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Minimal residual disease in acute myeloid leukemia is predicted by an apoptosis-resistant protein profile at diagnosis.

PURPOSE: Apoptosis is an important mechanism regulating survival of acute myeloid leukemia cells. The apoptosis-related protein profile at diagnosis is important for achieving complete remission thereby affecting survival variables such as disease-free survival (DFS) and overall survival (OS).To investigate the role of the apoptosis protein profile in further response to therapy and outgrowth of disease. EXPERIMENTAL DESIGN: We studied whether Bcl-2, Bcl-xL, Mcl-1, Bax as well as the Bcl-2/Bax ratio and a combination of all (antiapoptosis index, AAI) are related to the frequency of malignant cells surviving the chemotherapy (i.e., minimal residual disease, MRD). MRD cells were identified by leukemia-associated aberrant phenotypes established at diagnosis by flow cytometry. RESULTS: We found that Bcl-2 (R = 0.55, P = 0.002), Bcl-2/Bax (R = 0.42, P = 0.02), and AAI (R = 0.47, P = 0.01) at diagnosis directly correlated with MRD after the first cycle of chemotherapy. In turn, MRD frequency after first cycle correlated with DFS (P = 0.04). Taken together, these results directly explain why Bcl-2/Bax and especially AAI (P = 0.007) at diagnosis correlate with DFS. CONCLUSION: Our results show that apoptosis resistance plays an important role in the first stage of the therapy (i.e., to eliminate the bulk of malignant cells), in terms of achievement of complete remission and frequency of MRD after first cycle of therapy.

Acute Disease↗

Human pancreatic secretory protein profiles in the search for tumor markers.

Pancreatic cancer secretory protein profiles were shown to be different from normals by a microcomputer-assisted analysis of isoelectric focusing patterns. Two acidic protein band regions (pI 3.0 and 4.5) of the pancreatic carcinoma profiles were significantly increased, and eight protein bands (pI 3.7, 5.0, 5.5, 6.5, 6.9, 7.3, 8.0, greater than 10.0) were significantly decreased. Highly significant decreases occurred at pH 7.3 (chymotrypsinogen) and at pH 5.0 (procarboxypeptidase Al, DNase I) in nonactivated specimens. The ratio between the absorbance points at 2.08 and 2.63 cm from the anode in each protein pattern differentiated the pancreas cancer specimens from the control group. Profiles found for the control group gave pI values similar to those found in the literature. The potential value of these findings in the search for tumor markers warrants further investigation as to whether these specimens can be differentiated from those from chronic pancreatitis patients.

Carcinoma↗

Quantitative protein profiling in heart mitochondria from diabetic rats.

Quantitative protein profiling based on in vitro stable isotope labeling, two-dimensional polyacrylamide gel electrophoresis, and mass spectrometry is an accurate and reliable approach to measure simultaneously the relative abundance of many individual proteins within two different samples. In the present study, it was used to define a set of alterations caused by diabetes in heart mitochondria from streptozotocin-treated rats. We demonstrated that the expression of proteins from the myocardial tricarboxylic acid cycle was not altered in diabetes. However, up-regulation of the fatty acid beta-oxidation favored fatty acids over glucose as a source of acetyl CoA for the tricarboxylic acid cycle. Protein levels for several proteins involved in electron transport were modestly decreased. Whether this may depress overall ATP production remains to be established, since the protein level of ATP synthase seems to be unchanged. Other changes include down-regulation of protein levels for creatine kinase, voltage-dependent anion channel 1 (VDAC-1), HSP60, and Grp75. The mitochondria-associated level of albumin was decreased, while the level of catalase was substantially increased. All of the changes were evident as early as 1 week after streptozotocin administration. Taken together, these data point to a rapid and highly coordinated regulation of mitochondrial protein expression that occurs during the heart adaptation to diabetes.

Amino Acid Sequence↗

Comparison of major protein antigens and protein profiles of Treponema pallidum and Treponema pertenue.

