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PubMed2004

Protein profiling in respiratory disease: techniques and impact.

Multifactorial diseases such as respiratory disease call for a global analysis of such disorders. Recent advances in protein profiling techniques may allow for early diagnosis of respiratory disease, which is crucial for intervention and treatment. In order to reduce false-positive rates, clinical diagnosis requires a high degree of sensitivity and specificity to be an effective screening tool. Protein profiles identified by ProteinChip (Ciphergen Biosystems) technology coupled with mass spectrometry affords a global analysis of clinical samples and is beginning to reach acceptable levels of sensitivity and specificity. Combining the profile with another diagnostic tool enhances the effectiveness of protein profiles to classify disease. Although current efforts have centered on serum protein profiling, the local environment of the lung may be better reflected in proteins of bronchoalveolar lavage or sputum. Identification of biomarkers of disease by protein profiling analyses may lead to an understanding of the mechanisms of this disease and contribute to the discovery of new therapeutics for the prevention and treatment of disease. Advancing these analyses are techniques such as ProteinChip mass spectrometry, laser capture microdissection, tissue microarrays and fluorescently labeled antibody bead arrays, which enable the direct global analysis of complex mixtures. Effective high-throughput and ease of use of clinical testing will arrive with improvements in bioinformatics and decreases in instrumentation costs.

Antibodies↗

PubMed2001

Toward proteomics in uroscopy: urinary protein profiles after radiocontrast medium administration.

Previous attempts to use urinary protein profiles for diagnostic purposes have been rather disappointing with respect to their clinical validity, in part because of the insufficient reproducibility, sensitivity, and rapidity of available techniques. Therefore, a newly developed, high-throughput technique, namely surface-enhanced laser desorption/ionization (SELDI) ProteinChip array-time of flight mass spectrometry, was studied, to assess its applicability for protein profiling of urine and to exemplify its use for a group of patients receiving radiocontrast medium. Assessment of the accuracy, sensitivity, and reproducibility of SELDI in test urinary protein profiling was performed. Renal function was studied in 20 male Sprague-Dawley rats before and after intravenous administration of either 1.25 g/kg ioxilan (n = 10) or hypertonic saline solution (n = 10) as a control. Urine samples from 25 patients undergoing cardiac catheterization were obtained before, immediately after, and 6 to 12 h after the procedure. Administration of ioxilan to rats resulted in changes in the abundance of proteins of 9.9, 18.7, 21.0, and 66.3 kD. For patients, even in uncomplicated cases of radiocontrast medium infusion during cardiac catheterization, perturbations in the protein composition occurred but returned to baseline values after 6 to 12 h. Proteins with molecular masses of 9.75, 11.75, 23.5, and 66.4 kD changed in abundance. For patients with impaired renal function, these changes were not reversible within 6 to 12 h. As a proof of principle, one of the peaks, i.e., that at 11.75 kD, was identified as beta(2)-microglobulin. SELDI is a promising tool for the detection, identification, and characterization of trace amounts of proteins in urine. Even for patients without renal complications, proteins with a broad range of molecular masses either appear in or disappear from the urine. Some of these might represent markers of impending nephropathy.

Animals↗

PubMed2005

[Interest of performing systematically the protein profile in an internal medicine department. National survey and prospective study on 229 admissions].

INTRODUCTION: Despite a wide national use, the usefulness of the protein profile has only been evaluated in a small number of studies, essentially in patients with unknown diagnoses. METHODS: We conducted a survey on 339 french internal medicine departments to evaluate how the protein profile was used in these services. Concomitantly we achieved a prospective study on 229 patients in our department, with a mean follow up of 9 months, to evaluate how did the protein profile influence the diagnosis process. RESULTS: We received 183 responses to our national survey: the protein profile was available in 110/183 (60%) departments with 94/110 (85%) using it during hospitalisation and 20/94 (21.3%) using it systematically. Among the 229 protein profile analysed in our department, 44 (19.2%) were considered useful with 20 (8.7%) of them allowing the diagnosis of a new pathology. If the profile had not been done systematically, the physicians of our department would have performed the profile in 102/229 (44.5%) cases, whereas seven (3%) useful profiles would not have been done. CONCLUSION: We think that the profile has a consistent interest in hospitalized patients with a known or unknown pathology but performing systematically such a test appears to be of limited benefit.

