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1.
J Appl Lab Med ; 3(5): 788-798, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-31639754

RESUMO

BACKGROUND: We compared the rates of intraoperative parathyroid hormone (PTH) decline using the Siemens Immulite® Turbo PTH and Roche Elecsys® short turnaround time PTH assays in 95 consecutive surgical patients to investigate analytical and turnaround time (TAT) differences between the tests performed in the operating room (OR) vs the central clinical chemistry laboratory (CCL). METHODS: Serial blood samples from 95 patients undergoing parathyroidectomy were collected and measured using the 2 immunoassays. Specimens from the first 15 patients were measured simultaneously in the OR and CCL and used for the TAT study. In addition to 2 baseline samples, specimens were collected at 5, 10, and 15 min (for some patients, >15 min) after parathyroidectomy. RESULTS: In the TAT study, a significant difference was observed (OR median 20 min vs CCL median 27 min; P < 0.05). Of the 95 patient series, slower rates of parathyroid hormone decrease were observed in approximately 20% of the patients when comparing the Roche with the Immulite immunoassay. CONCLUSIONS: There was a slightly longer TAT in the CCL compared with running the assay directly within the OR (median difference of approximately 7 min). For a majority of the patients, both methods showed equivalent rates of PTH decline; however, for approximately 20% of the patients, there was a slower rate of PTH decline using the Roche assay.


Assuntos
Testes de Química Clínica/métodos , Hiperparatireoidismo Primário/sangue , Hiperparatireoidismo Primário/cirurgia , Imunoensaio/métodos , Hormônio Paratireóideo/sangue , Paratireoidectomia/métodos , Feminino , Humanos , Período Intraoperatório , Masculino , Pessoa de Meia-Idade
2.
NPJ Aging Mech Dis ; 3: 7, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28649425

RESUMO

Growing evidence suggests that many diseases of aging, including diseases associated with robust changes and adipose deports, may be caused by resident adult stem cell exhaustion due to the process called cellular senescence. Understanding how microRNA pathways can regulate cellular senescence is crucial for the development of novel diagnostic and therapeutic strategies to combat these pathologies. Herein, using integrated transcriptomic and semi-quantitative proteomic analysis, we provide a system level view of the regulation of human adipose-derived stem cell senescence by a subset of mature microRNAs (termed senescence-associated-microRNAs) produced by biogenesis of oncogenic MIR17HG and tumor-suppressive MIR100HG clusters. We demonstrate functional significance of these mature senescence-associated-microRNAs in the process of replicative senescence of human adipose-derived stem cells ex-vivo and define a set of senescence-associated-microRNA gene targets that are able to elicit, modulate and, most importantly, balance intimate connections between oncogenic and senescent events.

3.
Nat Methods ; 12(8): 725-31, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26121405

RESUMO

Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.


Assuntos
Anticorpos Monoclonais/química , Especificidade de Anticorpos , Cromatina/química , Imunoprecipitação/métodos , Proteômica/métodos , Clonagem Molecular , Biologia Computacional/métodos , Escherichia coli/metabolismo , Células HEK293 , Humanos , Fragmentos de Imunoglobulinas/química , Imunoglobulina G/química , Espectrometria de Massas/métodos , Biblioteca de Peptídeos , Proteínas/química , Proteoma , Reprodutibilidade dos Testes
4.
Methods ; 81: 66-73, 2015 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-25770357