The protein profiles of Treponema pallidum and Treponema pertenue, the causative agents of syphilis and yaws, respectively, were compared by one- and two-dimensional gel electrophoresis. One-dimensional gels showed essentially no differences in the protein patterns of these treponemes. On two-dimensional gels most radiolabeled protein species were shared; however, variations were noticed in several minor protein species. Antigenic comparison by radioimmunoprecipitation and Western blotting also demonstrated similarities between these spirochetes. However, lactoperoxidase-catalyzed iodination of T. pallidum and T. pertenue suggested differences in their surface proteins.

Animals↗

Protein profiles of benign hypertrophic prostate: stroma-abundant distribution of BPH-associated nonhistone proteins.

Mechanically isolated epithelium and stroma from benign hypertrophic prostates were highly pure on the basis of histochemical and biochemical criteria. By electrophoretic analyses, whole cellular and nuclear proteins were compared among whole tissues, epithelium, and stroma. The characteristic protein profiles of benign hypertrophic prostates were reflected in the electrophoretic patterns of the stroma. Two-dimensional gel electrophoretic patterns of the epithelium were different from those of the stroma with exception of about 18 major spots that were common to both fractions. Of the protein species separated, 35K/6.7 (molecular weight/pI) and 36K, which was composed of two species with pI of 4.4 and 4.6, were abundant in the epithelium and stroma, respectively. Nuclei prepared from whole tissues of benign hypertrophic prostates contained three kinds of nonhistone proteins (NHP) closely associated with benign prostatic hypertrophy (BPH); 42 K-NHP, 55 K-NHP and 190 K-NHP. Electrophoretic analysis of the nuclear proteins revealed that all the BPH-associated nonhistone proteins were abundantly distributed in the nuclei of the stroma.

DNA↗

Plasma protein profiling: unique and stable features of individuals.

Carefully controlled ZipTip extraction of diluted human plasma or serum was combined with MALDI-TOF-MS to produce highly reproducible protein profiles. Components detected included apolipoproteins CI, CII and CIII as well as transthyretin and several isoforms of each protein that are created by glycosylation or other modification and by proteolytic processing. Profiles of healthy individuals all contained the same 15 components. Others were found in plasma from individuals with disease. Profiles were analyzed by peak ratios within the same spectrum. Reproducibility for multiple assays was generally 4 to 10%. Within the healthy population, a given peak ratio occurred with a range of about fourfold. However, peak ratios of multiple samples from the same individual showed a much lower range, typically +/-10%. In fact, each individual displayed a personal protein profile that changed very little over time. Because of the stability of protein profiles over time within individuals, these results suggest further studies may discover that certain profile characteristics or changes in an individual's profile may be a sign of current or future disease, even when the altered profile remains within the range for healthy individuals.

Adult↗

Growth phase-dependent variations in the outer membrane protein profile of Brucella melitensis.

Changes in Brucella cell envelope protein profiles were investigated with batch cultures of B. melitensis strain 16M in a 2-litre fermenter. Analysis of expression of outer membrane proteins (OMP) (apparent molecular masses of 10, 16.5, 19, 25-27, 31-34, 36-38 and 89 kDa) and heat-shock protein DnaK (73 kDa) was performed with monoclonal antibodies (mAb) and immunoblotting techniques. Synthesis of the 89-kDa OMP and the heat-shock protein DnaK was invariant during B. melitensis growth. Expression of the 10-, 19- and 36-38-kDa minor OMPs was never detected. Variations in profiles of some OMPs, i.e. 25-27-kDa and 31-34-kDa major proteins and 16.5-kDa minor protein, occurred during growth stages, principally at the end of the exponential growth phase. These variations consisted of shifts in apparent molecular masses for the 25-27-kDa and 31-34-kDa OMPs and of peptidoglycan association for the 16.5-kDa OMP. Therefore, whereas the strong association of major OMPs with peptidoglycan was confirmed, results suggested that the 16.5-kDa minor OMP is also a peptidoglycan-associated protein.

Bacterial Outer Membrane Proteins↗

[Study of the protein profile of the Adélé tribe of Togo].