Blood Proteins↗

PubMedDate not supplied

Direct tandem mass spectrometry reveals limitations in protein profiling experiments for plasma biomarker discovery.

The low molecular weight plasma proteome and its biological relevance are not well defined; therefore, experiments were conducted to directly sequence and identify peptides observed in plasma and serum protein profiles. Protein fractionation, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) profiling, and liquid-chromatography coupled to MALDI tandem mass spectrometry (MS/MS) sequencing were used to analyze the low molecular weight proteome of heparinized plasma. Four fractionation techniques using functionally derivatized 96-well plates were used to extract peptides from plasma. Tandem TOF was successful for identifying peptides up to m/z 5500 with no prior knowledge of the sequence and was also used to verify the sequence assignments for larger ion signals. The peptides (n>250) sequenced in these profiles came from a surprisingly small number of proteins (n approximately 20), which were all common to plasma, including fibrinogen, complement components, antiproteases, and carrier proteins. The cleavage patterns were consistent with those of known plasma proteases, including initial cleavages by thrombin, plasmin and complement proteins, followed by aminopeptidase and carboxypeptidase activity. On the basis of these data, we discuss limitations in biomarker discovery in the low molecular weight plasma or serum proteome using crude fractionation coupled to MALDI-MS profiling.

Biomarkers↗

PubMed1994

Bacterial whole cell protein profiles of the rRNA group II pseudomonads.

Studies on bacterial whole cell protein profiles showed that members of the rRNA group II pseudomonads were distinct from other non-fluorescent and fluorescent pseudomonads, including Pseudomonas aeruginosa, the type species of the genus Pseudomonas. Strains of Ps. andropogonis, Ps. caryophylli, Ps. gladioli pv. gladioli, Ps. pickettii, Ps. pseudomallei and Ps. rubrisubalbicans showed uniform and distinct protein patterns, while strains of Ps. solanacearum and Ps. cepacia displayed differences within species. Numerical analysis of their protein profiles with GelManager and Taxan programs generated dendrograms comprising 16 clusters at 89% similarity. Each cluster included strains belonging to the same species with the exception of Ps. solanacearum, which fragmented into three clusters. Pseudomonas solanacearum showed different protein patterns correlating with different biovars and the two divisions of Cook et al. (1989), as well as the results of 16S rRNA gene sequencing. The whole cell protein profiles of a total of 83 strains belonging to 14 bacterial species were numerically analysed.

Bacterial Proteins↗

PubMed2001

Antibody fusions with fluorescent proteins: a versatile reagent for profiling protein expression.

We developed a system by which antibodies, fused to fluorescent proteins with different wavelengths, can be prepared within a month against various antigens. An antibody library composed of a large number of single-chain Fv-CL fragment was constructed by means of a phage-display system. The constructs were designed to facilitate changing of the protein forms by simple enzyme manipulation. In the present study, we adopted a molecular form of antibody in which a single-chain Fv-CL fragment is fused with a green fluorescent protein (GFP) or red fluorescent protein (RFP). In addition, a His-tag was inserted between CL and GFP (or RFP). We describe the utility of this system using Caenorhabditis elegans embryo as a model.

Amino Acid Sequence↗

PubMed2005

Serum proteins profile as an indicator of malignancy: multivariate logistic regression and ROC analyses.

