma between normal healthy donors and vivax malaria patients. The results showed that the expression level of haptoglobin had become significantly lower or undetectable in the plasma of vivax malaria patients due to proteolytic cleavage when compared to healthy donors on 2-DE gels. Meanwhile, serum amyloid A protein was significantly increased in vivax malaria patient''s plasma with high statistical values. These 2 proteins are common acute phase reactants and further large scale evaluation with a larger number of patient''s will be necessary to establish the possible clinical meaning of the existential changes of these proteins in vivax malaria patients. However, our proteomic analysis suggests the feasible values of some plasma proteins, such as haptoglobin and serum amyloid A, as associating factor candidates for vivax malaria.     

Amyloid-related serum protein SAA from three animal species: comparison with human SAA.

The amyloid-relates serum protein SAA has been isolated by gel filtration in 10% formic acid from three animal species: mink, mouse, rabbit. Sera used in the isolation procedure were obtained from animals in which high concentrations of SAA had been induced by treatment with LPS. The isolated SAA proteins had a subunit size similar to that of human SAA, with m.w. values ranging from 10,000 to 11,700 (estimated by gel filtration in 6 M guanidine-HC1) or 12,400 to 15,000 (estimated by SDS-PAGE). The m.w. studies and amino acid sequence data indicated that SAA and the amyloid fibril protein AA in the mouse, and probably also the mink, are related in the same way as in man, the two proteins having common NH2-terminal amino acid sequences and SAA being extended by 20 to 40 residues at the COOH-terminal end of the molecule.     

Identification of tumor-associated plasma biomarkers using proteomic techniques: from mouse to human

In an effort to identify tumor-associated proteins from plasma of tumor-bearing mice that may be used as diagnostic biomarkers, we developed a strategy that combines a tumor xenotransplantation model in nude mice with comparative proteomic technology. Five human cancer cell lines (SC-M1, HONE-1, CC-M1, OECM1, GBM 8401) derived from stomach, nasopharyngeal, colon, oral and brain cancers were subcutaneously inoculated into nude mice and compared to control nude mice injected with phosphate-buffered saline. One month later, plasma from mice inoculated with cancer cells was collected for proteomic analysis using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). Comparison of plasma 2-DE maps from tumor-bearing mice with those produced from control mice revealed the overexpression of several mouse acute phase proteins (APPs) such as haptoglobin. Another APP, serum amyloid A (SAA), was found only in mice bearing tumors induced by the stomach cancer cell line SC-M1, which has not previously been demonstrated in xenotransplatation experiment. Furthermore, by using immunohistochemistry, SAA and haptoglobin were found to originate from the mouse hosts and not from the human cancer cell line donors. The protein alterations were further confirmed on patients with stomach cancers where up-regulated levels of SAA were also observed. These results indicate that APPs may be used as nonspecific tumor-associated serum markers. SAA in particular may serve as a potential marker for detecting stomach cancer. Taken together, the combination of the xenotransplatation model in nude mice and proteomics analysis provided a valuable impact for clinical applications in cancer diagnostics. In addition, our findings demonstrate that a panel of APPs might serve as screening biomarkers for early cancer detection.     

MALDI-TOF/TOF-MS reveals elevated serum haptoglobin and amyloid A in Behcet's disease

Behcet's disease (BD) is a multisystemic autoimmune disease with unclear etiology and pathogenesis. To screen aberrant serum proteins in BD, serum samples were obtained from eight male BD patients with active uveitis and eight male healthy volunteers with informed consent. The serum samples from active BD patients and normal controls were pooled. Highly abundant serum proteins (albumin and IgG) were depleted from these two samples using an affinity capture based kit. The obtained samples were subjected to two-dimensional gel electrophoresis (2-DE). Protein spots were visualized with the "blue silver" staining. Differently expressed proteins were subsequently identified by matrix-assisted laser desorption /ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Western blot and enzyme-linked immunosorbent assay (ELISA) were performed using the serum samples from 18 patients with active BD, 6 patients with inactive BD, 22 patients with Vogt-Koyanagi-Harada (VKH) syndrome, and 20 healthy volunteers to validate the results of 2-DE and MS. Proteomic profiles of the pooled samples were compared, and approximately 800 protein spots were observed in each of the gels. Expression levels of four of the protein spots in active BD were significantly higher than those in the normal controls. Mass spectrometric protein identification revealed that the four protein spots corresponded to two proteins: haptoglobin (Hp) and serum amyloid A (SAA). Western blot and ELISA showed that Hp was only overexpressed in active BD but not in inactive BD, VKH syndrome, or healthy controls. An obvious band of SAA was detected in 72.2% of the serum samples from BD patients, whereas a vague band of this protein was found in 10.0% of the tested normal samples and 9.1% of VKH samples. Our results revealed a significantly increased expression of Hp and SAA in serum of active BD patients. These two proteins may be involved in the development of BD.     

