An analysis of all US Food and Drug Administration (FDA) approvals for protein-based assays through 2008 reveals 109 unique protein targets in plasma or serum, as well as 62 additional tests for ...peptides, protein posttranslational modifications, protein complexes, autoantibodies against endogenous proteins, and blood cell proteins. A further 96 unique protein targets are assayed in plasma by laboratory-developed tests available for clinical use in the US, yielding a total of 205 proteins that include products of approximately 211 genes (excluding immunoglobulins). These tests provide quantitative measurements for approximately 1% of the human protein gene products, defining a practical clinical plasma proteome. The rate of introduction of new protein analytes has remained essentially flat over the past 15 years, averaging 1.5 new proteins per year (median of 1 per year). This rate falls far short of that needed to support projected medical needs and indicates serious deficiencies in the protein biomarker pipeline, from which no proteomics-discovered analytes have yet emerged.
•We use surveys of husbands and wives in Tanzania to explore rural household decision-making.•We find perceptions of household decision-making authority differ depending on the spouse asked.•Factors ...associated with a wife’s authority include age, education, health, children, and labor hours.•The allocation of intra-household authority also varies across thirteen different decision questions.•A lack of “intra-household accord” over authority may be a barrier to empowerment efforts.
We use OLS and logistic regression to investigate variation in husband and wife perspectives on the division of authority over agriculture-related decisions within households in rural Tanzania. Using original data from husbands and wives (interviewed separately) in 1,851 Tanzanian households, the analysis examines differences in the wife’s authority over 13 household and farming decisions. The study finds that the level of decision-making authority allocated to wives by their husbands, and the authority allocated by wives to themselves, both vary significantly across households. In addition to commonly considered assets such as women’s age and education, in rural agricultural households women’s health and labor activities also appear to matter for perceptions of authority. We also find husbands and wives interviewed separately frequently disagree with each other over who holds authority over key farming, family, and livelihood decisions. Further, the results of OLS and logistic regression suggest that even after controlling for various individual, household, and regional characteristics, husband and wife claims to decision-making authority continue to vary systematically by decision—suggesting that decision characteristics themselves also matter. The absence of spousal agreement over the allocation of authority (i.e., a lack of “intra-household accord”) over different farm and household decisions is problematic for interventions seeking to use survey data to develop and inform strategies for reducing gender inequalities or empowering women in rural agricultural households. Findings provide policy and program insights into when studies interviewing only a single spouse or considering only a single decision may inaccurately characterize intra-household decision-making dynamics.
The key concept of proteomics (looking at many proteins at once) opens new avenues in the search for clinically useful biomarkers
of disease, treatment response and ageing. As the number of proteins ...that can be detected in plasma or serum (the primary
clinical diagnostic samples) increases towards 1000, a paradoxical decline has occurred in the number of new protein markers
approved for diagnostic use in clinical laboratories. This review explores the limitations of current proteomics protein discovery
platforms, and proposes an alternative approach, applicable to a range of biological/physiological problems, in which quantitative
mass spectrometric methods developed for analytical chemistry are employed to measure limited sets of candidate markers in
large sets of clinical samples. A set of 177 candidate biomarker proteins with reported associations to cardiovascular disease
and stroke are presented as a starting point for such a âdirected proteomicsâ approach.
Donors and governments increasingly seek to deliver development projects through community‐based organizations such as self‐help groups (SHGs), but little is known about the effectiveness of such ...arrangements. This article briefly summarizes hypotheses regarding the effectiveness of interventions using SHGs and presents the results of an evidence review on the impacts of interventions delivered through SHGs on health, finance, agriculture and empowerment outcomes in South Asia and sub‐Saharan Africa. Though the impacts of SHG‐based interventions are generally positive, the evidence base is limited and does not generally test whether alternative delivery mechanisms might be more effective.
