Proteomic Characterization of Cerebrospinal Fluid from Ataxia-Telangiectasia (A-T) Patients Using a LC/MS-Based Label-Free Protein Quantification Technology

Cerebrospinal fluid (CSF) has been used for biomarker discovery of neurodegenerative diseases in humans since biological changes in the brain can be seen in this biofluid. Inactivation of A-T-mutated protein (ATM), a multifunctional protein kinase, is responsible for A-T, yet biochemical studies have not succeeded in conclusively identifying the molecular mechanism(s) underlying the neurodegeneration seen in A-T patients or the proteins that can be used as biomarkers for neurologic assessment of A-T or as potential therapeutic targets. In this study, we applied a high-throughput LC/MS-based label-free protein quantification technology to quantitatively characterize the proteins in CSF samples in order to identify differentially expressed proteins that can serve as potential biomarker candidates for A-T. Among 204 identified CSF proteins with high peptide-identification confidence, thirteen showed significant protein expression changes. Bioinformatic analysis revealed that these 13 proteins are either involved in neurodegenerative disorders or cancer. Future molecular and functional characterization of these proteins would provide more insights into the potential therapeutic targets for the treatment of A-T and the biomarkers that can be used to monitor or predict A-T disease progression. Clinical validation studies are required before any of these proteins can be developed into clinically useful biomarkers

LC/MS-Based Quantitative Proteomic Analysis of Paraffin-Embedded Archival Melanomas Reveals Potential Proteomic Biomarkers Associated with Metastasis

BACKGROUND: Melanoma metastasis status is highly associated with the overall survival of patients; yet, little is known about proteomic changes during melanoma tumor progression. To better understand the changes in protein expression involved in melanoma progression and metastasis, and to identify potential biomarkers, we conducted a global quantitative proteomic analysis on archival metastatic and primary melanomas. METHODOLOGY AND FINDINGS: A total of 16 metastatic and 8 primary cutaneous melanomas were assessed. Proteins were extracted from laser captured microdissected formalin fixed paraffin-embedded archival tissues by liquefying tissue cells. These preparations were analyzed by a LC/MS-based label-free protein quantification method. More than 1500 proteins were identified in the tissue lysates with a peptide ID confidence level of >75%. This approach identified 120 significant changes in protein levels. These proteins were identified from multiple peptides with high confidence identification and were expressed at significantly different levels in metastases as compared with primary melanomas (q-Value<0.05). CONCLUSIONS AND SIGNIFICANCE: The differentially expressed proteins were classified by biological process or mapped into biological system networks, and several proteins were implicated by these analyses as cancer- or metastasis-related. These proteins represent potential biomarkers for tumor progression. The study successfully identified proteins that are differentially expressed in formalin fixed paraffin-embedded specimens of metastatic and primary melanoma

Quantification of cellular NEMO content and its impact on NF-κB activation by genotoxic stress.

NF-κB essential modulator, NEMO, plays a key role in canonical NF-κB signaling induced by a variety of stimuli, including cytokines and genotoxic agents. To dissect the different biochemical and functional roles of NEMO in NF-κB signaling, various mutant forms of NEMO have been previously analyzed. However, transient or stable overexpression of wild-type NEMO can significantly inhibit NF-κB activation, thereby confounding the analysis of NEMO mutant phenotypes. What levels of NEMO overexpression lead to such an artifact and what levels are tolerated with no significant impact on NEMO function in NF-κB activation are currently unknown. Here we purified full-length recombinant human NEMO protein and used it as a standard to quantify the average number of NEMO molecules per cell in a 1.3E2 NEMO-deficient murine pre-B cell clone stably reconstituted with full-length human NEMO (C5). We determined that the C5 cell clone has an average of 4 x 10(5) molecules of NEMO per cell. Stable reconstitution of 1.3E2 cells with different numbers of NEMO molecules per cell has demonstrated that a 10-fold range of NEMO expression (0.6-6x10(5) molecules per cell) yields statistically equivalent NF-κB activation in response to the DNA damaging agent etoposide. Using the C5 cell line, we also quantified the number of NEMO molecules per cell in several commonly employed human cell lines. These results establish baseline numbers of endogenous NEMO per cell and highlight surprisingly normal functionality of NEMO in the DNA damage pathway over a wide range of expression levels that can provide a guideline for future NEMO reconstitution studies

Effects of NEMO amount on NF-κB activation in response to etoposide.

<p>(A) Time course analysis. The indicated 1.3E2 clones were left untreated or treated with 5 μM VP16 for indicated times and analyzed by EMSA. Three independent experiments were performed as above on different days. The NF-kB bands were quantified and fold inductions of NF-κB DNA binding activity were graphed below. Error bars represent S.E.M. (B) Dose response analysis. The indicated 1.3E2 clones were left untreated or treated with the indicated concentration of etoposide (VP16) for 1 hr. The quantification and graph were done as in A.</p

Purification of full length recombinant human NEMO protein.

<p>(A) SDS–PAGE gel stained with Coomassie Brilliant Blue R-250 for the protein purification profile. (B) 10 μl of purified recombinant NEMO proteins were loaded with a serial dilution of BSA at known concentrations (C) GST or GST-NEMO protein was incubated with <i>in vitro</i> translated mock or IKKβ for GST pull-down assay (lane 1–4), or GST or GST-cleaved NEMO protein was incubated with <i>in vitro</i> translated mock or flag-IKKβ for co-immunoprecipitation with anti-NEMO antibody (lane 7–10). Flag-IKKβ in the complexes or inputs (lanes 5 and 6) were separated on SDS-PAGE gel and immunoblotted with flag antibodies.</p