The electrophoretic pattern of serum proteins of 85 patients carrying different types of neoplasia and 85 matched healthy adults were comparatively studied by agarose gel electrophoresis, to find out if there is a specific protein pattern common to different types of cancer. Each protein fraction was analyzed quantitatively by densitometry. Multivariate logistic regression and receiver operating characteristic (ROC) analyses were performed using SPSS software. When total protein and albumin were measured by colorimetric methods, cancer patients, compared to controls, had a decreased concentration of total protein (66.0+/-11.5 g/L vs. 76.4+/-6.8 g/L, p< or =0.0001) and of albumin (39.0+/-8.1 g/L vs. 46.0+/-4.3 g/L, p< or =0.0001). The electrophoretic data of serum proteins showed that the ratio of albumin to globulin (0.92+/-0.30 vs. 1.21+/-0.16, p< or =0.0001), percent of the fractions albumin (46.7+/-8.5% vs. 54.4+/-3.5%, p< or =0.0001) and beta-globulin (11.6+/-4.4% vs. 13.0+/-1.9%, p< or =0.001) were decreased and alpha(1)- (5.3+/-2.5% vs. 2.9+/-0.8%, p< or =0.0001), alpha(2)- (13.5+/-4.8% vs. 11.3+/-2.1%, p< or =0.0001) and gamma-globulins (23.0+/-7.7% vs. 18.3+/-3.1%, p< or =0.0001) were significantly increased in cancer patients relative to controls. Cancer patients also had higher counts of leukocytes (7.98+/-3.11, x10(9) cells/L vs. 6.33+/-1.68 x10(9) cells/L, p< or =0.0001) and erythrocyte sedimentation rate (35.9+/-23.5 mm/h vs. 14.1+/-9.5 mm/h,p< / =0.0001). On the basis of univariate analysis, a protein profile out of the normal ranges was more prevalent in cancer patients than in controls. Analysis of the data using multiple logistic regression indicated that the prevalence of cancer was strongly associated with the serum proteins' profile, and alpha(1)-globulin, erythrocyte sedimentation rate, total protein and the ratio of albumin to globulin were the best parameters to discriminate between malignant and healthy states. The area under the ROC curves were the same for most components of the serum proteins' profile at about 0.75+/-0.09, p< or =0.001. We conclude that the profile of serum proteins indicates high diagnostic values for discriminating between cancer patients and healthy individuals and may be useful as an adjunct diagnosis for detection of malignancy.

Adult↗

PubMed2004

Urine protein profiling with surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry.

BACKGROUND: In the last few years there has been an increasing interest in exploring the human proteome. In particular, efforts have focused on developing strategies to generate reproducible protein maps of normal cells, tissues, and biologic fluids, from which studies can then compare protein expression between different groups (e.g., healthy individuals vs. those with a specific pathologic state). METHODS: Various extrinsic factors (instrument settings, matrix composition, urine storage post void, freeze-thaw cycles) and intrinsic factors (blood in urine, urine dilution, first-void vs. midstream urine) were analyzed with respect to their impact on urine protein profiling using surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). RESULTS: Extrinsic factors that critically influenced reproducibility and peak detection of urine protein profiling were matrix composition and instrument settings, while freeze-thaw cycles had minimal impact. Midstream urines samples did not undergo changes in their protein profile when stored for three days at 4 degrees C. Intrinsic factors that influenced normal urine protein profiling were blood in the urine and urine dilution. Female first-void urine had a significantly different ratio of proteins present compared to a midstream urine sample. Limitations of the SELDI-TOF-MS technique included ion suppression and quantification of individual proteins when protein composition was complex. CONCLUSION: SELDI-TOF-MS offers a unique platform for high throughput urine protein profiling; however, standardization of analysis conditions is critical, and both extrinsic and intrinsic factors must be taken into account for accurate data interpretation.

Female↗

PubMed2000

Protein profiling of rat cerebella during development.