RESUMO

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of circulating low density lipoprotein cholesterol (LDL-C) levels. Besides its full-length mature form, multiple variants of PCSK9 have been reported such as forms that are truncated, mutated and/or with posttranslational modifications (PTMs). Previous studies have demonstrated that most of these variants affect PCSK9's function and thereby LDL-C levels. Commercial ELISA kits are available for quantification of PCSK9, but do not allow discrimination between the various forms and PTMs of the protein. To address this issue and given the complexity and wide dynamic range of the plasma proteome, we have developed a mass spectrometric immunoassay coupled to selected reaction monitoring (MSIA-SRM) for the multiplexed quantification of several forms of circulating PCSK9 in human plasma. Our MSIA-SRM assay quantifies peptides spanning the various protein domains and the S688 phosphorylation site. The assay was applied in two distinct cohorts of obese patients and healthy pregnant women stratified by their circulating LDL-C levels. Seven PCSK9 peptides were monitored in plasma samples: one in the prodomain prior to the autocleavage site at Q152, one in the catalytic domain prior to the furin cleavage site at R218, two in the catalytic domain following R218, one in the cysteine and histidine rich domain (CHRD) and the C-terminal peptide phosphorylated at S688 and unmodified. The latter was not detectable in sufficient amounts to be quantified in human plasma. All peptides were measured with high reproducibility and with LLOQ and LOD below the clinical range. The abundance of 5 of the 6 detectable PCSK9 peptides was higher in obese patients stratified with high circulating LDL-C levels as compared to those with low LDL-C (p < 0.05). The same 5 peptides showed good and statistically significant correlations with LDL-C levels (0.55 < r < 0.65; 0.0002 ⩽ p ⩽ 0.002), but not the S688 phosphorylated peptide. However, this phosphopeptide was significantly correlated with insulin resistance (r = 0.48; p = 0.04). In the pregnant women cohort, none of the peptides were associated to LDL-C levels. However, the 6 detectable PCSK9 peptides, but not PCSK9 measured by ELISA, were significantly correlated with serum triglyceride levels in this cohort. Our results also suggest that PCSK9 circulates with S688 phosphorylated at high stoichiometry. In summary, we have developed and applied a robust and sensitive MSIA-SRM assay for the absolute quantification of all PCSK9 domains and a PTM in human plasma. This assay revealed novel relationships between PCSK9 and metabolic phenotypes, as compared to classical ELISA assays.


Assuntos
Imunoensaio/métodos , Espectrometria de Massas/métodos , Pró-Proteína Convertases/sangue , Serina Endopeptidases/sangue , Adolescente , Adulto , Feminino , Humanos , Resistência à Insulina , Lipoproteínas LDL/sangue , Masculino , Pessoa de Meia-Idade , Obesidade/sangue , Fenótipo , Gravidez , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Serina Endopeptidases/metabolismo , Triglicerídeos/sangue , Adulto Jovem
5.
Clin Proteomics ; 12(1): 2, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25678897

RESUMO

BACKGROUND: The anatomy of PFO suggests that it can allow thrombi and potentially harmful circulatory factors to travel directly from the venous to the arterial circulation - altering circulatory phenotype. Our previous publication using high-resolution LC-MS/MS to profile protein and peptide expression patterns in plasma showed that albumin was relatively increased in donor samples from PFO-related than other types of ischemic strokes. Since albumin binds a host of molecules and acts as a carrier for lipoproteins, small molecules and drugs, we decided to investigate the albumin-bound proteins (in a similar sample cohort) in an effort to unravel biological changes and potentially discover biomarkers related to PFO-related stroke and PFO endovascular closure. METHODS: The method used in this study combined albumin immuno-enrichment with high resolution LC-MS in order to specifically capture and quantify the albumin-bound proteins. Subsequently, we measured cholesterol and HDL in a larger, separate cohort of PFO stroke patients, pre and post closure. RESULTS: The results demonstrated that a number of proteins were specifically associated with albumin in samples with and without endovascular closure of the PFO, and that the protein profiles were very different. Eight proteins, typically associated with HDL were common to both sample sets and quantitatively differently abundant. Pathway analysis of the MS results suggested that enhanced cholesterol efflux and reduced lipid oxidation were associated with PFO closure. Measurement of total cholesterol and HDL in a larger cohort of PFO closure samples using a colorimetric assay was consistent with the proteomic predictions. CONCLUSIONS: The collective data presented in this study demonstrate that analysis of albumin-bound proteins could provide a valuable tool for biomarker discovery on the effects of PFO endovascular closure. In addition, the results suggest that PFO endovascular closure can potentially have effects on HDL, cholesterol and albumin-bound ApoA-I abundance, therefore possibly providing benefits in cardioprotective functions.

