Biomedical subjects

Vanitha Thulasiraman

Publications and source records attributed to Vanitha Thulasiraman.

5 recordsLinked to original sources

PubMed2006

Reducing protein concentration range of biological samples using solid-phase ligand libraries.

The discovery of specific polypeptides of diagnostic relevance from a biological liquid is complicated by the overall vast number and the large concentration range of all polypeptides/proteins in the sample. Depletion or fractionation methodologies have been used for selectively removing abundant proteins; however, they failed to significantly enrich trace proteins. Here we expand upon a new method that allows the reduction of the protein concentration range within a complex mixture, like neat serum, through the simultaneous dilution of high abundance proteins and the concentration of low abundance ones in a single, simple step. This methodology utilizes solid-phase ligand libraries of large diversity. With a controlled sample-to-ligand ratio it is possible to modulate the relative concentration of proteins such that a large number of peptides or proteins that are normally not detectable by classical analytical methods become, easily detectable. Application of this method for reducing the dynamic range of unfractionated serum is specifically described along with treatment of other biological extracts. Analytical surface enhanced laser desorption/ionization mass spectrometry (SELDI-MS) technology and mono- and two-dimensional electrophoresis (1-DE and 2-DE) demonstrate the increase in the number of proteins detected. Examples linking this approach with additional fractionation methods demonstrate a further increase in the number of detectable species using either the so-called "top down" or "bottom up" approaches for proteomics analysis. By enabling the detection of a greater proportion of polypeptides/proteins within a sample, this method may contribute significantly towards the discovery of new biomarkers of diagnostic relevance.

Chromatography, Ion Exchange↗

PubMed2005

Reduction of the concentration difference of proteins in biological liquids using a library of combinatorial ligands.

The discovery of polypeptides and proteins with relevance to a particular biological state is complicated by their vast number and concentration range in most biological mixtures. Depletion methodologies are frequently used to remove the most abundant species; however, this removal not only fails significantly to enrich trace proteins, it may also nonspecifically deplete them due to their interactions with the removed high-abundance proteins. Here we report a simple-to-use methodology that reduces the protein concentration range of a complex mixture like whole serum through the simultaneous dilution of high-abundance proteins and the concentration of low-abundance proteins. This methodology utilizes solid-phase ligand libraries of immense diversity, generated by "split, couple, recombine" combinatorial chemistry, that are used for affinity-based binding to the proteins of a given mixture. With a controlled sample-to-ligand ratio it is possible to modulate the relative concentration of proteins such that many peptides or proteins that are undetectable by classical analytical methods become easily accessible. The reduction in the dynamic range of unfractionated serum is specifically described along with treatment of other proteomes such as extracts from Escherichia coli, chicken egg white and cell culture supernatant. Mono- and bi-dimensional electrophoresis (1-DE and 2-DE respectively) and surface-enhanced laser desorption/ionization-mass spectrometry (SELDI-TOF-MS) technology demonstrate the reduction in protein concentration range. Combining this approach with additional fractionation methods further increased the number of detectable species.

Adsorption↗

PubMed2004

Identification of serum amyloid a protein as a potentially useful biomarker to monitor relapse of nasopharyngeal cancer by serum proteomic profiling.

PURPOSE: Nasopharyngeal cancer (NPC) is a common cancer in Hong Kong, and relapse can occur frequently. Using protein chip profiling analysis, we aimed to identify serum biomarkers that were useful in the diagnosis of relapse in NPC. EXPERIMENTAL DESIGN: Profiling analysis was performed on 704 sera collected from 42 NPC patients, 39 lung cancer patients, 30 patients with the benign metabolic disorder thyrotoxicosis (TX), and 35 normal individuals (NM). Protein profile in each NPC patient during clinical follow up was correlated with the relapse status. RESULTS: Profiling analysis identified two biomarkers with molecular masses of 11.6 and 11.8 kDa, which were significantly elevated in 22 of 31 (71%) and 21 of 31 (68%) NPC patients, respectively, at the time of relapse (RP) as compared with 11 patients in complete remission (CR; RP versus CR, P = 0.009), 30 TX (RP versus TX, P < 0.001), or 35 NM (RP versus NM, P < 0.001). The markers were also elevated in 16 of 39 (41%) lung cancer patients at initial diagnosis. By tryptic digestion, followed by tandem mass spectrometry fragmentation, the markers were identified as two isoforms of serum amyloid A (SAA) protein. Monitoring the patients longitudinally for SAA level both by protein chip and immunoassay showed a dramatic SAA increase, which correlated with relapse and a drastic fall correlated with response to salvage chemotherapy. Serum SAA findings were compared with those of serum Epstein-Barr virus DNA in three relapsed patients showing a similar correlation with relapse and chemo-response. CONCLUSIONS: SAA could be a useful biomarker to monitor relapse of NPC.

Adult↗

PubMed2004

Simultaneous monitoring of multiple kinase activities by SELDI-TOF mass spectrometry.

Cellular response to the external environment is often controlled by one or more protein kinases. We report a methodology for simultaneously monitoring multiple kinase activities across multiple signal-transduction pathways using ProteinChip Array technology. Based on the addition of specific peptide reporters, kinase activity is detected by the presence of a mass shift of 80 Da (or multiple thereof) corresponding to the addition of one or more phosphate groups. These phosphorylated peptide substrates are then enriched using an immobilized metal affinity capture (IMAC)-Ga array and detected directly by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). SELDI-TOF MS is sensitive, tagless (nonradioactive, nonfluorescent), can be easily multiplexed for the analysis of several different kinases in a single reaction mixture (limited only by the specificity of the kinase for its substrate peptides), and is directly scalable through the use of robotic sample processing. By multiplexing kinase assays, one can dramatically increase the amount of information obtained from rare or volume-limited samples. More important, results reflect closely the complex interrelationships between kinases and show high correlation with in vivo assays.

Enzyme Inhibitors↗

PubMed2002

Differential inhibition of Hsc70 activities by two Hsc70-binding peptides.

The ability of two high-affinity Hsc70-binding peptides [FYQLALT (peptide-Phi) and NIVRKKK (peptide-K)] to differentially inhibit Hsc70-dependent processes in rabbit reticulocyte lysate (RRL) was examined. Both peptide-Phi and peptide-K inhibited chaperone-dependent renaturation of luciferase in RRL. Peptide-Phi, but not peptide-K, blocked Hsp90/Hsc70-dependent transformation of the heme-regulated eIF2 alpha kinase (HRI) into an active, heme-regulatable kinase. In contrast, peptide-K, but not peptide-Phi, inhibited Hsc70-mediated suppression of the activation of mature-transformed HRI. Furthermore, HDJ2 (Human DnaJ homologue 2), but not HDJ1, potentiated the ability of Hsc70 to suppress the activation of HRI in RRL. Mechanistically, peptide-K inhibited, while peptide-Phi enhanced, HDJ2-induced stimulation of Hsc70 ATPase activity in vitro. The data presented support the hypotheses that peptide-Phi acts to inhibit Hsc70 function by binding to the hydrophobic peptide-binding cleft of Hsc70, while peptide-K acts through binding to a site that modulates the interaction of Hsc70 with DnaJ homologues. Overall, the data indicate that peptide-Phi and peptide-K have differential effects on Hsc70 functions under quasi-physiological conditions in RRL, and suggest that therapeutically valuable peptide mimetics can be designed to inhibit specific functions of Hsc70.

Amino Acid Sequence↗