Jinsam You

Design, Implementation and Multisite Evaluation of a System Suitability Protocol for the Quantitative Assessment of Instrument Performance in Liquid Chromatography-Multiple Reaction Monitoring-MS (LC-MRM-MS)

Multiple reaction monitoring (MRM) mass spectrometry coupled with stable isotope dilution (SID) and liquid chromatography (LC) is increasingly used in biological and clinical studies for precise and reproducible quantification of peptides and proteins in complex sample matrices. Robust LC-SID-MRM-MS-based assays that can be replicated across laboratories and ultimately in clinical laboratory settings require standardized protocols to demonstrate that the analysis platforms are performing adequately. We developed a system suitability protocol (SSP), which employs a predigested mixture of six proteins, to facilitate performance evaluation of LC-SID-MRM-MS instrument platforms, configured with nanoflow-LC systems interfaced to triple quadrupole mass spectrometers. The SSP was designed for use with low multiplex analyses as well as high multiplex approaches when software-driven scheduling of data acquisition is required. Performance was assessed by monitoring of a range of chromatographic and mass spectrometric metrics including peak width, chromatographic resolution, peak capacity, and the variability in peak area and analyte retention time (RT) stability. The SSP, which was evaluated in 11 laboratories on a total of 15 different instruments, enabled early diagnoses of LC and MS anomalies that indicated suboptimal LC-MRM-MS performance. The observed range in variation of each of the metrics scrutinized serves to define the criteria for optimized LC-SID-MRM-MS platforms for routine use, with pass/fail criteria for system suitability performance measures defined as peak area coefficient of variation <0.15, peak width coefficient of variation <0.15, standard deviation of RT <0.15 min (9 s), and the RT drift <0.5min (30 s). The deleterious effect of a marginally performing LC-SID-MRM-MS system on the limit of quantification (LOQ) in targeted quantitative assays illustrates the use and need for a SSP to establish robust and reliable system performance. Use of a SSP helps to ensure that analyte quantification measurements can be replicated with good precision within and across multiple laboratories and should facilitate more widespread use of MRM-MS technology by the basic biomedical and clinical laboratory research communities.

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.

Searching for potential biomarkers of cisplatin resistance in human ovarian cancer using a label-free LC/MS-based protein quantification method.

Platinum‐based chemotherapy, such as cisplatin, is the primary treatment for ovarian cancer. However, drug resistance has become a major impediment to the successful treatment of ovarian cancer. To date, the molecular mechanisms of resistance to platinum‐based chemotherapy remain unclear. In this study, we applied an LC/MS‐based protein quantification method to examine the global protein expression of two pairs of ovarian cancer cell lines, A2780/A2780‐CP (cisplatin‐sensitive/cisplatin‐resistant) and 2008/2008‐C13*5.25 (cisplatin‐sensitive/cisplatin‐resistant). We identified and quantified over 2000 proteins from these cell lines and 760 proteins showed significant expression changes with a false discovery rate of less than 5% between paired groups. Based on the results we obtained, we suggest several potential pathways that may be involved in cisplatin resistance in human ovarian cancer. This study provides not only a new proteomic platform for large‐scale quantitative protein analysis, but also important information for discovery of potential biomarkers of cisplatin resistance in ovarian cancer. Furthermore, these results may be clinically relevant for diagnostics, prognostics, and therapeutic improvement for ovarian cancer treatment.

Chk1 phosphorylation of Metnase enhances DNA repair but inhibits replication fork restart.

Chk1 both arrests replication forks and enhances repair of DNA damage by phosphorylating downstream effectors. Although there has been a concerted effort to identify effectors of Chk1 activity, underlying mechanisms of effector action are still being identified. Metnase (also called SETMAR) is a SET and transposase domain protein that promotes both DNA double-strand break (DSB) repair and restart of stalled replication forks. In this study, we show that Metnase is phosphorylated only on Ser495 (S495) in vivo in response to DNA damage by ionizing radiation. Chk1 is the major mediator of this phosphorylation event. We had previously shown that wild-type (wt) Metnase associates with chromatin near DSBs and methylates histone H3 Lys36. Here we show that a Ser495Ala (S495A) Metnase mutant, which is not phosphorylated by Chk1, is defective in DSB-induced chromatin association. The S495A mutant also fails to enhance repair of an induced DSB when compared with wt Metnase. Interestingly, the S495A mutant demonstrated increased restart of stalled replication forks compared with wt Metnase. Thus, phosphorylation of Metnase S495 differentiates between these two functions, enhancing DSB repair and repressing replication fork restart. In summary, these data lend insight into the mechanism by which Chk1 enhances repair of DNA damage while at the same time repressing stalled replication fork restart.

