RESUMO
BACKGROUND: Cetuximab (CTX) is a chimeric IgG1 Kappa monoclonal antibody used to treat head and neck cancer and colorectal cancer. Previous clinical studies indicated that the pharmacokinetics of CTX influences patient survival. Thus, individualizing CTX treatment by measuring trough levels of the drug in plasma could have a major impact on clinical efficacy. METHODS: To measure these levels, a full-length stable isotope-labeled CTX standard was used in a generic, rapid, and high-throughput sample preparation protocol based on IgG capture followed by trypsin digestion, on-line solid-phase extraction cleanup, and liquid chromatography-high resolution mass spectrometry (LC-HRMS). RESULTS: The optimized method displayed good analytical performance and was linear over a range from 5 to 150 mcg/mL. The within-run and between-run imprecision of the assay were equal to or less than 10%, for 6 replicates at 3 different concentrations and for runs performed on 5 separate days. The plasma CTX concentrations in 19 patients were also determined. CONCLUSIONS: The results showed that quantification of mAb in clinical samples does not strictly require a tandem mass spectrometry system, and LC-HRMS is also relevant in this context. This first study implementing a quantitative LC-HRMS assay with a specific stable isotope-labeled mAb internal standard paves the way for more robust clinical monitoring of anticancer mAbs.
Assuntos
Anticorpos Monoclonais/sangue , Isótopos de Carbono/sangue , Cetuximab/sangue , Isótopos de Nitrogênio/sangue , Plasma/química , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Marcação por Isótopo/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
Infliximab (IFX) is a chimeric monoclonal antibody targeting tumor necrosis factor-alpha. It is currently approved for the treatment of certain rheumatic diseases or inflammatory bowel diseases. Clinical studies have suggested that monitoring IFX concentrations could improve treatment response. However, in most studies, IFX was quantified using ELISA assays, the resulting discrepancies of which raised concerns about their reliability. Here, we describe the development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for IFX quantification in human plasma. Full-length stable-isotope-labeled antibody (SIL-IFX) was added to plasma samples as internal standard. Samples were then prepared using Mass Spectrometry Immuno Assay (MSIA™) followed by trypsin digestion and submitted to multiple reaction monitoring (MRM) for quantification of IFX. The chromatographic run lasted 13 min. The range of quantification was 1 to 26 mg/L. For two internal quality controls spiked with 6 and 12 mg/L of IFX, the method was reproducible (coefficients of variation (CV%): 12.7 and 2.1), repeatable (intra-day CV%: 5.5 and 5.0), and accurate (inter-day and intra-day deviations from nominal values: +6.4 to +3.7 % and 5.5 to 9.2 %, respectively). There was no cross - contamination effect. Samples from 45 patients treated with IFX were retrospectively analyzed by LC-MS/MS and results were compared to those obtained with an in-house ELISA assay and the commercial Lisa Tracker® method. Good agreement was found between LC-MS/MS and in-house ELISA (mean underestimation of 13 % for in-house ELISA), but a significant bias was found with commercial ELISA (mean underestimation of 136 % for commercial ELISA). This method will make it possible to standardize IFX quantification between laboratories. Graphical Abstract Interassay comparison of the three methods: LC-MS/MS vs inhouse ELISA assay or vs Lisa Tracker® ELISA assays, Passing & Bablok (a) and Bland & Altman (b) for the comparison of LC-MS/MS vs in-house ELISA assay; Passing & Bablok
Assuntos
Cromatografia Líquida/métodos , Infliximab/sangue , Espectrometria de Massas em Tandem/métodos , Ensaio de Imunoadsorção Enzimática , HumanosRESUMO
In bottom-up mass spectrometry-based proteomics analyses, variability at any step of the process, particularly during sample proteolysis, directly affects the sensitivity, accuracy, and precision of peptide detection and quantification. Currently, no generic internal standards are available to control the quality of sample processing steps. This makes it difficult to assess the comparability of MS proteomic data obtained under different experimental conditions. Here, we describe the design, synthesis, and validation of a universal protein standard, called DIGESTIF, that can be added to any biological sample. The DIGESTIF standard consists of a soluble recombinant protein scaffold to which a set of 11 artificial peptides (iRT peptides) with good ionization properties has been incorporated. In the protein scaffold, the amino acids flanking iRT peptide cleavage sites were selected either to favor or hinder protease cleavage. After sample processing, the retention time and relative intensity pattern of the released iRT peptides can be used to assess the quality of sample workup, the extent of digestion, and the performance of the LC-MS system. Thus, DIGESTIF can be used to standardize a broad spectrum of applications, ranging from simple replicate measurements to large-scale biomarker screening in biomedical applications.