Plasma proteins provide precise information about the physiological status of an individual. In this study, we compared the plasma protein profiles of 168 individuals from the Adélé ethnic group, from an isolated rural area of Togo, with those of 159 individuals from an urban population from the capital, Lomé. The Adélé villages are located in the Atakora mountains. The subjects were volunteers, all apparently healthy and aged between 18 and 65 years. We separated serum proteins by electrophoresis and identified proteins specific for nutritional, inflammatory and immune status. The Adélé significantly higher total serum protein concentrations than the urban individuals, with higher concentrations of a1 globulins (2.35 +/- 0.57 g/L versus 1.94 +/- 0.52 g/L) and g globulins (22.19 +/- 5.67 g/L versus 16.98 +/- 5.23 g/L) and lower concentrations of b globulins (6.83 +/- 1.56 g/L versus 7.34 +/- 1. 52 g/L). The Adélé also had lower plasma concentrations of albumin (41.91 +/- 5.74 g/L versus 44.56 +/- 6.32 g/L), tranferrin (2.5 +/- 0.52 g/L versus 3.03 +/- 0.6 g/L), haptoglobin (0.57 +/- 0.59 g/L versus 1.32 +/- 0.89 g/L) and IgA (2.3 +/- 0.89 g/L versus 2.88 +/- 1.12 g/L) and higher plasma concentrations of orosomucoid (0.85 +/- 0.26 g/L versus 0.69 +/- 0.27 g/L); IgG (25.3 +/- 7.11 g/L versus 21. 79 +/- 6.5 g/L) and IgM (4.25 +/- 2.83 g/L versus 2.25 +/- 1.0 g/L). The data obtained for the Adélé and urban populations were similar to those obtained for European populations except for IgM (higher in the Adélé than in the urban and European populations), IgG and CRP (higher for the Adélé and urban populations than for European populations). Nutritional status, as estimated by albumin and transferrin concentrations, was higher in the urban population of Lomé than in the Adélé population but the Adélé population suffered no malnutrition problems. These results are consistent with those of a previous study, using apo A-I concentrations as an index of nutritional status. Apo A-I has also been shown to be a reliable indicator of nutritional status, as prealbumin concentration alone is sufficient for the early diagnosis of protein malnutrition. The very high concentrations of plasma CRP obtained indicate the presence of an inflammatory syndrome in the Adélé and urban populations, as this protein is the first acute phase protein to be detected. However, the orosomucoid concentrations obtain-ed provide no evidence of significant inflammation. The high affinity of haptoglobin (Hp) for hemoglobin (Hb) results in the formation of soluble Hp-Hb complexes, reducing the value of Hp as a marker of the acute phase of inflammation. The frequency os sickle cell disease was higher in the Adélé population than in the urban population (10-25% versus 2-6%). Hemoglobinopathies are correlated with haptoglobin concentration and thus plasma haptoglobin concentration was lower in the Adélé population than in the urban population. The plasma concentrations of a1-antitrypsin in this study were similar to those reported for Europeans. The plasma concentration of protease inhibitors, such as a1-antitrypsin, increased as protease levels increased. These data confirm that the Adélé and urban populations suffer no disease due to high levels of protease release into the bloodstream. They also show that a1-antitrypsin is of some value as an acute phase marker protein. The acute nature of the inflammatory syndrome (as assessed by CRP concentration) in the Adélé and urban populations was confirmed by the hyperglobulinemia (high levels of production of IgM and IgG antibodies) observed in these populations. The Adélé and Lomé urban populations live in a tropical environment in which they are continuously in contact with infectious agents. This results in repeated stimulation of the immune system in both these populations. This study of plasma proteins in the Adélé provides insight into the physiological conditions of this ethnic group, w

Adolescent↗

Urinary protein profiles in patients with urothelial bladder tumours.

