RESUMO
Previous work from our laboratories utilized a novel skin taping method and mass spectrometry-based proteomics to discover clinical biomarkers of skin conditions; these included atopic dermatitis, Staphylococcus aureus colonization, and eczema herpeticum. While suitable for discovery purposes, semi-quantitative proteomics is generally time-consuming and expensive. Furthermore, depending on the method used, discovery-based proteomics can result in high variation and inadequate sensitivity to detect low abundant peptides. Therefore, we strove to develop a rapid, sensitive, and reproducible method to quantitate disease-related proteins from skin tapings. We utilized isotopically-labeled peptides and tandem mass spectrometry to obtain absolute quantitation values on 14 peptides from 7 proteins; these proteins had shown previous importance in skin disease. The method demonstrated good reproducibility, dynamic range, and linearity (R2â¯>â¯0.993) when nâ¯=â¯3 standards were analyzed across 0.05-2.5â¯pmol. The method was used to determine if differences exist between skin proteins in a small group of atopic versus non-atopic individuals (nâ¯=â¯12). While only minimal differences were found, peptides were detected in all samples and exhibited good correlation between peptides for 5 of the 7 proteins (R2â¯=â¯0.71-0.98). This method can be applied to larger cohorts to further establish the relationships of these proteins to skin disease.
Assuntos
Marcação por Isótopo/métodos , Peptídeos/análise , Pele/química , Espectrometria de Massas em Tandem/métodos , Estudos de Casos e Controles , Dermatite Atópica/metabolismo , Humanos , Limite de Detecção , Modelos Lineares , Proteômica/métodos , Reprodutibilidade dos TestesRESUMO
Advances in proteomics technology over the past decade offer forensic serologists a greatly improved opportunity to accurately characterize the tissue source from which a DNA profile has been developed. Such information can provide critical context to evidence and can help to prioritize downstream DNA analyses. Previous proteome studies compiled panels of "candidate biomarkers" specific to each of five body fluids (i.e., peripheral blood, vaginal/menstrual fluid, seminal fluid, urine, and saliva). Here, a multiplex quadrupole time-of-flight mass spectrometry assay has been developed in order to verify the tissue/body fluid specificity the 23 protein biomarkers that comprise these panels and the consistency with which they can be detected across a sample population of 50 humans. Single-source samples of these human body fluids were accurately identified by the detection of one or more high-specificity biomarkers. Recovery of body fluid samples from a variety of substrates did not impede accurate characterization and, of the potential inhibitors assayed, only chewing tobacco juice appeared to preclude the identification of a target body fluid. Using a series of 2-component mixtures of human body fluids, the multiplex assay accurately identified both components in a single-pass. Only in the case of saliva and peripheral blood did matrix effects appear to impede the detection of salivary proteins.
Assuntos
Líquidos Corporais/química , Proteínas/análise , Proteoma/análise , Biomarcadores/química , Cromatografia Líquida de Alta Pressão , Feminino , Medicina Legal , Humanos , Masculino , Espectrometria de Massas , Peptídeos/análise , RNA Mensageiro/análiseRESUMO
Although most cases of chronic obstructive pulmonary disease (COPD) occur in smokers, only a fraction of smokers develop the disease. We hypothesized distinct molecular signatures for COPD and emphysema in the peripheral blood mononuclear cells (PBMCs) of current and former smokers. To test this hypothesis, we identified and validated PBMC gene expression profiles in smokers with and without COPD. We generated expression data on 136 subjects from the COPDGene study, using Affymetrix U133 2.0 microarrays (Affymetrix, Santa Clara, CA). Multiple linear regression with adjustment for covariates (gender, age, body mass index, family history, smoking status, and pack-years) was used to identify candidate genes, and ingenuity pathway analysis was used to identify candidate pathways. Candidate genes were validated in 149 subjects according to multiplex quantitative real-time polymerase chain reaction, which included 75 subjects not previously profiled. Pathways that were differentially expressed in subjects with COPD and emphysema included those that play a role in the immune system, inflammatory responses, and sphingolipid (ceramide) metabolism. Twenty-six of the 46 candidate genes (e.g., FOXP1, TCF7, and ASAH1) were validated in the independent cohort. Plasma metabolomics was used to identify a novel glycoceramide (galabiosylceramide) as a biomarker of emphysema, supporting the genomic association between acid ceramidase (ASAH1) and emphysema. COPD is a systemic disease whose gene expression signatures in PBMCs could serve as novel diagnostic or therapeutic targets.
Assuntos
Gangliosídeos/sangue , Regulação da Expressão Gênica , Leucócitos Mononucleares/metabolismo , Doença Pulmonar Obstrutiva Crônica/sangue , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Feminino , Humanos , Masculino , Metabolômica/métodos , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Enfisema Pulmonar/sangue , Enfisema Pulmonar/diagnóstico , Reação em Cadeia da Polimerase em Tempo RealRESUMO
A new analytical method suitable for high throughput measurements of LTE(4) in human urine is described. The methodology utilizes on-line enrichment and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The novel LC/MS/MS method is rapid, linear from 5 to 500pg/ml in spiked urine samples of both healthy and asthmatic subjects and more accurate and precise than enzyme immunoassay (EIA) and previous LC/MS/MS methods. Results from sample integrity experiments and preliminary values of urinary LTE(4) from healthy adults and children are reported.