7.
J Proteomics ; 114: 115-24, 2015 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-25462431

RESUMO

Generation of monoclonal antibody (mAb) libraries against antigens in complex matrices can prove a valuable analytical tool. However, delineating the specificity of newly generated antibodies is the limiting step of the procedure. Here, we propose a strategy for mAb production by injecting mice with complex biological fluid and mAb characterization by coupling immunoaffinity techniques with Mass spectrometry (immuno-MS). Mice were immunized against fractionated seminal plasma and mAbs were produced. Different immuno-MS protocols based on four types of solid support (i.e. polystyrene microtiter plates, NHS-activated agarose beads, tosyl-activated magnetic beads and MSIA™ pipette tips) were established. A well-characterized mouse monoclonal anti-KLK3 (PSA) Ab was used as a model to evaluate each protocol's robustness and reproducibility and to establish a set of criteria which would allow antigen characterization of newly developed Abs. Three of the newly generated Abs were analyzed using our optimized protocols. Analysis revealed that all assay configurations used were capable of antibody characterization. Furthermore, low-abundance antigens (e.g. ribonuclease T2) could be identified as efficiently as the high-abundance ones. Our data suggest that complex biological samples can be used for the production of mAbs, which will facilitate the analysis of their proteome, while the established immuno-MS protocols can offer efficient mAb characterization. BIOLOGICAL SIGNIFICANCE: The inoculation of animals with complex biological samples is aiming at the discovery of novel disease biomarkers, present in the biological specimens, as well as the production of rare reagents that will facilitate the ultra-sensitive analysis of the biomolecules' native form. In the present study, we initially propose a general workflow concerning the handling of biological samples, as well as the monoclonal antibody production. Furthermore, we established protocols for the reliable and reproducible identification of antibody specificity using various immuno-affinity purification techniques coupled to mass spectrometry. Our data suggest that processed biological fluids can be used for the production of mAbs targeting proteins of varying abundance, and that various immuno-MS protocols can offer great capabilities for the mAb characterization procedure.


Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Espectrometria de Massas/métodos , Adipocinas , Animais , Anticorpos Monoclonais/química , Proteínas de Transporte/química , Proteínas de Transporte/imunologia , Cromatografia Líquida , Epitopos/imunologia , Feminino , Glicoproteínas/química , Glicoproteínas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Antígeno Prostático Específico/imunologia , Espectrometria de Massas em Tandem
8.
PLoS Genet ; 10(7): e1004508, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25078964

RESUMO

The phenotypic effect of some single nucleotide polymorphisms (SNPs) depends on their parental origin. We present a novel approach to detect parent-of-origin effects (POEs) in genome-wide genotype data of unrelated individuals. The method exploits increased phenotypic variance in the heterozygous genotype group relative to the homozygous groups. We applied the method to >56,000 unrelated individuals to search for POEs influencing body mass index (BMI). Six lead SNPs were carried forward for replication in five family-based studies (of ∼4,000 trios). Two SNPs replicated: the paternal rs2471083-C allele (located near the imprinted KCNK9 gene) and the paternal rs3091869-T allele (located near the SLC2A10 gene) increased BMI equally (beta = 0.11 (SD), P<0.0027) compared to the respective maternal alleles. Real-time PCR experiments of lymphoblastoid cell lines from the CEPH families showed that expression of both genes was dependent on parental origin of the SNPs alleles (P<0.01). Our scheme opens new opportunities to exploit GWAS data of unrelated individuals to identify POEs and demonstrates that they play an important role in adult obesity.