Extrahepatic production of acute phase serum amyloid A

Amyloidosis is a group of diseases characterized by the extracellular deposition of protein that contains non-branching, straight fibrils on electron microscopy (amyloid fibrils) that have a high content of beta-pleated sheet conformation. Various biochemically distinct proteins can undergo transformation into amyloid fibrils. The precursor protein of amyloid protein A (AA) is the acute phase protein serum amyloid A (SAA). The concentration of SAA in plasma increases up to 1000-fold within 24 to 48 h after trauma, inflammation or infection. Individuals with chronically increased SAA levels may develop AA amyloidosis. SAA has been divided into two groups according to the encoding genes and the source of protein production. These two groups are acute phase SAA (A-SAA) and constitutive SAA (C-SAA). Although the liver is the primary site of the synthesis of A-SAA and C-SAA, extrahepatic production of both SAAs has been observed in animal models and cell culture experiments of several mammalian species and chicken. The functions of A-SAA are thought to involve lipid metabolism, lipid transport, chemotaxis and regulation of the inflammatory process. There is growing evidence that extrahepatic A-SAA formation may play a crucial role in amyloidogenesis and enhances amyloid formation at the site of SAA production.     

Degradation of serum amyloid A and apolipoproteins by serum proteases

We have investigated the protease activity, present in human serum, that digests the serum amyloid A (SAA) protein. SAA radiolabeled with 125I was incubated at 37 degrees C with serum and plasma and analyzed for degradation products by alkaline urea-polyacrylamide gel electrophoresis and gel filtration chromatography. Serum initially digested SAA to intermediates of 3000-5000 in molecular weight, and these were further degraded to smaller peptides with prolonged incubation. SAA was not degraded by plasma anticoagulated with ethylenediaminetetraacetic acid (EDTA) or heparin. Recalcification of plasma anticoagulated with EDTA led to the generation of protease activity against SAA whereas EDTA plasma defibrinated with thrombin was inactive. We employed both nonselective and selective protease inhibitors and synthetic substrates for kallikrein and plasmin to further characterize the serum protease. These studies demonstrated that degradation of SAA is not directly attributable to enzymes involved in coagulation, kinin formation, or fibrinolysis, but the unidentified protease may be activated by one of the clotting factors. The specificity of the SAA degradation was demonstrated in experiments with three of the well-characterized apolipoproteins. Apolipoproteins A-I, C-I, and C-III-1, which also associate with the plasma high-density lipoproteins, were not degraded by serum although they were good substrates for purified thrombin and plasmin.     

Maternal and cord blood levels of serum amyloid A, C-reactive protein, tumor necrosis factor-alpha, interleukin-1beta, and interleukin-8 during and after delivery

C-reactive protein (CRP) and serum amyloid A (SAA) are acute-phase proteins mainly synthesized by the liver in response to some cytokines. They are potentially useful to diagnosing infection and monitoring different clinical conditions. The aim of this study was to measure SAA and CRP in maternal and cord blood during and after delivery and try to correlate these proteins with tumor necrosis factor-alpha, interleukin-1beta, and interleukin-8. Acute-phase proteins and cytokines were measured by ELISA in 24 healthy pregnant women undergoing vaginal delivery or Cesarean section. Cord blood samples in addition to maternal blood were collected. SAA and CRP reached the maximum maternal serum levels 24 hours after delivery, while cytokines remained constant over time. SAA and CRP were significantly higher in maternal serum than in newborn's (P< .001) at the moment of delivery. SAA and CRP, regardless of the type of delivery, reproduce the common pattern observed in most inflammatory conditions. Proinflammatory cytokine serum levels do not mirror the increase in SAA and CRP levels.     

Targeted protein quantitation and profiling using PVDF affinity probe and MALDI-TOF MS

Development of a rapid, effective, and highly specific platform for target identification in complex biofluids is one of the most important tasks in proteomic research. Taking advantage of the natural hydrophobic interaction of PVDF with probe protein, a simple and effective method was developed for protein quantitation and profiling. Using antibody-antigen interactions as a proof-of-concept system, the targeted plasma proteins, serum amyloid P (SAP), serum amyloid A (SAA), and C-reactive protein (CRP), could be selectively isolated and enriched from human plasma by antibody-immobilized PVDF membrane and directly identified by MALDI-TOF MS without additional elution step. The approach was successfully applied to human plasma for rapid quantitation and variant screening of SAP, SAA, and CRP in healthy individuals and patients with gastric cancer. The triplexed on-probe quantitative analysis revealed significant overexpression of CRP and SAA in gastric cancer group, consistent with parallel ELISA measurements and pathological progression and prognostic significance reported in previous literatures. Furthermore, the variant mass profiling of the post-translationally modified forms revealed a high occurrence of de-sialic acid SAP in patients with gastric cancer. Due to the versatile assay design, ease of probe preparation without chemical synthesis, and compatibility with MALDI-TOF MS analysis, the methodology may be useful for target protein characterization, functional proteomics, and screening in clinical proteomics.     