Protein profiles of developing rat cerebella were analyzed by means of two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). The analysis of adult rat cerebellum gave rise to a protein map comprising approximately 3000 spots detectable by silver staining following high resolution 2-DE with a pH range of 3-10 and a mass range of 8-100 kDa. To obtain landmarks for comparison of developmental profiles of cerebellar proteins, 100 spots were subjected to peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and 67 spots were assigned on the map. Analysis of profiles of the developing cerebella revealed significant changes in the expression of proteins during development. In most cases the expression levels of proteins increased as the cerebellum matured, while the expression of 42 spots appeared specific or remarkably abundant in the immature cerebellum. Peptide mass fingerprinting of these spots allowed us to identify 29 proteins, which include, in addition to proteins of unknown function, many proteins known to have roles in the development of the central nervous system. These results suggest that the proteomic approach is valuable for mass identification of proteins involved in cerebellar morphogenesis.

Animals↗

PubMedDate not supplied

The antigenic composition and protein profiles of eumycetoma agents.

The protein profiles of different eumycetoma agents were compared by SDS gel electrophoresis. Dendrograms confirmed the homogeneity of isolates of Pseudallescheria boydii but amongst Madurella species, particularly isolates identified as M. grisea, there were substantial differences in protein composition. However using Western blotting reference isolates of the different species showed distinct antigen patterns in response to immune rabbit sera. In particular there was little evidence of cross reactivity between M. mycetomatis and M. grisea. However this specificity was not apparent when human sera from patients with different eumycetoma infections were compared in an ELISA system using the same antigens. It is possible that the formation of a mycetoma grain may limit a patient's exposure to antigens which confer specificity, an explanation which may also account for the variability in antibody responses seen.

Antigens, Fungal↗

PubMed1994

Glycolipid and protein profiles in trypanosomatids.

A comparative study of glycolipid and protein composition in Trypanosoma cruzi and non-pathogenic trypanosomatids was carried out using Triton X-114 extraction. Protein profiles were analysed by polyacrylamide gel electrophoresis in sodium dodecyl sulphate (SDS-PAGE), and glycolipids were detected using high-performance thin-layer chromatography (HPTLC). Hydrophilic protein profiles were similar in non-pathogenic protozoa. Endotrypanum schaudinni, Crithidia luciliae and T. mega showed five characteristic protein bands ranging between 30 and 66 kDa. In the hydrophobic phase, a band of 50 kDa was present only in T. mega. Strain-specific protein distribution was detected in T. cruzi clone Dm28c and T. cruzi G and Y strains; clone Dm28c had five typical hydrophilic proteins at between 24 and 45 kDa, the G strain had two bands at 45 kDa in the hydrophilic phase and the Y strain had a major protein band at 24 kDa in both phases. T. dionisii and T. cruzi clone Dm28c showed a characteristic distribution of three hydrophilic proteins of approx. 45 kDa. Qualitative analysis of glycolipid composition showed that the T. cruzi strains and Dm28c clone and T. dionisii had four orcinol-positive spots, whereas in the other non-pathogenic trypanosomatids only three glycolipids were detected.

Animals↗

PubMed2006

[Application of SELDI-TOF MS technology analyzing serum protein profiling for distinguishing laryngeal carcinoma from ordinary people].

OBJECTIVE: To analyze the spectrometric serum protein profiling of laryngeal carcinoma and healthy controls by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), to establish serum marker pattern for the diagnosis of laryngeal carcinoma. METHOD: The serum samples of 46 cases of laryngeal carcinoma, 51 cases of healthy controls on WCX2 proteinchip, were collected. the spectrometric protein profiling was defected by SELDI-TOF-MS, the data were analyzed by Biomarker Patterns Software provided by Ciphergen Corp. A primary diagnosis model of laryngeal carcinoma was set up. This model was further evaluated by blind test with other l2 cases patients and 13 cases healthy controls. RESULT: Seventy-six protein peaks were detected at the molecular range of 2000-20000 Da., among which 27 ones were significantly different between laryngeal carcinoma and controls( P < 0.05). A diagnostic pattern consisting of 3 protein peaks was established with of accuracy 88.7% (86/97), sensitivity 87% (40/46), specificity 90.2% (46/51), Blind test generated a sensitivity of 83.3% (10/12)and specificity of 84.6% (11/13) respectively. CONCLUSION: It is successful to develop and evaluate the different spectrometric protein profiling patterns of laryngeal carcinoma and healthy control by SELDI-TOF-MS. This affords possibly a new method to early diagnosis of laryngeal carcinoma.

