Numerous previously uncharacterized molecules resident within the low molecular weight circulatory proteome may provide a picture of the ongoing pathophysiology of an organism. Recently, proteomic ...signatures composed of low molecular weight molecules have been identified using mass spectrometry combined with bioinformatic algorithms. Attempts to sequence and identify the molecules that underpin the fingerprints are currently underway. The finding that many of these low molecular weight molecules may exist bound to circulating carrier proteins affords a new opportunity for fractionation and separation techniques prior to mass spectrometry-based analysis. In this study we demonstrate a method whereby nanoporous substrates may be used for the facile and reproducible fractionation and selective binding of the serum-based biomarker material, including subcellular proteins found within the serum. Aminopropyl-coated nanoporous silicon, when exposed to serum, can deplete serum of proteins and yield a serum with a distinct, altered MS profile. Additionally, aminopropyl-coated, nanoporous controlled-pore glass beads are able to bind a subset of serum proteins and release them with stringent elution. The eluted proteins have distinct MS profiles, gel electrophoresis profiles, and differential peptide sequence identities, which vary based on the size of the nanopores. These material surfaces could be employed in strategies for the harvesting and preservation of labile and carrier-protein-bound molecules in the blood.
We tested the hypothesis that differences in the low molecular weight (500–20.000 Da) proteomic profile of plasma may be detectable between members of a protein C-deficient thrombophilic family who ...have suffered thrombotic events before age <40 compared to family members without a history of venous thrombosis.
Unfractionated plasma samples from members of a previously described large thrombophilic kindred with type I protein C deficiency were applied to ProteinChip weak cation exchange interaction chips (WCX2; Ciphergen Biosystems, Fremont, CA) and subjected to SELDI-TOF (surface-enhanced laser desorption/ionization time-of-flight) mass spectrometry using the Ciphergen PBSII ProteinChip System (Ciphergen Biosystems, Freemont, CA, USA). Profiles were analyzed by a boosted decision-tree algorithm. When individuals who had presented with deep venous thrombosis before the age of 40 (n=21) were compared to age-matched, healthy family members (n=50), the proteomic patterns defined by the decision-tree analysis could classify the entity of DVT before age 40 with 66% sensitivity, at a specificity of 86%. When a small group of cases with history of superficial venous thrombosis (n=6) was added to the case group, the sensitivity was 87.5% at a specificity of 80%. These data support the hypothesis that members of the protein C deficient Vermont kindred II who suffer a thrombotic event before age 40 display significant differences in low-molecular weight proteomics profile compared to those who remain disease-free. This is the first study to apply SELDI-TOF technology in conjunction with a bioinformatics tool to analyze low molecular weight proteomic patterns in patients with venous thrombosis. We are presently pursuing higher resolution analysis with the prOTOF, MALDI orthogonal time of flight mass spectrometer system in order to identify specific peptides
Purpose: There is significant need for well‐characterized antibodies to the spectrum of human proteins encoded by the genome. Advances in tissue‐based proteomic profiling have led to the discovery of ...many candidate molecular biomarkers and therapeutic targets for which development of clinical assays is depending on high quality antibodies. We developed an antibody validation approach for screening of new mAbs.
Experimental design: We utilized a multi‐stage approach of protein array and immunohistochemistry. In the first phase, we screened the NCI60 panel of cell lines by means of protein array and select antibodies based on concordance of mRNA expression to protein array signal. Results of this assay are used to predict antibody titer for immunohistochemistry on the NCI60 cell lines, presented as a tissue microarray. In the final stage, we created a tissue‐based protein expression map by performing immunohistochemistry on a multi‐tumor tissue microarray.
Results: The success rate of this systematic antibody‐screening tool was approximately 93% as measured by the results from the protein array. Data from the NCI60 protein array could be used to predict antibody titer for immunohistochemistry, improving the success rate of immunohistochemical assay development.
Conclusions and clinical relevance: The presented strategy of antibody validation and characterization can be provided a new tool for exploration of human proteome.