Inflammation protein SAA2.2 spontaneously forms marginally stable amyloid fibrils at physiological temperature

For nearly four decades, the formation of amyloid fibrils by the inflammation-related protein serum amyloid A (SAA) has been pathologically linked to the disease amyloid A (AA) amyloidosis. However, here we show that the nonpathogenic murine SAA2.2 spontaneously forms marginally stable amyloid fibrils at 37 °C that exhibit cross-beta structure, binding to thioflavin T, and fibrillation by a nucleation-dependent seeding mechanism. In contrast to the high stability of most known amyloid fibrils to thermal and chemical denaturation, experiments monitored by glutaraldehyde cross-linking/SDS-PAGE, thioflavin T fluorescence, and light scattering (OD(600)) showed that the mature amyloid fibrils of SAA2.2 dissociate upon incubation in >1.0 M urea or >45 °C. When considering the nonpathogenic nature of SAA2.2 and its ~1000-fold increased concentration in plasma during an inflammatory response, its extreme in vitro amyloidogenicity under physiological-like conditions suggest that SAA amyloid might play a functional role during inflammation. Of general significance, the combination of methods used here is convenient for exploring the stability of amyloid fibrils that are sensitive to urea and temperature. Furthermore, our studies imply that analogous to globular proteins, which can possess structures ranging from intrinsically disordered to extremely stable, amyloid fibrils formed in vivo might have a broader range of stabilities than previously appreciated with profound functional and pathological implications.     

Serum amyloid A (SAA)-induced remodeling of CSF-HDL

Inflammation is a risk factor for Alzheimer's disease. Serum amyloid A (SAA) is an acute phase protein that dissociates apolipoprotein AI (apoAI) from plasma HDL. In cerebrospinal fluid (CSF), the SAA concentration is much higher in subjects with Alzheimer's disease than in controls. CSF-HDL is rich in apoE, which plays an important role as a ligand for lipoprotein receptors in the central nervous system (CNS). To clarify whether SAA dissociates apoE from CSF-HDL, we added recombinant SAA to CSF and determined the apoE distribution in the CSF using native two-dimensional gel electrophoresis. We found that SAA dissociated apoE from CSF-HDL in a dose-dependent manner. This effect was more evident in apoE4 carriers than in apoE3 or apoE2 carriers. After a 24-h incubation at 37 degrees C, SAA continuously dissociated apoE from CSF-HDL. Amyloid beta (Abeta) fragments (1-42) were bound to large CSF-HDL but not to apoE dissociated by SAA. In conclusion, SAA dissociates apoE from CSF-HDL. We postulate that inflammation in the CNS may impair Abeta clearance due to the loss of apoE from CSF-HDL.     

Identification of a plasma proteomic signature to distinguish pediatric osteosarcoma from benign osteochondroma

Osteosarcoma (OS) is the most common malignant bone tumor in children. To identify a plasma proteomic signature that can detect OS, we used SELDI MS to perform proteomic profiling on plasma specimens from 29 OS and 20 age-matched osteochondroma (OC) patients. Nineteen statistically significant ion peaks that were differentially expressed in OS when compared with OC patients were identified (p < 0.001 and false discovery rate < 10%). Using the proteomic profiles, we constructed a multivariate 3-nearest neighbors classifier to distinguish OS from OC patients with a sensitivity of 97% and a specificity of 80% based on external leave-one-out crossvalidation. Permutation test showed that the classification result was statistically significant (p < 0.00005). One of the proteins (m/z 11 704) in the proteomic signature was identified as serum amyloid protein A (SAA) by PMF. The higher plasma level of SAA in OS patients was further validated by Western blotting when compared to that of osteochrondroma patients and normal subjects as reference. The classifier based on this plasma proteomic signature may be useful to differentiate malignant bone cancer from benign bone tumors and for early detection of OS in high-risk individuals.     