Adult↗

PubMed2006

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry serum protein profiling to identify nasopharyngeal carcinoma.

BACKGROUND: Diagnosis of nasopharyngeal carcinoma (NPC) at an early disease stage is important for successful treatment and improving the outcome of patients. The use of serum protein profiles and a classification tree algorithm were explored to distinguish NPC from noncancer. METHODS: Serum samples were applied to metal affinity protein chips to generate mass spectra by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Protein peak identification and clustering were performed using the Biomarker Wizard software. Proteomic spectra of serum samples from 50 NPC patients and 54 noncancer controls were used as a training set and a classification tree with 6 distinct protein masses was generated by using Biomarker Pattern software. The validity of the classification tree was then challenged with a blind test set including another 20 NPC patients and 25 noncancer controls. RESULTS: The software identified an average of 93 mass peaks/spectrum and 6 of the identified peaks were used to construct the classification tree. The classification tree correctly determined 83% (123 of 149) of the test samples with 83% (58 of 70) of the NPC samples and 82% (65 of 79) of the noncancer samples. In a combination of the serum protein profiles with Epstein-Barr (EBV) nuclear antigen 1 (EBNA1 IgA) test, the diagnostic sensitivity and specificity were increased to 99% and 96%, respectively. CONCLUSIONS: The results suggest that SELDI-TOF-MS serum protein profiles could discriminate NPC from noncancer. The combination of serum protein profiles with an EBV antibody serology test could further improve the accuracy of NPC screening.

Adult↗

PubMed1987

Comparative studies on soluble protein profiles and isozyme patterns of seven Trichinella isolates.

Soluble protein profiles and isozyme patterns of eight enzymes were compared for extracts of muscle stage larvae of the seven Trichinella isolates, using isoelectric focusing in polyacrylamide gel. Soluble protein profiles and isozyme patterns of four enzymes: malic enzyme, glucosephosphate isomerase, phosphoglucomutase, superoxide dismutase of them were clearly divided into four types. T. pseudospiralis from a racoon and the Polar strain from a polar bear formed type 1 and type 2. The Iwasaki strain from a Japanese black bear and the Yamagata strain from a racoon dog, both from Japan, were type 3. Type 4 consisted of three remaining strains, the Polish strain from a wild pig, the USA strain from a pig and the Thai strain from a human case, which have similar infectivities to pigs. The Thai strain varied a bit electrophoretically from other members of type 4. Zymograms of adenylate kinase and malate dehydrogenase were similar in types 2 and 3. The 6-phosphogluconate dehydrogenase zymogram of type 3, similar to that of type 4, was different from that of type 2. It is assumed from the data that type 3 (Japanese strain) was genetically intermediate to types 2 and 4. T. pseudospiralis and the Polar strain had a common main isozyme of 6-phosphogluconate dehydrogenase. The zymogram of lactate dehydrogenase was common except for T. pseudospiralis.

Animals↗

PubMed1997

[Limitations of the protein profile for diagnostic orientation in initial internal medicine consultation. Prospective study on 76 patients].