Urinary protein profiles were studied in 32 patients with urothelial bladder tumours, using immunonephelometry. In patients without residual or recurrent tumours a glomerular type of proteinuria was demonstrated. In patients with grade III-IV transitional cell carcinoma the glomerular proteinuria was more severe than in patients with grade I-II tumours. Increased excretion of low molecular weight proteins was observed only in patients with grade III-IV tumours. The use of the increased relative clearance of albumin, transferrin, and haptoglobin as a diagnostic test was attempted in detecting residual or recurrent tumours. In patients with grade III-IV tumours the predictive value of a positive or negative test was 92% and 100% respectively, whereas the relative protein clearance was without predictive value in patients with grade I-II tumours.

Adult↗

Age-related changes in protein profiles of the normal human trabecular meshwork.

The protein profile of the trabecular meshwork from 44 normal human eyes of various ages, ranging from 5 months to 87 years, was investigated by using sodium dodecyl sulfate-polyacrylamide-gel (SDS-PAGE) electrophoresis and a highly sensitive silver-stain technique. Samples from each eye were analysed individually to avoid anomalies that may occur with tissue pooling. More than 20 protein bands were consistently visible at all ages and varied in molecular weight from 20,000 to 290,000 MW. The shifts which occurred in band prominence with increasing age included a consistent decrease in the intensity of bands at MWs 42,000 (tentatively identified as G-actin) and 58,000; a noticeable increase in the intensity of the bands at MWs 140,000 and 160,000 that correspond to alpha I and II components of type IV collagen; and a gradual disappearance of a faint band at MW 68,000 in eyes older than 31 years. Our study shows that molecular aging does occur in the trabecular meshwork. The data presented also provides a basis for comparison of the polypeptide alterations that may occur in primary open-angle glaucoma and in other disease processes.

Adolescent↗

Developmental changes in the protein profiles of human cardiac and skeletal muscle.

1. The use of SDS electrophoresis as a tool for the analysis of development processes in man has been evaluated. 2. The protein profiles of cardiac and skeletal muscle from foetal (10--24 weeks gestation) infant and adult specimens have been analysed and striking developmental changes were found which involved all the major proteins. 3. Before 20 weeks gestation the soluble protein profile of skeletal muscle appears to consist largely of extracellular proteins. 4. Myoglobin was found in foetal cardiac muscle from 20 weeks gestation but was not demonstrable in foetal (greater than 24 weeks) skeletal muscle. Foetal and adult myoglobin were indistinguishable. 5. A limited survey of the protein patterns of brain, liver and kidney was carried out. In general these tissues show less developmental change than skeletal or cardiac muscle.

Adult↗

Protein profiles associated with survival in lung adenocarcinoma.

Morphologic assessment of lung tumors is informative but insufficient to adequately predict patient outcome. We previously identified transcriptional profiles that predict patient survival, and here we identify proteins associated with patient survival in lung adenocarcinoma. A total of 682 individual protein spots were quantified in 90 lung adenocarcinomas by using quantitative two-dimensional polyacrylamide gel electrophoresis analysis. A leave-one-out cross-validation procedure using the top 20 survival-associated proteins identified by Cox modeling indicated that protein profiles as a whole can predict survival in stage I tumor patients (P = 0.01). Thirty-three of 46 survival-associated proteins were identified by using mass spectrometry. Expression of 12 candidate proteins was confirmed as tumor-derived with immunohistochemical analysis and tissue microarrays. Oligonucleotide microarray results from both the same tumors and from an independent study showed mRNAs associated with survival for 11 of 27 encoded genes. Combined analysis of protein and mRNA data revealed 11 components of the glycolysis pathway as associated with poor survival. Among these candidates, phosphoglycerate kinase 1 was associated with survival in the protein study, in both mRNA studies and in an independent validation set of 117 adenocarcinomas and squamous lung tumors using tissue microarrays. Elevated levels of phosphoglycerate kinase 1 in the serum were also significantly correlated with poor outcome in a validation set of 107 patients with lung adenocarcinomas using ELISA analysis. These studies identify new prognostic biomarkers and indicate that protein expression profiles can predict the outcome of patients with early-stage lung cancer.

Adenocarcinoma↗

Abscence of changes in the protein profile of erythrocyte membranes in Duchenne patients.