Quantitative LC-MS/MS assays were designed for tryptic peptides representing 53 high and medium abundance proteins in human plasma using a multiplexed multiple reaction monitoring (MRM) approach. Of ...these, 47 produced acceptable quantitative data, demonstrating within-run coefficients of variation (CVs) (n = 10) of 2–22% (78% of assays had CV <10%). A number of peptides gave CVs in the range 2–7% in five experiments (10 replicate runs each) continuously measuring 137 MRMs, demonstrating the precision achievable in complex digests. Depletion of six high abundance proteins by immunosubtraction significantly improved CVs compared with whole plasma, but analytes could be detected in both sample types. Replicate digest and depletion/digest runs yielded correlation coefficients (R2) of 0.995 and 0.989, respectively. Absolute analyte specificity for each peptide was demonstrated using MRM-triggered MS/MS scans. Reliable detection of L-selectin (measured at 0.67 μg/ml) indicates that proteins down to the μg/ml level can be quantitated in plasma with minimal sample preparation, yielding a dynamic range of 4.5 orders of magnitude in a single experiment. Peptide MRM measurements in plasma digests thus provide a rapid and specific assay platform for biomarker validation, one that can be extended to lower abundance proteins by enrichment of specific target peptides (stable isotope standards and capture by anti-peptide antibodies (SISCAPA)).
Mass spectrometry-based multiple reaction monitoring (MRM) quantitation of proteins can dramatically impact the discovery and quantitation of biomarkers via rapid, targeted, multiplexed protein ...expression profiling of clinical samples. A mixture of 45 peptide standards, easily adaptable to common plasma proteomics work flows, was created to permit absolute quantitation of 45 endogenous proteins in human plasma trypsin digests. All experiments were performed on simple tryptic digests of human EDTA-plasma without prior affinity depletion or enrichment. Stable isotope-labeled standard peptides were added immediately following tryptic digestion because addition of stable isotope-labeled standard peptides prior to trypsin digestion was found to generate elevated and unpredictable results. Proteotypic tryptic peptides containing isotopically coded amino acids (13C6Arg or 13C6Lys) were synthesized for all 45 proteins. Peptide purity was assessed by capillary zone electrophoresis, and the peptide quantity was determined by amino acid analysis. For maximum sensitivity and specificity, instrumental parameters were empirically determined to generate the most abundant precursor ions and y ion fragments. Concentrations of individual peptide standards in the mixture were optimized to approximate endogenous concentrations of analytes and to ensure the maximum linear dynamic range of the MRM assays. Excellent linear responses (r > 0.99) were obtained for 43 of the 45 proteins with attomole level limits of quantitation (<20% coefficient of variation) for 27 of the 45 proteins. Analytical precision for 44 of the 45 assays varied by <10%. LC-MRM/MS analyses performed on 3 different days on different batches of plasma trypsin digests resulted in coefficients of variation of <20% for 42 of the 45 assays. Concentrations for 39 of the 45 proteins are within a factor of 2 of reported literature values. This mixture of internal standards has many uses and can be applied to the characterization of trypsin digestion kinetics and plasma protein expression profiling because 31 of the 45 proteins are putative biomarkers of cardiovascular disease.
Lipoprotein-associated phospholipase A2 (Lp-PLA
), an enzyme associated with inflammation, is used as a biomarker for cardiovascular disease risk. Both the concentration and activity of Lp-PLA
have ...been shown to be clinically relevant. However, there is a discordance between the serum concentration of Lp-PLA
measured by the standard ELISA-based immunoassays and the activity of this enzyme, leading to substantial discordance in risk categorization depending on assay format.
We developed 2 LC-MS/MS-based assays to quantify serum Lp-PLA
activity (multiple reaction monitoring detection of product) and concentration stable isotope standards and capture by antipeptide antibody (SISCAPA) immunoaffinity, and we investigated their correlation to commercially offered colorimetric activity and immunometric concentrations assays. Associations between Lp-PLA
and lipoproteins and the effect of selected detergents in liberating Lp-PLA
were evaluated by use of immunoprecipitation and Western blot analyses.