Assuntos
Proteínas Facilitadoras de Transporte de Glucose/genética , Obesidade/genética , Polimorfismo de Nucleotídeo Único/genética , Canais de Potássio de Domínios Poros em Tandem/genética , Adulto , Índice de Massa Corporal , Feminino , Regulação da Expressão Gênica , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Impressão Genômica , Genótipo , Humanos , Masculino , Obesidade/patologia , População Branca/genética
9.
Clin Chem Lab Med ; 52(9): 1251-63, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24762644

RESUMO

The analysis of intact parathyroid hormone (PTH) (PTH1-84) is useful in the diagnosis of hyper- and hypocalcaemia, hyperparathyroidism, and in the prevention of bone mineral disorders in renal patients. The analysis is complicated by the presence of PTH fragments, which may accumulate in renal failure and cross-react in immunoassays, including the most recent third-generation immunoassays. Large variability exists between different commercially available assays. This article reviews the current literature on PTH testing, with emphasis on the use of mass spectrometry-based methods, and considers the important sources of variation which still need to be addressed prior to the development of much needed candidate reference methods for PTH analysis. Recently, mass spectrometric methods have been developed for the quantitation of PTH1-84 using surrogate tryptic peptides, but even these methods are subject to significant interferences due to the presence of newly observed modified PTH species, such as oxidised and phosphorylated PTH variants, which can accumulate in patient samples. Further work, including: 1) the use of high-resolution mass spectrometry; and 2) the analysis of PTH without prior protease digestion, is required before these approaches can be considered as reference methods against which other methods should be harmonised.


Assuntos
Análise Química do Sangue/métodos , Espectrometria de Massas/métodos , Hormônio Paratireóideo/sangue , Análise Química do Sangue/tendências , Cromatografia Líquida/métodos , Variação Genética , Humanos , Imunoensaio/métodos , Oxirredução , Hormônio Paratireóideo/química , Hormônio Paratireóideo/genética , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fosforilação , Proteólise , Valores de Referência
10.
Expert Rev Proteomics ; 11(3): 371-81, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24754513

RESUMO

Apolipoprotein E (ApoE) is an abundant plasma protein that interacts with low density lipoprotein receptors and other proteins, participating in the transport of cholesterol and lipids. Research has revealed many other roles for this multifunctional protein. ApoE is polymorphic and exists in three major isoforms: ApoE2, ApoE3 (the most common isoform) and ApoE4, which differ by only one amino acid, at positions 112 and 158. The altered binding to lipids and receptors by ApoE isoforms E2 and E4 results in an elevated risk for neurological, cerebrovascular and cardiovascular pathologies. Most notably, ApoE4 is associated with an elevated risk (relative to E3) for Alzheimer's disease. The application of mass spectrometry for genotyping and also direct measurement of ApoE protein isoforms is a recent development and is well suited to high-throughput applications. The precise quantification of protein isoforms will allow better characterization of effects resulting from heterozygous APOE genotypes.


Assuntos
Doença de Alzheimer/metabolismo , Apolipoproteínas E/metabolismo , Doenças Cardiovasculares/metabolismo , Doenças Neurodegenerativas/metabolismo , Apolipoproteínas E/genética , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteômica
11.
Proteomics ; 14(12): 1445-56, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24668948

RESUMO

The detection and quantification of insulin and its therapeutic analogs is important for medical, sports doping, and forensic applications. Synthetic variants contain slight sequence variations to affect bioavailability. To reduce sample handling bias, a universal extraction method is required for simultaneous extraction of endogenous and variant insulins with subsequent targeted quantification by LC-MS. A mass spectrometric immunoassay (MSIA), a multiplexed assay for intact insulin and its analogues that couples immunoenrichment with high resolution and accurate mass (HR/AM) spectrometric detection across the clinical range is presented in this report. The assay is sensitive, selective, semi-automated and can potentially be applied to detect new insulin isoforms allowing their further incorporation into second or third generation assays.