Alkaptonuria is a novel human secondary amyloidogenic disease

Alkaptonuria (AKU) is an ultra-rare disease developed from the lack of homogentisic acid oxidase activity, causing homogentisic acid (HGA) accumulation that produces a HGA-melanin ochronotic pigment, of unknown composition. There is no therapy for AKU. Our aim was to verify if AKU implied a secondary amyloidosis. Congo Red, Thioflavin-T staining and TEM were performed to assess amyloid presence in AKU specimens (cartilage, synovia, periumbelical fat, salivary gland) and in HGA-treated human chondrocytes and cartilage. SAA and SAP deposition was examined using immunofluorescence and their levels were evaluated in the patients' plasma by ELISA. 2D electrophoresis was undertaken in AKU cells to evaluate the levels of proteins involved in amyloidogenesis. AKU osteoarticular tissues contained SAA-amyloid in 7/7 patients. Ochronotic pigment and amyloid co-localized in AKU osteoarticular tissues. SAA and SAP composition of the deposits assessed secondary type of amyloidosis. High levels of SAA and SAP were found in AKU patients' plasma. Systemic amyloidosis was assessed by Congo Red staining of patients' abdominal fat and salivary gland. AKU is the second pathology after Parkinson's disease where amyloid is associated with a form of melanin. Aberrant expression of proteins involved in amyloidogenesis has been found in AKU cells. Our findings on alkaptonuria as a novel type II AA amyloidosis open new important perspectives for its therapy, since methotrexate treatment proved to significantly reduce in vitro HGA-induced A-amyloid aggregates.     

Serum amyloid A (SAA) protein-interaction with itself and serum albumin.

Serum amyloid A (SAA) protein is a 12,000 dalton protein that exists in serum under physiologic conditions as an 85,000 dalton complex and under certain conditions, as a 170,000 dalton component. To study the reason for this finding, the behavior of 125I-SAA was studied in the presence of cold SAA and several serum proteins. SAA caused a shift of some of the radioactivity to the region of albumin. Addition of normal human serum or albumin caused a shift of a significant fraction of the radioactivity to a peak eluting slightly ahead of albumin (80.000 daltons). This interaction could be blocked by the addition of cold SAA. No shift was noted when IgG or Bence Jones proteins were added. Thus, it appears that low molecular SAA protein has a tendency to aggregate with itself and to bind to albumin but not to human IgG or Bence Jones proteins.     

Identification of two novel amyloid A protein subsets coexisting in an individual patient of AA-amyloidosis.

Amyloid A protein (AA), the major fibril protein in AA-amyloidosis, is an N-terminal cleavage product of the precursor protein, serum amyloid A (SAA). Using mass spectrometry and amino-acid sequencing, we identified and characterized two novel AA protein subsets co-deposited as amyloid fibrils in an patient having AA-amyloidosis associated with rheumatoid arthritis. One of the AA proteins corresponded to positions 2-76 (or 75) of SAA2 alpha and the other corresponded to positions 2-76 (or 75) of known SAA1 subsets, except for position 52 or 57, where SAA1 alpha has valine and alanine and SAA1 beta has alanine and valine in position 52 and 57, respectively, whereas the AA protein had alanine at the both positions. Our findings (1), demonstrate that not only one but two SAA subsets could be deposited together as an AA-amyloid in a single individual and (2), support the existence of a novel SAA1 allotype, i.e., SAA152,57Ala.     

Urea-induced denaturation of apolipoprotein serum amyloid A reveals marginal stability of hexamer

Serum Amyloid A (SAA) is an acute phase reactant protein that is predominantly found bound to high-density lipoprotein in plasma. Upon inflammation, the plasma concentration of SAA can increase dramatically, occasionally leading to the development of amyloid A (AA) amyloidosis, which involves the deposition of SAA amyloid fibrils in major organs. We previously found that the murine isoform SAA2.2 exists in aqueous solution as a hexamer containing a central channel. Here we show using various biophysical and biochemical techniques that the SAA2.2 hexamer can be totally dissociated into monomer by approximately 2 M urea, with the concerted loss of its alpha-helical structure. However, limited trypsin proteolysis experiments in urea showed a conserved digestion profile, suggesting the preservation of major backbone topological features in the urea-denatured state of SAA2.2. The marginal stability of hexameric SAA2.2 and the presence of residual structure in the denatured monomeric protein suggest that both forms may interconvert in vivo to exert different functions to meet the various needs during normal physiological conditions and in response to inflammatory stimuli.     

Identification of two novel amyloid A protein subsets coexisting in an individual patient of AA-amyloidosis

Amyloid A protein (AA), the major fibril protein in AA-amyloidosis, is an N-terminal cleavage product of the precursor protein, serum amyloid A (SAA). Using mass spectrometry and amino-acid sequencing, we identified and characterized two novel AA protein subsets co-deposited as amyloid fibrils in an patient having AA-amyloidosis associated with rheumatoid arthritis. One of the AA proteins corresponded to positions 2–76 (or 75) of SAA2α and the other corresponded to positions 2–76 (or 75) of known SAA1 subsets, except for position 52 or 57, where SAA1α has valine and alanine and SAA1β has alanine and valine in position 52 and 57, respectively, whereas the AA protein had alanine at the both positions. Our findings (1), demonstrate that not only one but two SAA subsets could be deposited together as an AA-amyloid in a single individual and (2), support the existence of a novel SAA1 allotype, i.e., SAA1^52,57Ala.