Assuntos
Proteínas/química , Proteômica , Sequência de Aminoácidos , Animais , Biomarcadores/química , Cromatografia Líquida , Humanos , Cinética , Masculino , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Proteólise , Controle de QualidadeRESUMO
Development of new biomarkers needs to be significantly accelerated to improve diagnostic, prognostic, and toxicity monitoring as well as therapeutic follow-up. Biomarker evaluation is the main bottleneck in this development process. Selected Reaction Monitoring (SRM) combined with stable isotope dilution has emerged as a promising option to speed this step, particularly because of its multiplexing capacities. However, analytical variabilities because of upstream sample handling or incomplete trypsin digestion still need to be resolved. In 2007, we developed the PSAQ™ method (Protein Standard Absolute Quantification), which uses full-length isotope-labeled protein standards to quantify target proteins. In the present study we used clinically validated cardiovascular biomarkers (LDH-B, CKMB, myoglobin, and troponin I) to demonstrate that the combination of PSAQ and SRM (PSAQ-SRM) allows highly accurate biomarker quantification in serum samples. A multiplex PSAQ-SRM assay was used to quantify these biomarkers in clinical samples from myocardial infarction patients. Good correlation between PSAQ-SRM and ELISA assay results was found and demonstrated the consistency between these analytical approaches. Thus, PSAQ-SRM has the capacity to improve both accuracy and reproducibility in protein analysis. This will be a major contribution to efficient biomarker development strategies.
Assuntos
Biomarcadores/sangue , Proteínas Sanguíneas/análise , Doença das Coronárias/sangue , Creatina Quinase Forma MB/sangue , L-Lactato Desidrogenase/sangue , Infarto do Miocárdio/sangue , Mioglobina/sangue , Troponina I/sangue , Estudos de Casos e Controles , Cromatografia Líquida , Doença das Coronárias/diagnóstico , Ensaio de Imunoadsorção Enzimática , Humanos , Isoenzimas/sangue , Espectrometria de Massas , Infarto do Miocárdio/diagnósticoRESUMO
Absolute quantification of proteins using isotope dilution mass spectrometry requires the selection of proteotypic peptides. When choosing these peptides, a certain number of rules must be respected. Several of these were established to safeguard against quantification errors resulting from the isotopically labeled standard peptides not behaving in the same way as the peptides to be quantified. Of all absolute quantification methods using isotope dilution, Protein Standard for Absolute Quantification (PSAQ(TM) ) offers the maximal protein sequence coverage. In the present study, we show that the PSAQ method presents a previously unreported advantage for protein quantification as it makes use of Met/Cys-containing peptides and peptides-containing miscleavages in addition to proteotypic peptides. By increasing the total number of peptides that can be considered, robustness of quantification is improved, paving the way for a facilitated quantification of low abundant and/or low-molecular-weight proteins.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Peptídeos/análise , Proteômica/métodos , Motivos de Aminoácidos , Arginina/química , Cisteína/química , Humanos , Marcação por Isótopo , Lisina/química , Metionina/química , Dados de Sequência Molecular , Peptídeos/sangue , Proteólise , Técnica de Diluição de Radioisótopos , Padrões de Referência , Tripsina/químicaRESUMO
Nivolumab is a fully human immunoglobulin G4 used for the treatment of several advanced solid cancers as immune checkpoint inhibitors. There are some challenges for the quantification of mAb in plasma because IgG are present intrinsically in complex biologic matrices and this determination must be based on reliable, selective, and accurate analytical methods. This study described two validated methods carried out in two separate laboratories, one developed with a triple quadrupole tandem mass spectrometry (LC-MS/MS) and the other with high resolution mass spectrometry with an orbitrap system (LC-MS/HRMS). Both methods used full-length stable isotope-labeled nivolumab-like (Arginine 13C6-15N4 and Lysine 13C6-15N2) as internal standard. The sample preparation was based on IgG immunocapture, then trypsin digestion was performed and one surrogate peptide was quantified in positive mode. Assays showed good linearity over the range of 5-100 µg/mL and 5-150 µg/mL for LC-MS/HRMS and LC-MS/MS, respectively. The limit of quantification was set at 2 and 5 µg/mL for LC-MS/HRMS and LC-MS/MS, respectively. Acceptable accuracy (from - 13.6% to 3.0%) and precision (within 20%) values were also obtained with both methods. The two LC-MS methods showed a very different matrix effect linked to the use of different analytical columns and elution gradients. Nivolumab plasma concentrations from 60 cancer outpatients were compared with the two mass spectrometry methods and also with a home-made ELISA method. The Bland-Altman analysis did not show any significant bias between the three methods. The Passing-Bablock linear regression analysis showed a good agreement between the three methods with a better correlation between the two mass spectrometry methods.
