Article

Development of a Multiplex Selected Reaction Monitoring Assay for Quantification of Biochemical Markers of Down Syndrome in Amniotic Fluid Samples

Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Journal of Proteome Research (Impact Factor: 5). 06/2012; 11(7):3880-7. DOI: 10.1021/pr300355a
Source: PubMed

ABSTRACT Down syndrome (DS) is one of the most common chromosomal abnormalities affecting about 1 of every 700 fetuses. Current screening strategies have detection rates of 90-95% at a 5% false positive rate. The aim of this study was to discover new biomarkers of DS in amniotic fluid by using a multiplex selected reaction monitoring assay. Nine proteins were analyzed: CEL, CPA1, MUC13, CLCA1, MUC5AC, PLUNC, and HAPLN1, and CGB as positive control and serotransferrin as negative control. One proteotypic peptide for each protein was selected, and internal heavy isotope-labeled peptide standards were spiked into the samples. Fifty-four samples from pregnant women carrying normal (n = 37) or DS-affected (n = 17) fetuses were analyzed. The median protein concentrations for DS and normal samples, respectively, were as follows: 20 and 49 ng/mL (p < 0.01) for CEL; 3.7 and 14 ng/mL (p < 0.001) for CPA1; 80 and 263 ng/mL (p < 0.001) for MUC13; 46 and 135 ng/mL (p < 0.001) for CLCA1; 0.65 and 0.93 μg/mL (p < 0.05) for MUC5AC; 61 and 73 ng/mL (p > 0.05) for PLUNC; 144 and 86 ng/mL (p < 0.01) for HAPLN1; 0.89 and 0.54 μg/mL (p = 0.05) for CGB; 91 and 87 μg/mL (p > 0.05) for serotransferrin. Statistically significant differences were found in six out of the seven candidate proteins analyzed, reflecting a different regulation in DS.

Download full-text

Full-text

Available from: Eduardo Martínez-Morillo, Dec 15, 2014
0 Followers
 · 
151 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Protein biomarker development is a multidisciplinary task involving basic, translational and clinical research. Integration of multidisciplinary efforts in a single pipeline is challenging, but crucial to facilitate rational discovery of protein biomarkers and alleviate existing disappointments in the field. In this review, we discuss in detail individual phases of biomarker development pipeline, such as biomarker candidate identification, verification and validation. We focus on mass spectrometry as a principal technique for protein identification and quantification, and discuss complementary -omics approaches for selection of biomarker candidates. Proteomic samples, protein-based clinical laboratory tests and limitations of biomarker development are reviewed in detail, and critical assessment of all phases of biomarker development pipeline is provided. This article is part of a Special Issue entitled: Medical Proteomics.
    Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 09/2014; DOI:10.1016/j.bbapap.2014.09.006 · 3.19 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Psoriatic arthritis (PsA) is a distinct inflammatory arthritis occurring in 30% of psoriasis patients. There is a high prevalence of undiagnosed PsA in psoriasis patients; therefore, identifying soluble biomarkers for PsA could help in screening psoriasis patients for appropriate referral to a rheumatologist. Potential PsA biomarkers likely originate in sites of inflammation, such as the skin, and subsequently enter systemic circulation. Our goal was to identify candidate PsA biomarkers by comparing the proteome of skin biopsies obtained from patients with PsA to that from patients with psoriasis without PsA. Skin biopsies were obtained from involved and uninvolved skin of 10 PsA and 10 age/gender-matched psoriasis patients without PsA (PsC). Using strong cation exchange chromatography, followed by label-free quantitative tandem mass spectrometry, we characterized the proteomes of pooled skin samples. Extracted ion current intensities were used to calculate protein abundance ratios, and these were utilized to identify differentially regulated proteins. Forty-seven proteins were elevated in PsA-derived skin compared to PsC-derived skin. Selected reaction monitoring assays were developed to quantify these potential PsA markers in individual skin samples, and 8 markers were confirmed in an independent sample set. ITGB5 and POSTN were measured in serum samples from 33 PsA and 15 PsC patients, using enzyme-linked immunosorbent assays. ITGB5 was significantly elevated in PsA serum (P < 0.01), and POSTN showed a trend. ITGB5 and POSTN correlated significantly in both patient groups (r = 0.472, P < 0.001). Proteomic analysis of PsA and PsC skin identified eight new candidate biomarkers. These markers need to be validated with a larger and independent cohort, in order to delineate their clinical utility in PsA patients. These proteins may also uncover unknown aspects of PsA pathobiology.
  • [Show abstract] [Hide abstract]
    ABSTRACT: The study of interactions between genes and their environment, thus of molecules that form the "functional genome", has been greatly enhanced throughout the course of post-genomic era by the expansion of the -omic technologies. Proteomic technologies and mass spectrometry in particular, form a well-armoured strategy towards high-thoughput simultaneous analysis of thousands of proteins and protein-related molecules. Proteomics have in the past, and are currently offering novel perspectives in the understanding over the pathophysiology of malicious conditions as well as in the development of molecular targeted-therapeutics against them. This review examines the offerings of proteomics research over the unique physiological phenomenon of pregnancy and its related complications, which affect both maternal and perinatal health.
    Clinical biochemistry 10/2012; 46(6). DOI:10.1016/j.clinbiochem.2012.10.011 · 2.23 Impact Factor