Assuntos
Cromatografia Líquida/métodos , Ensaios de Triagem em Larga Escala , Imunoensaio/métodos , Insulina/análogos & derivados , Insulina/sangue , Proteômica , Espectrometria de Massas em Tandem/métodos , Humanos , Isoformas de Proteínas
12.
J Proteome Res ; 13(2): 1077-87, 2014 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-24392642

RESUMO

Apolipoprotein E (ApoE) is a polymorphic protein that plays a major role in lipid metabolism in the central nervous system and periphery. It has three common allelic isoforms, ApoE2, ApoE3, and ApoE4, that differ in only one or two amino acids. ApoE isoforms have been associated with the occurrence and progression of several pathological conditions, such as coronary atherosclerosis and Alzheimer's disease. The aim of this study was to develop a mass spectrometry (MS)-based assay for absolute quantification of ApoE isoforms in cerebrospinal fluid and plasma samples using isotope-labeled peptides. The assay included five tryptic peptides: CLAVYQAGAR (ApoE2), LGADMEDVCGR (ApoE2 and 3), LAVYQAGAR (ApoE3 and 4), LGADMEDVR (ApoE4), and LGPLVEQGR (total ApoE). Both cerebrospinal fluid and plasma samples were assayed to validate the method. The digestion yield and the extension of chemical modifications in selected amino acid residues (methionine oxidation, glutamine deamidation, and cyclization of N-terminus carbamidomethylcysteine) were also studied. The ApoE phenotype was successfully assigned to all samples analyzed in a blinded manner. The method showed good linearity (R(2) > 0.99) and reproducibility (within laboratory imprecision <13%). The comparison of the MS-based assay with an ELISA for total ApoE concentration showed a moderate correlation (R(2) = 0.59). This MS-based assay can serve as an important tool in clinical studies aiming to elucidate the association between ApoE genotype, total ApoE, and ApoE isoform concentrations in various disorders related to ApoE polymorphisms.


Assuntos
Apolipoproteínas E/química , Peptídeos/química , Isoformas de Proteínas/química , Sequência de Aminoácidos , Apolipoproteínas E/análise , Cromatografia Líquida , Espectrometria de Massas , Dados de Sequência Molecular , Oxirredução , Isoformas de Proteínas/análise , Tripsina/química
13.
PLoS One ; 8(11): e81125, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24278387

RESUMO

Insulin-like growth factor 1 (IGF1) is an important biomarker of human growth disorders that is routinely analyzed in clinical laboratories. Mass spectrometry-based workflows offer a viable alternative to standard IGF1 immunoassays, which utilize various pre-analytical preparation strategies. In this work we developed an assay that incorporates a novel sample preparation method for dissociating IGF1 from its binding proteins. The workflow also includes an immunoaffinity step using antibody-derivatized pipette tips, followed by elution, trypsin digestion, and LC-MS/MS separation and detection of the signature peptides in a selected reaction monitoring (SRM) mode. The resulting quantitative mass spectrometric immunoassay (MSIA) exhibited good linearity in the range of 1 to 1,500 ng/mL IGF1, intra- and inter-assay precision with CVs of less than 10%, and lowest limits of detection of 1 ng/mL. The linearity and recovery characteristics of the assay were also established, and the new method compared to a commercially available immunoassay using a large cohort of human serum samples. The IGF1 SRM MSIA is well suited for use in clinical laboratories.


Assuntos
Imunoensaio/métodos , Fator de Crescimento Insulin-Like I/metabolismo , Espectrometria de Massas/métodos , Biomarcadores/sangue , Humanos , Imunoensaio/normas , Fator de Crescimento Insulin-Like I/química , Espectrometria de Massas/normas , Padrões de Referência , Valores de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
14.
Arthritis Rheum ; 65(4): 981-92, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23400684