Role of Zinc-Finger Motif in Redox Regulation of Human Replication Protein A

Replication protein A (RPA) is a heterotrimeric zinc-finger protein complex involved in DNA replication, repair, and genetic recombination. Unlike other zinc-finger proteins, RPAs zinc-finger motif is not essential for its single-stranded DNA (ssDNA) binding activity, but is involved in redox regulation of its single-stranded DNA (ssDNA) binding activity. To get an insight into the regulation of RPA-ssDNA interaction, wild-type RPA (wt-RPA) and zinc-finger mutant were examined for ssDNA binding activity using surface plasmon resonance technique. Interaction of wt-RPA with ssDNA under nonreducing conditions was very weak (KD x 2.33 x 10(-8) M) compared with that under reducing conditions (KD = 7.35 x 10(-11) M), whereas ssDNA binding affinity of the zinc-finger mutant was not affected by redox. The divalent ion chelator, o-phenanthroline, significantly reduced wt-RPA-ssDNA interaction, but had no effect on the zinc-finger mutant. The inhibitory effect of o-phenanthroline on RPA-ssDNA interaction was reversed by Zn(II), but not by other divalent cations, suggesting that Zn(II) is the unique metal coordinating the zinc-finger cysteines in redox regulation of RPA-ssDNA interaction. In DNA repair, redox affected RPAs interaction with damaged DNA, but not its role in stabilizing the xeroderma pigmentosum group A (XPA)-damaged DNA complex, suggesting that the zinc-finger motif may mediate the transition of RPA-XPA interaction to a stable RPA-XPA-damaged DNA complex in a redox-dependent manner.

Ethanol Exposure Alters Protein Expression in a Mouse Model of Fetal Alcohol Spectrum Disorders

Alcohol exposure during development can result in variable growth retardation and facial dysmorphology known as fetal alcohol spectrum disorders. Although the mechanisms underlying the disorder are not fully understood, recent progress has been made that alcohol induces aberrant changes in gene expression and in the epigenome of embryos. To inform the gene and epigenetic changes in alcohol-induced teratology, we used whole-embryo culture to identify the alcohol-signature protein profile of neurulating C6 mice. Alcohol-treated and control cultures were homogenized, isoelectrically focused, and loaded for 2D gel electrophoresis. Stained gels were cross matched with analytical software. We identified 40 differentially expressed protein spots (P < 0.01), and 9 spots were selected for LC/MS-MS identification. Misregulated proteins include serotransferrin, triosephosphate isomerase and ubiquitin-conjugating enzyme E2 N. Misregulation of serotransferrin and triosephosphate isomerase was confirmed with immunologic analysis. Alteration of proteins with roles in cellular function, cell cycle, and the ubiquitin-proteasome pathway was induced by alcohol. Several misregulated proteins interact with effectors of the NF-κB and Myc transcription factor cascades. Using a whole-embryo culture, we have identified misregulated proteins known to be involved in nervous system development and function.

Proteomics of Cerebrospinal Fluid: Methods for Sample Processing

Cerebrospinal fluid (CSF) provides an important source of potential biomarkers for brain disorders and therapeutic drug development. Applications of proteomic technology to the identification and quantification of proteins in CSF are increasing rapidly. Key to obtaining reproducible and reliable data about protein levels in CSF are standardization of methods for sample collection, storage, and subsequent sample processing. Methods are described here for all steps of sample processing for a number of different proteomic approaches.

Mass Spectrometry for Protein Quantification in Biomarker Discovery

Major technological advances have made proteomics an extremely active field for biomarker discovery in recent years due primarily to the development of newer mass spectrometric technologies and the explosion in genomic and protein bioinformatics. This leads to an increased emphasis on larger scale, faster, and more efficient methods for detecting protein biomarkers in human tissues, cells, and biofluids. Most current proteomic methodologies for biomarker discovery, however, are not highly automated and are generally labor-intensive and expensive. More automation and improved software programs capable of handling a large amount of data are essential to reduce the cost of discovery and to increase throughput. In this chapter, we discuss and describe mass spectrometry-based proteomic methods for quantitative protein analysis.

Utility of formalin-fixed, paraffin-embedded liver biopsy specimens for global proteomic analysis in nonalcoholic steatohepatitis.

Purpose: To compare the proteomic profiles of formalin‐fixed, paraffin‐embedded (FFPE) liver biopsy material and matched frozen liver tissue from patients with nonalcoholic steatohepatitis (NASH).

Protein Target Quantification Decision Tree

The utility of mass spectrometry-(MS-) based proteomic platforms and their clinical applications have become an emerging field in proteomics in recent years. Owing to its selectivity and sensitivity, MS has become a key technological platform in proteomic research. Using this platform, a large number of potential biomarker candidates for specific diseases have been reported. However, due to lack of validation, none has been approved for use in clinical settings by the Food and Drug Administration (FDA). Successful candidate verification and validation will facilitate the development of potential biomarkers, leading to better strategies for disease diagnostics, prognostics, and treatment. With the recent new developments in mass spectrometers, high sensitivity, high resolution, and high mass accuracy can be achieved. This greatly enhances the capabilities of protein biomarker validation. In this paper, we describe and discuss recent developments and applications of targeted proteomics methods for biomarker validation.