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1.
J Proteome Res ; 11(3): 1868-78, 2012 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-22257466

RESUMO

We have investigated the precision of peptide quantitation by MALDI-TOF mass spectrometry (MS) using six pairs of proteotypic peptides (light) and same-sequence stable isotope labeled synthetic internal standards (heavy). These were combined in two types of dilution curves spanning 100-fold and 2000-fold ratios. Coefficients of variation (CV; standard deviation divided by mean value) were examined across replicate MALDI spots using a reflector acquisition method requiring 100 000 counts for the most intense peak in each summed spectrum. The CV of light/heavy peptide centroid peak area ratios determined on four replicate spots per sample, averaged across 11 points of a 100-fold dilution curve and over all six peptides, was 2.2% (ranging from 1.5 to 3.7% among peptides) at 55 fmol total (light + heavy) of each peptide applied per spot, and 2.5% at 11 fmol applied. The average CV of measurements at near-equivalence (light = heavy, the center of the dilution curve) for the six peptides was 1.0%, about 17-fold lower CV than that observed when five peptides were ratioed to a sixth peptide (i.e., a different-sequence internal standard). Response curves across the 100-fold range were not completely linear but could be closely modeled by a power law fit giving R(2) values >0.998 for all peptides. The MALDI-TOF MS method was used to determine the endogenous level of a proteotypic peptide (EDQYHYLLDR) of human protein C inhibitor (PCI) in a plasma digest after enrichment by capture on a high affinity antipeptide antibody, a technique called stable isotope standards and capture by anti-peptide antibodies (SISCAPA). The level of PCI was determined to be 770 ng/mL with a replicate measurement CV of 1.5% and a >14 000-fold target enrichment via SISCAPA-MALDI-TOF. These results indicate that MALDI-TOF technology can provide precise quantitation of high-to-medium abundance peptide biomarkers over a 100-fold dynamic range when ratioed to same-sequence labeled internal standards and enriched to near purity by specific antibody capture. The robustness and throughput of MALDI-TOF in comparison to conventional nano-LC-MS technology could enable currently impractical large-scale verification studies of protein biomarkers.


Assuntos
Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Calibragem , Humanos , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/sangue , Inibidor da Proteína C/sangue , Inibidor da Proteína C/química , Proteólise , Proteômica , Padrões de Referência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/normas , Tripsina/química
2.
J Proteome Res ; 8(7): 3239-51, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19402737

RESUMO

Currently, the precursor ion selection strategies in LC-MS mainly choose the most prominent peptide signals for MS/MS analysis. Consequently, high-abundance proteins are identified by MS/MS of many peptides, whereas proteins of lower abundance might elude identification. We present a novel, iterative and result-driven approach for precursor ion selection that significantly increases the efficiency of an MS/MS analysis by decreasing data redundancy and analysis time. By simulating different strategies for precursor ion selection on an existing data set, we compare our method to existing result-driven strategies and evaluate its performance with regard to mass accuracy, database size, and sample complexity.


Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Proteômica/métodos , Algoritmos , Linhagem Celular , Simulação por Computador , Bases de Dados de Proteínas , Escherichia coli/metabolismo , Humanos , Íons , Modelos Estatísticos , Peptídeos/análise , Proteínas/química , Reprodutibilidade dos Testes , Software
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