Assuntos
Nivolumabe , Espectrometria de Massas em Tandem , Cromatografia Líquida , Ensaio de Imunoadsorção Enzimática , Humanos , PlasmaRESUMO
A major class of clinical biomarkers is constituted of intracellular proteins which are leaking into the blood following ischemia, exposure to toxic xenobiotics or mechanical aggression. Their ectopic presence in plasma/serum is an indicator of tissue damage and raises a warning signal. These proteins, referred to as cytolysis biomarkers, are generally of cytoplasmic origin and as such, are devoid of glycosylation. In contrast, most plasma/serum proteins originate from the hepatic secretory pathway and are heavily glycosylated (at the exception of albumin). Recent advances in targeted proteomics have supported the parallelized evaluation of new blood biomarkers. However, these analytical methods must be combined with prefractionation strategies that reduce the complexity of plasma/serum matrix. In this article, we present the glycodepletion method, which reverses the hydrazide-based glycocapture concept to remove plasma/serum glycoproteins from plasma/serum matrix and facilitates the detection of cytolysis biomarkers. Glycodepletion was integrated to a targeted proteomics pipeline to evaluate 4 liver cytolysis biomarker candidates in the context of acetaminophen-induced acute hepatitis.
Assuntos
Proteínas Sanguíneas/isolamento & purificação , Glicoproteínas/isolamento & purificação , Proteínas/análise , Proteômica/métodos , Sequência de Aminoácidos , Biomarcadores/análise , Biomarcadores/sangue , Fracionamento Químico/métodos , Cromatografia Líquida/métodos , Glicoproteínas/sangue , Glicosilação , Hepatite/sangue , Humanos , Espectrometria de Massas em Tandem/métodosRESUMO
There is a need for multiplex, specific and quantitative methods to speed-up the development of acute kidney injury biomarkers and allow a more specific diagnosis. Targeted proteomic analysis combined with stable isotope dilution has recently emerged as a powerful option for the parallelized evaluation of candidate biomarkers. This article presents the development of a targeted proteomic assay to quantify 4 acute kidney injury biomarker candidates in urine samples. The proteins included in the assessed panel consisted of myo-inositol oxygenase (MIOX), phosphoenolpyruvate carboxykinase 1 (PCK1), neutrophil gelatinase-associated lipocalin (NGAL) and liver fatty acid-binding protein (L-FABP). The proteomic assay combined an antibody-free sample preparation and a liquid chromatography-selected reaction monitoring (LC-SRM) analysis pipeline. For accurate quantification of the selected candidates, we used PSAQ (Protein Standard Absolute Quantification) standards which are isotopically labeled versions of the target proteins. When added directly to the biological samples, these standards improve detection specificity and quantification accuracy. The multiplexed assay developed for the 4 biomarker candidates showed excellent analytical performance, in line with the recommendations of health authorities. Tests on urine from two small patient cohorts and a group of healthy donors confirmed the relevance of NGAL and L-FABP as biomarkers for AKI diagnosis. The assay is readily adaptable to other biomarker candidates and should be very useful for the simultaneous and accurate quantification of multiple biomarkers.
Assuntos
Injúria Renal Aguda/urina , Proteômica/métodos , Proteômica/normas , Biomarcadores/urina , Proteínas de Ligação a Ácido Graxo/urina , Humanos , Limite de Detecção , Lipocalina-2/urina , Padrões de ReferênciaRESUMO
BACKGROUND: In preclinical studies, monoclonal antibodies (mAbs) are traditionally assayed by ligand-binding-assays. Recently, quantitative liquid chromatography mass spectrometry (MS)-based assays have emerged which circumvent a number of challenges. These assays may also be multiplex, making them potentially compatible with pharmacokinetic assays for combined antibody therapies. MATERIALS & METHODS: We combined a quantitative MS-based approach with the protein standard for absolute quantification (PSAQ™) strategy to simultaneously quantify three mAb variants presenting minor sequence differences. Stable isotopically labeled mAbs were produced and used as quantification standards. Titration curves were performed to assess the analytical performances of the method. LC-MS/MS and ELISA data were cross-compared. RESULTS: The approach presented provides similar accuracy and precision than ELISA, while being multiplex and faster to develop. It has applications at all stages of the pharmaceutical development.
Assuntos
Anticorpos Monoclonais/sangue , Cromatografia Líquida/métodos , Imunoglobulina G/sangue , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G/química , Dados de Sequência Molecular , RatosRESUMO
Absolute protein quantification, i.e. determining protein concentrations in biological samples, is essential to our understanding of biological and physiopathological phenomena. Protein quantification methods based on the use of antibodies are very effective and widely used. However, over the last ten years, absolute protein quantification by mass spectrometry has attracted considerable interest, particularly for the study of systems biology and as part of biomarker development. This interest is mainly linked to the high multiplexing capacity of MS analysis, and to the availability of stable-isotope-labelled standards for quantification. This article describes the details of how to produce, control the quality and use a specific type of standard: Protein Standard Absolute Quantification (PSAQ™) standards. These standards are whole isotopically labelled proteins, analogues of the proteins to be assayed. PSAQ standards can be added early during sample treatment, thus they can correct for protein losses during sample prefractionation and for incomplete sample digestion. Because of this, quantification of target proteins is very accurate and precise using these standards. To illustrate the advantages of the PSAQ method, and to contribute to the increase in its use, selected applications in the biomedical field are detailed here.