Shotgun proteomics coupled to nanoparticle-based biomarker enrichment reveals a novel panel of extracellular matrix proteins as candidate serum protein biomarkers for early-stage breast cancer detection.

Background: The lack of specificity and high degree of false positive and false negative rates when using mammographic screening for detecting early-stage breast cancer is a critical issue. Blood-based molecular assays that could be used in adjunct with mammography for increased specificity and sensitivity could have profound clinical impact. Our objective was to discover and independently verify a panel of candidate blood-based biomarkers that could identify the earliest stages of breast cancer and complement current mammographic screening approaches.

Quantitative Proteomic Approach for MicroRNA Target Prediction Based on O/O Labeling.

Motivation: Among many large-scale proteomic quantification methods, O/O labeling requires neither specific amino acid in peptides nor label incorporation through several cell cycles, as in metabolic labeling; it does not cause significant elution time shifts between heavy- and light-labeled peptides, and its dynamic range of quantification is larger than that of tandem mass spectrometry-based quantification methods. These properties offer O/O labeling the maximum flexibility in application. However, O/O labeling introduces large quantification variations due to varying labeling efficiency.

Immunoprecipitation and mass spectrometry defines an extensive RBM45 protein-protein interaction network.

The pathological accumulation of RNA-binding proteins (RBPs) within inclusion bodies is a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). RBP aggregation results in both toxic gain and loss of normal function. Determining the protein binding partners and normal functions of disease-associated RBPs is necessary to fully understand molecular mechanisms of RBPs in disease.

In Vitro Transcription/Translation System: A Versatile Tool in the Search for Missing Proteins.

Approximately 18% of all human genes purported to encode proteins have not been directly evidenced at the protein level, according to the validation criteria established by neXtProt, and are considered to be "missing" proteins. One of the goals of the Chromosome-Centric Human Proteome Project (C-HPP) is to identify as many of these missing proteins as possible in human samples using mass spectrometry-based methods. To further this goal, a consortium of C-HPP teams (chromosomes 5, 10, 16, and 19) has joined forces to devise new strategies to identify missing proteins by use of a cell-free in vitro transcription/translation system (IVTT).

Accurate LC peak boundary detection for ¹⁶O/¹⁸O labeled LC-MS data.

In liquid chromatography-mass spectrometry (LC-MS), parts of LC peaks are often corrupted by their co-eluting peptides, which results in increased quantification variance. In this paper, we propose to apply accurate LC peak boundary detection to remove the corrupted part of LC peaks. Accurate LC peak boundary detection is achieved by checking the consistency of intensity patterns within peptide elution time ranges.

Isolation and characterization of muscle fatigue substance with anti-tumor activities.

Research during the 1950's indicated that exercise played a role in the reduction of tumor growth. In the 1960's our studies confirmed that tumor-bearing rats, exercised to fatigue, demonstrated tumor inhibition. Our further studies isolated an extract (Fatigue Substance, or F-Substance) from rectus femoris muscles of rats which had been electrically stimulated to fatigue.

Differential effects of AKT1(p.E17K) expression on human mammary luminal epithelial and myoepithelial cells.

Recently, we identified a somatic mutation in AKT1, which results in a glutamic acid to lysine substitution (p.Glu17Lys or E17K). E17K mutations appear almost exclusively in breast cancers of luminal origin.

Targeted quantitative analysis of superoxide dismutase 1 in cisplatin-sensitive and cisplatin-resistant human ovarian cancer cells.

Protein quantification in a complex protein mixture presents a daunting task in biochemical analysis. Antibody-based immunoassays are traditional methods for protein quantification. However, there are issues associated with accuracy and specificity in these assays, especially when the changes are small (e.

Repeatability and reproducibility in proteomic identifications by liquid chromatography-tandem mass spectrometry.

The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random.

Interlaboratory study characterizing a yeast performance standard for benchmarking LC-MS platform performance.

Optimal performance of LC-MS/MS platforms is critical to generating high quality proteomics data. Although individual laboratories have developed quality control samples, there is no widely available performance standard of biological complexity (and associated reference data sets) for benchmarking of platform performance for analysis of complex biological proteomes across different laboratories in the community. Individual preparations of the yeast Saccharomyces cerevisiae proteome have been used extensively by laboratories in the proteomics community to characterize LC-MS platform performance.

Performance metrics for liquid chromatography-tandem mass spectrometry systems in proteomics analyses.

A major unmet need in LC-MS/MS-based proteomics analyses is a set of tools for quantitative assessment of system performance and evaluation of technical variability. Here we describe 46 system performance metrics for monitoring chromatographic performance, electrospray source stability, MS1 and MS2 signals, dynamic sampling of ions for MS/MS, and peptide identification. Applied to data sets from replicate LC-MS/MS analyses, these metrics displayed consistent, reasonable responses to controlled perturbations.

Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma.

Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated. We describe a multilaboratory study to assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRM-based assays, conducted by NCI-CPTAC.

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.

Label-free mass spectrometry-based protein quantification technologies in proteomic analysis.

Major technological advances have made proteomics an extremely active field for biomarker discovery and validation in recent years. These improvements have lead to an increased emphasis on larger scale, faster and more efficient methods for protein biomarker discoveries in human tissues, cells and biofluids. However, most current proteomic methodologies for biomarker discovery and validation are not highly automated and generally labour intensive and expensive.

A multiple reaction monitoring method for absolute quantification of the human liver alcohol dehydrogenase ADH1C1 isoenzyme.

Although significant progress has been made in protein quantification using mass spectrometry during recent years, absolute protein quantification in complex biological systems remains a challenging task in proteomics. The use of stable isotope-labeled standard peptide is the most commonly used strategy for absolute quantification, but it might not be suitable in all instances. Here we report an alternative strategy that employs a stable isotope-labeled intact protein as an internal standard to absolutely quantify the alcohol dehydrogenase (ADH) expression level in a human liver sample.

Microdeposition device interfacing capillary electrochromatography and microcolumn liquid chromatography with matrix-assisted laser desorption/ionization mass spectrometry.

A sample deposition device has been constructed and optimized for interfacing CEC and capillary LC columns to MALDI mass spectrometry. For CEC analysis, the device is composed of an inlet buffer reservoir and an outlet buffer reservoir connected to a matrix reservoir through a connection sleeve. The matrix reservoir is connected to a deposition capillary via another connection sleeve.

Surfactant-mediated capillary electrochromatography with octadecyl-silica- packed capillary columns for the separation of nonpolar compounds. Case of pyrethroid insecticides.

Capillary electrochromatography (CEC) with octadecyl-silica-packed capillary columns was evaluated in the separation of nonpolar compounds, e.g., pyrethroid insecticides, using surfactant-rich mobile phases.

On-column trace enrichment by sequential frontal and elution electrochromatography II. Enhancement of sensitivity by segmented capillaries with z-cell configuration--application to the detection of dilute samples of moderately polar and nonpolar pesticides.

An on-column trace enrichment method for capillary electrochromatography of dilute samples is described. It involves the sequential use of frontal and elution electrochromatography on a segmented capillary column comprising of two contiguous segments each packed with a different sorbent. While the entering segment is for preconcentration by frontal electrochromatography the second segment is much longer and is meant for separation of the enriched analytes in the subsequent elution electrochromatography step.