RESUMO

OBJECTIVE: The pathophysiology of the most common joint disease, osteoarthritis (OA), remains poorly understood. Since synovial fluid (SF) bathes joint cartilage and synovium, we reasoned that a comparative analysis of its protein constituents in health and OA could identify pathways involved in joint damage. We undertook this study to perform a proteomic analysis of knee SF from OA patients and control subjects and to compare the results to microarray expression data from cartilage and synovium. METHODS: Age-matched knee SF samples from 10 control subjects, 10 patients with early-stage OA, and 10 patients with late-stage OA were compared using 2-dimensional difference-in-gel electrophoresis and mass spectrometry (MS). MS with a multiplexed peptide selected reaction monitoring assay was used to confirm differential expression of a subset of proteins in an independent OA patient cohort. Proteomic results were analyzed by Ingenuity Pathways Analysis and compared to published synovial tissue and cartilage messenger RNA profiles. RESULTS: Sixty-six proteins were differentially present in healthy and OA SF. Three major pathways were identified among these proteins: the acute-phase response signaling pathway, the complement pathway, and the coagulation pathway. Differential expression of 5 proteins was confirmed by selected reaction monitoring assay. A focused analysis of transcripts corresponding to the differentially present proteins indicated that both synovial and cartilage tissues may contribute to the OA SF proteome. CONCLUSION: Proteins involved in the acute-phase response signaling pathway, the complement pathway, and the coagulation pathway are differentially regulated in SF from OA patients, suggesting that they contribute to joint damage. Validation of these pathways and their utility as biomarkers or therapeutic targets in OA is warranted.


Assuntos
Cartilagem/metabolismo , Osteoartrite do Joelho/metabolismo , Proteoma/análise , RNA Mensageiro/análise , Líquido Sinovial/metabolismo , Membrana Sinovial/metabolismo , Proteínas de Fase Aguda/genética , Proteínas de Fase Aguda/metabolismo , Reação de Fase Aguda/metabolismo , Idoso , Fatores de Coagulação Sanguínea/genética , Fatores de Coagulação Sanguínea/metabolismo , Estudos de Casos e Controles , Proteínas do Sistema Complemento/genética , Proteínas do Sistema Complemento/metabolismo , Eletroforese em Gel Bidimensional , Feminino , Perfilação da Expressão Gênica , Humanos , Articulação do Joelho/metabolismo , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Osteoartrite do Joelho/genética , Líquido Sinovial/química
15.
Clin Biochem ; 46(6): 399-410, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23313081

RESUMO

OBJECTIVES: The aim of this study was to develop high-throughput, quantitative and highly selective mass spectrometric, targeted immunoassays for clinically important proteins in human plasma or serum. DESIGN AND METHODS: The described method coupled mass spectrometric immunoassay (MSIA), a previously developed technique for immunoenrichment on a monolithic microcolumn activated with an anti-protein antibody and fixed in a pipette tip, to selected reaction monitoring (SRM) detection and accurate quantification of targeted peptides, including clinically relevant sequence or truncated variants. RESULTS: In this report, we demonstrate the rapid development of MSIA-SRM assays for sixteen different target proteins spanning seven different clinically important areas (including neurological, Alzheimer's, cardiovascular, endocrine function, cancer and other diseases) and ranging in concentration from pg/mL to mg/mL. The reported MSIA-SRM assays demonstrated high sensitivity (within published clinical ranges), precision, robustness and high-throughput as well as specific detection of clinically relevant isoforms for many of the target proteins. Most of the assays were tested with bona-fide clinical samples. In addition, positive correlations, (R2 0.67-0.87, depending on the target peptide), were demonstrated for MSIA-SRM assay data with clinical analyzer measurements of parathyroid hormone (PTH) and insulin growth factor 1 (IGF1) in clinical sample cohorts. CONCLUSIONS: We have presented a practical and scalable method for rapid development and deployment of MS-based SRM assays for clinically relevant proteins and measured levels of the target analytes in bona fide clinical samples. The method permits the specific quantification of individual protein isoforms and addresses the difficult problem of protein heterogeneity in clinical proteomics applications.