Determination of the protein profile of orientation (PPO) is now considered by some authors as a means of improving the diagnosis in internal medicine. The feasibility of systematizing this practice was investigated in 76 outpatients (79 included, three excluded secondarily) seen for pathology of undetermined diagnosis. The 79 patients (mean age: 52 years) underwent the classical biological explorations plus PPO. The physicians were divided into two groups (seniors and assistants). Two complete clinical files were established for each patient, with one difference concerning inflammatory and immunologic data: one file included the minimum number of tests considered necessary by the physician and the other the complete PPO (nine proteins). Each file (with or without PPO) was randomly distributed to one of two physicians in the same group. Each physician filled in a diagnostic evaluation sheet indicating whether there was organic pathology or not, the main diagnosis (inflammatory, neoplastic, infectious or other), the secondary diagnosis and the hypothesis of probability. The relevance of the clinical opinion was analyzed by an internal medicine specialist from outside the department with 40 years of clinical experience. The duration of symptoms before the medical visit was from 3 weeks to 5 years (mean 6 months). A diagnosis of organic pathology was reached for three out of four patients. Sixty-seven patients were seen again after a minimum of 6 months, and nine were lost to follow-up. Diagnostic efficiency was no greater for cases with PPO, which appears to be a biological examination of second intention. We suggest that the term "protein profile of orientation" be replaced by "broad protein profile."

Adult↗

PubMed2005

The effect of paternal heat stress on protein profiles of pre-implantation embryos in the mouse.

The study was undertaken to compare the protein profiles of [35S]-methionine-labelled control-sired embryos with heat-sired embryos at 7, 14 or 21 days after mature fertile B6CBF F1 male mice were kept at 36 +/- 0.3 degrees C and 62 +/- 2.7% relative humidity for 24 h. One-dimensional gel electrophoresis and autoradiographs were used to examine the protein profiles between the two-cell embryos and the blastocysts. The results obtained demonstrate that paternal heat stress 7 or 14 days earlier did not apparently affect protein patterns of two-cell embryos, four-cell to eight-cell embryos, morulae or blastocysts. However, 21 days earlier, there were changes in protein patterns of two-cell embryos and abnormal embryos, but not the morulae. To further support and extend these results, two-dimensional gel electrophoresis and phosphorimaging were employed and the results obtained show that paternal heat stress 21 days before mating affected protein profiles of two-cell embryos and morulae in the mouse. Together, these findings have indicated that paternal heat stress affects most but not all protein patterns of pre-implantation embryos, which strongly supports our previous results demonstrating that paternal heat stress significantly reduced the developmental proportion of pre-implantation embryos in the mouse.

Animals↗

PubMed1996

Determination of bacterial protein profiles by matrix-assisted laser desorption/ionization mass spectrometry with high-performance liquid chromatography.

A rapid method for profiling bacterial and cellular proteins has been developed using a combination of capillary high-performance liquid chromatography separation followed by (MALDI-MS) matrix-assisted laser desorption/ionization mass spectrometric analysis. In this method, bacteria are sonicated, the cell walls broken, and the water-soluble proteins precipitated for analysis. The proteins are separated by capillary liquid chromatography and detected on-line by a UV absorption detector. The eluents are then collected for off-line analysis by MALDI-MS. Using this method, it is demonstrated that bacteria can be discriminated based upon their protein profiles to the species level with only pmol level detection of proteins. It has also proved to be a fast and accurate means for monitoring the expression of Hsp27 in an insect cell system.

Animals↗

PubMed2000

Study of protein profile in the visceral leishmaniasis.

A study of the ten proteins of the protein profile in 7 children infected with visceral leishmaniasis leads to obtain an evocating diagram of the protein profile in this disease. In these patients, the authors have found the association of a polyconal hypergammaglobulinaemia, an inflammatory syndrom (increase of orosomucoid, CRP, alpha-1-antitrypsin, decrease of albumin, prealbumin and transferrin), and an haemolytic syndrom (decrease of haptoglobin). Such a diagram may be an supplementary help to establish the diagnosis of visceral leishmaniasis, in some cases when it is difficult to find the parasites.

Blood Proteins↗