Numerous authors have suggested an impairement in protein synthesis as a possible cause for the failure in growth with degeneration of the skeletal muscles protein content present in Duchenne muscular dystrophy (DMD). Others have supported the use of in vitro erythrocyte membranes as a model of the sarcolemmal changes in DMD. Very recently, in vitro decrease in protein synthesis of ribosomal proteins of skeletal muscle cells, fibroblasts and erythrocytes of DMD patients have been reported. We studied the protein profile of erythrocytic membranes of 5 patients with confirmed DMD by means of SDS-polyacrylamide discontinuous gels electrophoresis (method with which we detected changes in the electrophoretic profile of erythrocyte membrane proteins induced by myotonizing drugs) and compared it with the profile of 5 healthy subjects of a similar age range. No differences between the two groups were apparent, rising doubts about the appropriatness of the erythrocyte membrane as an in vivo model of molecular changes in DMD and about the limitations of unidimensional electrophoresis as a suitable method for the detection of such changes.

Erythrocyte Membrane↗

Protein profile in bronchoalveolar lavage fluid from patients with sarcoidosis and idiopathic pulmonary fibrosis as revealed by SDS-PAGE electrophoresis and western blot analysis.

So far bronchoalveolar lavage (BAL)-protein in interstitial lung disease (ILD) is evaluated by measuring concentrations of single proteins. Due to the high dilution of most proteins in BAL, analysis of protein profile has been disappointing. This study describes a new method to overcome this problem and to reveal a highly differentiated picture of BAL proteins. Eighteen patients with pulmonary sarcoidosis, 18 patients with idiopathic pulmonary fibrosis (IPF) and 22 patients with no clinical, roentgenologic or functional evidence of ILD underwent BAL. Total and differential cell count was performed. Normal values for the control group, a lymphocytic alveolitis in sarcoidosis and a granulocytic alveolitis in IPF-patients were found. Median total protein concentration in sarcoidosis showed an increase five times higher than that of the controls (150 mg 1(-1) and 27 mg 1(-1), respectively) with p < 0.001, IPF protein concentration (58 mg 1(-1)) exceeded twice the control values (0.01 > p > 0.001). Analysis of electrophoretic protein profile in controls with Western blot analysis and the biotin/streptavidin staining system revealed a highly differentiated range of bands. Staining with immunoglobulin antibody identified six bands. Four proteins with molecular weight < 21.000 dalton were present only in sarcoidosis patients. These proteins may be identical with fragmented serum proteins or different cell mediators detected in alveolar cell supernatants. Furthermore, in sarcoidosis the intensity and number of bands with molecular weight more than 67.000 dalton was increased. This gives strong evidence for an injury of the alveolar membrane integrity in the alveolitis during the course of sarcoidosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Sepsis plasma protein profiling with immunodepletion, three-dimensional liquid chromatography tandem mass spectrometry, and spectrum counting.

Sepsis is a systemic, often fatal inflammatory response whose biochemical pathways are not fully understood and with no single biomarker capable of its reliable prediction. Increased interest in protein profiling to reveal fundamental biochemical events as well as disease diagnosis has grown considerably, largely due to advances in mass spectrometry and related front-end technologies. In this study, patients with sepsis and systemic inflammatory response syndrome (SIRS) were examined using plasma protein profiling following immunodepletion treatment to remove the most abundant proteins, serum albumin, transferrin, haptoglobin, anti-trypsin, IgG, and IgA. These proteins cause significant signal suppression, and their removal allows for lower abundance proteins to be examined through improved ion signal. Analyses after immunodepletion were performed using 3-dimensional reverse phase/strong cation exchange/reverse phase liquid chromatography with electrospray ion trap mass spectrometry (3D LC-MS/MS) and spectrum counting for comparative quantitation. The results revealed a major theme in immune system activity, including activation of the complement and coagulation pathways. Additionally, lipid transport may prove to be important in distinguishing sepsis from SIRS. Specifically, significant multi-fold changes were observed in 10 proteins and are now being investigated for the early diagnosis of sepsis.