Assuntos
Proteínas Sanguíneas/isolamento & purificação , Ensaios de Triagem em Larga Escala , Imunoensaio/métodos , Espectrometria de Massas/métodos , Doença de Alzheimer/sangue , Doenças Cardiovasculares/sangue , Transtornos do Crescimento/sangue , Humanos , Neoplasias/sangue , Insuficiência Renal/sangue
16.
Aging (Albany NY) ; 4(11): 823-42, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23235539

RESUMO

Cellular senescence is associated with global chromatin changes, altered gene expression, and activation of chronic DNA damage signaling. These events ultimately lead to morphological and physiological transformations in primary cells. In this study, we show that chronic DNA damage signals caused by genotoxic stress impact the expression of histones H2A family members and lead to their depletion in the nuclei of senescent human fibroblasts. Our data reinforce the hypothesis that progressive chromatin destabilization may lead to the loss of epigenetic information and impaired cellular function associated with chronic DNA damage upon drug-evoked senescence. We propose that changes in the histone biosynthesis and chromatin assembly may directly contribute to cellular aging. In addition, we also outline the method that allows for quantitative and unbiased measurement of these changes.


Assuntos
Senescência Celular/genética , Dano ao DNA/genética , Histonas/genética , Transdução de Sinais/genética , Sequência de Aminoácidos , Antibióticos Antineoplásicos , Bleomicina , Western Blotting , Senescência Celular/efeitos dos fármacos , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Histonas/metabolismo , Humanos , Imuno-Histoquímica , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos
17.
J Investig Med ; 60(8): 1122-30, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23147404

RESUMO

Patent foramen ovale (PFO) is highly prevalent and associated with more than 150,000 strokes per year. Traditionally, it is thought that PFOs facilitate strokes by allowing venous clots to travel directly to the brain. However, only a small portion of PFO stroke patients have a known tendency to form blood clots, and the optimal treatment for this multiorgan disease is unclear. Therefore, mapping the changes in systemic circulation of PFO-related stroke is crucial in understanding the pathophysiology to individualize the best clinical treatment for each patient. We initiated a study using a novel quantitative, 2-pass discovery workflow using high-resolution liquid chromatography-mass spectrometry/mass spectrometry coupled with label-free analysis to track protein expression in PFO patients before and after endovascular closure of the PFO. Using this approach, we were able to demonstrate quantitative differences in protein expression between both PFO-related and non-PFO-related ischemic stroke groups as well as before and after PFO closure. As an initial step in understanding the molecular landscape of PFO-related physiology, our methods have yielded biologically relevant information on the synergistic and functional redundancy of various cell-signaling molecules with respect to PFO circulatory physiology. The resulting protein expression patterns were related to canonical pathways including prothrombin activation, atherosclerosis signaling, acute-phase response, LXR/RXR activation, and coagulation system. In particular, after PFO closure, numerous proteins demonstrated reduced expression in stroke-related canonical pathways such as acute inflammatory response and coagulation signaling. These findings demonstrate the feasibility and robustness of using a proteomic approach for biomarker discovery to help gauge therapeutic efficacy in stroke.


Assuntos
Forame Oval Patente/sangue , Regulação da Expressão Gênica , Proteômica/métodos , Transdução de Sinais/fisiologia , Acidente Vascular Cerebral/sangue , Espectrometria de Massas em Tandem , Adulto , Encéfalo/fisiologia , Cromatografia Líquida/métodos , Estudos de Coortes , Feminino , Forame Oval Patente/epidemiologia , Forame Oval Patente/cirurgia , Coração/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Acidente Vascular Cerebral/epidemiologia , Espectrometria de Massas em Tandem/métodos , Adulto Jovem
18.
Mob Genet Elements ; 2(1): 26-35, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-22754750

RESUMO

Recent analyses suggest that transposable element-derived transcripts are processed to yield a variety of small RNA species that play critical functional roles in gene regulation and chromatin organization as well as genome stability and maintenance. Here we report a mass spectrometry analysis of an RNA-affinity complex isolation using a piRNA homologous sequence derived from Alu retrotransposal RNA. Our data point to potential roles for piALU RNAs in DNA repair, cell cycle and chromatin regulations.