We tested the hypothesis that differences in the low-molecular-weight (500-20,000 Da) proteomic profile of plasma may be detectable between members of a protein C-deficient family who have suffered ...thrombotic events before age 40 compared to family members without a history of venous thrombosis. Unfractionated plasma samples from members of a previously described large thrombophilic kindred with type I protein C deficiency were applied to ProteinChip weak cation exchange interaction arrays (WCX2; Ciphergen Biosystems, Fremont, CA, USA) and subjected to SELDI-TOF (surface-enhanced laser desorption/ionization time-of-flight) mass spectrometry using the Ciphergen PBSII ProteinChip System (Ciphergen Biosystems). Profiles were analyzed by a boosted decision-tree algorithm. When individuals who had presented with deep venous thrombosis (DVT) before the age of 40 (n = 21) were compared to age-matched, healthy family members (n = 50), the proteomic patterns defined by the decision-tree analysis could classify the entity of DVT before age 40 with 67% sensitivity, at a specificity of 86%. When a small group of cases with history of superficial venous thrombosis (n = 6) was added to the case group, the sensitivity was 87.5% at a specificity of 80%. These data support the hypothesis that members of the protein C deficient Vermont kindred II who suffer a thrombotic event before age 40 display significant differences in low-molecular-weight proteomics profile compared to those who remain disease-free. This is the first study to apply SELDI-TOF technology in conjunction with a bioinformatics tool to analyze low-molecular-weight proteomic patterns in patients with venous thrombosis.
Both urease-positive and urease-negative Proteeae isolated from cross-infected patients in the same hospitals and, in three cases, from the same patients were examined for their biochemical reactions ...and somatic (O-) antigens. All isolates gave the same reactions in 17 biochemical tests and possessed a-antigens characteristic of Providencia O-type strains 4 or 17. Study of the isolates indicated that endemic strains are capable of undergoing variation in urease activity. In the current classification urease-positive and urease-negative strains are classified as Proteus rettgeri and Providencia stuartii, respectively. The observed variation in urease activity of nosocomial isolates of Proteeae suggests that taxonomy should be modified so that all such strains would be accommodated in a single group.
The somatic (O-) antigens of the type strains of the providencia antigenic scheme were examined for their biochemical reactions and their O-specificities. The scheme of 62 O-antigens was ...reconstituted from 52 original type strains and 10 strains substituted for originals that either were biochemically atypical of the genus or showed inappropriate serological reactions. Thirty-six type strains showed no significant relations with other type strains, and antisera could be used for typing without absorption. Among 26 type strains, significant reciprocal relations were demonstrated, and each cross-reacting antigen was examined for specificity and for its distribution among the type strains. Antisera to these strains required absorption with cell suspensions of other type strains for production of specificity in O-typing. Each typing antiserum, at low dilution, was shown to agglutinate homologous, but not heterologous, cell suspensions of type strains, and this result demonstrated the required specificity for typing on the basis of the O-antigens.
Next generation sequencing (NGS) has rapidly become an invaluable tool for the detection, identification and relative quantification of environmental microorganisms. Here, we demonstrate two new 16S ...rDNA primer sets, which are compatible with NGS approaches and are primarily for use in water quality studies. Compared to 16S rRNA gene based universal primers, in silico and experimental analyses demonstrated that the new primers showed increased specificity for the Cyanobacteria and Proteobacteria phyla, allowing increased sensitivity for the detection, identification and relative quantification of toxic bloom-forming microalgae, microbial water quality bioindicators and common pathogens. Significantly, Cyanobacterial and Proteobacterial sequences accounted for ca. 95% of all sequences obtained within NGS runs (when compared to ca. 50% with standard universal NGS primers), providing higher sensitivity and greater phylogenetic resolution of key water quality microbial groups. The increased selectivity of the new primers allow the parallel sequencing of more samples through reduced sequence retrieval levels required to detect target groups, potentially reducing NGS costs by 50% but still guaranteeing optimal coverage and species discrimination.
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