Biomarkers↗

Depletion of multiple high-abundance proteins improves protein profiling capacities of human serum and plasma.

Systematic detection of low-abundance proteins in human blood that may be putative disease biomarkers is complicated by an extremely wide range of protein abundances. Hence, depletion of major proteins is one potential strategy for enhancing detection sensitivity in serum or plasma. This study compared a recently commercialized HPLC column containing antibodies to six of the most abundant blood proteins ("Top-6 depletion") with either older Cibacron blue/Protein A or G depletion methods or no depletion. In addition, a prototype spin column version of the HPLC column and an alternative prototype two antibody spin column were evaluated. The HPLC polyclonal antibody column and its spin column version are very promising methods for substantially simplifying human serum or plasma samples. These columns show the lowest nonspecific binding of the depletion methods tested. In contrast other affinity methods, particularly dye-based resins, yielded many proteins in the bound fractions in addition to the targeted proteins. Depletion of six abundant proteins removed about 85% of the total protein from human serum or plasma, and this enabled 10- to 20-fold higher amounts of depleted serum or plasma samples to be applied to 2-D gels or alternative protein profiling methods such as protein array pixelation. However, the number of new spots detected on 2-D gels was modest, and most newly visualized spots were minor forms of relatively abundant proteins. The inability to detect low-abundance proteins near expected 2-D staining limits was probably due to both the highly heterogeneous nature of most plasma or serum proteins and masking of many low-abundance proteins by the next series of most abundant proteins. Hence, non2-D methods such as protein array pixelation are more promising strategies for detecting lower abundance proteins after depleting the six abundant proteins.

Animals↗

Changes in serum protein profile of healthy adult Nigerians after three decades.

Serum protein profile was recently determined in 55 healthy adult Nigerians and compared with values obtained 3 decades ago. The total serum protein levels were similar, but an increase in titre was recorded in albumin, alpha 1-globulin and beta-globulin. The gamma-globulin level fell markedly in the present study, suggesting improvement in health care and quality of life in the last 3 decades. Changes seen in the serum protein pattern of healthy adult Nigerians were within the normal range compared with values recorded in other Africans and Black Americans. These changes should be considered in the interpretation of laboratory results.

Adult↗

The application of new software tools to quantitative protein profiling via isotope-coded affinity tag (ICAT) and tandem mass spectrometry: II. Evaluation of tandem mass spectrometry methodologies for large-scale protein analysis, and the application of statistical tools for data analysis and interpretation.

Proteomic approaches to biological research that will prove the most useful and productive require robust, sensitive, and reproducible technologies for both the qualitative and quantitative analysis of complex protein mixtures. Here we applied the isotope-coded affinity tag (ICAT) approach to quantitative protein profiling, in this case proteins that copurified with lipid raft plasma membrane domains isolated from control and stimulated Jurkat human T cells. With the ICAT approach, cysteine residues of the two related protein isolates were covalently labeled with isotopically normal and heavy versions of the same reagent, respectively. Following proteolytic cleavage of combined labeled proteins, peptides were fractionated by multidimensional chromatography and subsequently analyzed via automated tandem mass spectrometry. Individual tandem mass spectrometry spectra were searched against a human sequence database, and a variety of recently developed, publicly available software applications were used to sort, filter, analyze, and compare the results of two repetitions of the same experiment. In particular, robust statistical modeling algorithms were used to assign measures of confidence to both peptide sequences and the proteins from which they were likely derived, identified via the database searches. We show that by applying such statistical tools to the identification of T cell lipid raft-associated proteins, we were able to estimate the accuracy of peptide and protein identifications made. These tools also allow for determination of the false positive rate as a function of user-defined data filtering parameters, thus giving the user significant control over and information about the final output of large-scale proteomic experiments. With the ability to assign probabilities to all identifications, the need for manual verification of results is substantially reduced, thus making the rapid evaluation of large proteomic datasets possible. Finally, by repeating the experiment, information relating to the general reproducibility and validity of this approach to large-scale proteomic analyses was also obtained.

Amino Acid Sequence↗