19.
J Proteome Res ; 11(8): 3986-95, 2012 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-22639787

RESUMO

Over the past few years, mass spectrometry has emerged as a technology to complement and potentially replace standard immunoassays in routine clinical core laboratories. Application of mass spectrometry to protein and peptide measurement can provide advantages including high sensitivity, the ability to multiplex analytes, and high specificity at the amino acid sequence level. In our previous study, we demonstrated excellent reproducibility of mass spectrometry-selective reaction monitoring (MS-SRM) assays when applying standardized standard operating procedures (SOPs) to measure synthetic peptides in a complex sample, as lack of reproducibility has been a frequent criticism leveled at the use of mass spectrometers in the clinical laboratory compared to immunoassays. Furthermore, an important caveat of SRM-based assays for proteins is that many low-abundance analytes require some type of enrichment before detection with MS. This adds a level of complexity to the procedure and the potential for irreproducibility increases, especially across different laboratories with different operators. The purpose of this study was to test the interlaboratory reproducibility of SRM assays with various upfront enrichment strategies and different types of clinical samples (representing real-world body fluids commonly encountered in routine clinical laboratories). Three different, previously published enrichment strategies for low-abundance analytes and a no-enrichment strategy for high-abundance analytes were tested across four different laboratories using different liquid chromatography-SRM (LC-SRM) platforms and previously developed SOPs. The results demonstrated that these assays were indeed reproducible with coefficients of variation of less than 30% for the measurement of important clinical proteins across all four laboratories in real world samples.


Assuntos
Análise Química do Sangue/normas , Laboratórios/normas , Espectrometria de Massas/normas , Sequência de Aminoácidos , Cromatografia de Fase Reversa , Feminino , Hormônio do Crescimento Humano/urina , Humanos , Limite de Detecção , Masculino , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/sangue , Padrões de Referência , Reprodutibilidade dos Testes , Proteínas de Plasma Seminal/química
20.
Proteomics Clin Appl ; 6(3-4): 190-200, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22532455

RESUMO

PURPOSE: Typically, apolipoproteins are individually measured in blood by immunoassay. In this report, we describe the development of a multiplexed selected reaction monitoring (SRM) based assay for a panel of apolipoproteins and its application to a clinical cohort of samples derived from acute stroke patients. EXPERIMENTAL DESIGN: An SRM assay for a panel of nine apolipoproteins was developed on a triple quadrupole mass spectrometer. Quantitative data for each apolipoprotein were analyzed to determine expression ratio and receiver operating characteristic (ROC) values for ischemic versus hemorrhagic stroke. RESULTS: The optimized SRM assay was used to interrogate a small cohort of well-characterized plasma samples obtained from patients with acute ischemic and hemorrhagic strokes. The ROC analyses demonstrated good classification power for several single apolipoproteins, most notably apoC-III and apoC-I. When a novel multi-marker ROC algorithm was applied, the ischemic versus hemorrhagic groups were best differentiated by a combination of apoC-III and apoA-I with an area under the curve (AUC) value of 0.92. CONCLUSIONS AND CLINICAL RELEVANCE: This proof-of-concept study provides interesting and provocative data for distinguishing ischemic versus hemorrhage within first week of symptom onset. However, the observations are based on one cohort of patient samples and further confirmation will be required.


Assuntos
Algoritmos , Apolipoproteínas/sangue , Proteínas Sanguíneas/análise , Transtornos Hemorrágicos/diagnóstico , Espectrometria de Massas/métodos , Curva ROC , Acidente Vascular Cerebral/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Apolipoproteínas/classificação , Biomarcadores/sangue , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Transtornos Hemorrágicos/patologia , Humanos , Isquemia/diagnóstico , Isquemia/patologia , Limite de Detecção , Masculino , Espectrometria de Massas/normas , Pessoa de Meia-Idade , Dados de Sequência Molecular , Acidente Vascular Cerebral/patologia , Adulto Jovem
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