Serum Lp-PLA
concentrations measured by quantitative SISCAPA-mass spectrometry were substantially higher than concentrations typically measured by immunoassay and showed an improved agreement with Lp-PLA
activity. With detergents, liberation of Lp-PLA
from lipoprotein complexes dramatically increased the amount of protein detected by immunoassay and improved the agreement with activity measurements.
Quantitative analysis of Lp-PLA
concentration and activity by LC-MS/MS assays provided key insight into resolving the well-documented discordance between Lp-PLA
concentration (determined by immunoassay) and activity. Quantitative detection of Lp-PLA
by immunoassay appears to be strongly inhibited by interaction of Lp-PLA
with lipoprotein. Together, the results illustrate the advantages of quantitative LC-MS/MS for measurement of Lp-PLA
concentration (by SISCAPA) and activity (by direct product detection).
There is an urgent need for quantitative assays in verifying and validating the large numbers of protein biomarker candidates produced in modern “-omics” experiments. Stable isotope standards with ...capture by anti-peptide antibodies (SISCAPA) has shown tremendous potential to meet this need by combining peptide immunoaffinity enrichment with quantitative mass spectrometry. In this study, we describe three significant advances to the SISCAPA technique. First, we develop a method for an automated magnetic bead-based platform capable of high throughput processing. Second, we implement the automated method in a multiplexed SISCAPA assay (nine targets in one assay) and assess the performance characteristics of the multiplexed assay. Using the automated, multiplexed platform, we demonstrate detection limits in the physiologically relevant ng/ml range (from 10 µl of plasma) with sufficient precision (median coefficient of variation, 12.6%) for quantifying biomarkers. Third, we demonstrate that enrichment of peptides from larger volumes of plasma (1 ml) can extend the limits of detection to the low pg/ml range of protein concentration. The method is generally applicable to any protein or biological specimen of interest and holds great promise for analyzing large numbers of biomarker candidates.
Cancer has profound effects on gene expression, including a cell's glycosylation machinery. Thus, tumors produce glycoproteins that carry oligosaccharides with structures that are markedly different ...from the same protein produced by a normal cell. A single protein can have many glycosylation sites that greatly amplify the signals they generate compared with their protein backbones.
In this article, we survey clinical tests that target carbohydrate modifications for diagnosing and treating cancer. We present the biological relevance of glycosylation to disease progression by highlighting the role these structures play in adhesion, signaling, and metastasis and then address current methodological approaches to biomarker discovery that capitalize on selectively capturing tumor-associated glycoforms to enrich and identify disease-related candidate analytes. Finally, we discuss emerging technologies--multiple reaction monitoring and lectin-antibody arrays--as potential tools for biomarker validation studies in pursuit of clinically useful tests.
The future of carbohydrate-based biomarker studies has arrived. At all stages, from discovery through verification and deployment into clinics, glycosylation should be considered a primary readout or a way of increasing the sensitivity and specificity of protein-based analyses.
Clinical Pulse-Labeling of Plasma Proteins and Sample Analysis Recently, a unique pulse-chase study was described in which blood samples collected from human subjects infused with stable isotope ...labeled leucine were analyzed using an immunoaffinity mass-spectrometric approach to determine protein turnover rates (3). Importantly, of the 24 proteins, 19 had turnover rates shorter than 5 days, and of those, 8 proteins approximately 1 day or less. ...for proteins with shorter half-lives, accurate measurement of variations in protein con- centration on a personal level would require a daily sampling frequency. Author Contributions: Allauthors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation ofdata; (b) drafting or revising the article for intellectual content; (c) final approval ofthe published article; and (d) agreement to be accountable for all aspects ofthe article thus ensuringthatquestions related to the accuracy or integrity ofanypart ofthe article are appropriately investigated and resolved. 1 SISCAPA Assay Technologies, Inc. Washington, DC 2 Biomedicine Design, Worldwide Research & Development, Pfizer Inc. Andover, MA * Address correspondence to this author at: SISCAPA Assay Technologies, Inc. Box 53309 Washington, DC 20009 Fax 250-721-8855 E-mail mrazavi@siscapa.com † M. Razavi and V. Farrokhi contributed equally to this work.
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