RESUMEN
Rituximab is a chimeric immunoglobulin G1-kappa (IgG1κ) antibody targeting the CD20 antigen on B-lymphocytes. Its applications are various, such as for the treatment of chronic lymphoid leukemia or non-Hodgkin's lymphoma in oncology, and it can also be used in the treatment of certain autoimmune diseases. Several studies support the interest in therapeutic drug monitoring to optimize dosing regimens of rituximab. Thus, two different laboratories have developed accurate and reproductive methods to quantify rituximab in human plasma: one using liquid chromatography quadripolar tandem mass spectrometer (LC-MS/MS) and the other, liquid chromatography orbitrap tandem mass spectrometer (LC-MS/HRMS). For both assays, quantification was based on albumin depletion or IgG-immunocapture, surrogate peptide analysis, and full-length stable isotope-labeled rituximab. With LC-MS/MS, the concentration range was from 5 to 500 µg/mL, the within- and between-run precisions were <8.5%, and the limit of quantitation was 5 µg/mL. With LC-MS/HRMS, the concentration range was from 10 to 200 µg/mL, the within- and between-run accuracy were <11.5%, and the limit of quantitation was 2 µg/mL. Rituximab plasma concentrations from 63 patients treated for vasculitis were compared. Bland-Altman analysis and Passing-Bablok regression showed the interchangeability between these two methods. Overall, these methods were robust and reliable and could be applied to routine clinical samples.
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Antineoplásicos Inmunológicos/sangre , Cromatografía Liquida/métodos , Linfoma/sangre , Rituximab/sangre , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masas en Tándem/métodos , Vasculitis/sangre , Antineoplásicos Inmunológicos/administración & dosificación , Monitoreo de Drogas , Humanos , Marcaje Isotópico , Linfoma/tratamiento farmacológico , Linfoma/patología , Reproducibilidad de los Resultados , Rituximab/administración & dosificación , Vasculitis/tratamiento farmacológico , Vasculitis/patologíaRESUMEN
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.
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Anticuerpos Monoclonales/sangre , Isótopos de Carbono/sangre , Cetuximab/sangre , Isótopos de Nitrógeno/sangre , Plasma/química , Cromatografía Líquida de Alta Presión/métodos , Humanos , Marcaje Isotópico/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
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
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Cromatografía Liquida/métodos , Infliximab/sangre , Espectrometría de Masas en Tándem/métodos , Ensayo de Inmunoadsorción Enzimática , HumanosRESUMEN
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.
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Proteínas/química , Proteómica , Secuencia de Aminoácidos , Animales , Biomarcadores/química , Cromatografía Liquida , Humanos , Cinética , Masculino , Espectrometría de Masas , Ratones , Datos de Secuencia Molecular , Proteolisis , Control de CalidadRESUMEN
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.
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Biomarcadores/sangre , Proteínas Sanguíneas/análisis , Enfermedad Coronaria/sangre , Forma MB de la Creatina-Quinasa/sangre , L-Lactato Deshidrogenasa/sangre , Infarto del Miocardio/sangre , Mioglobina/sangre , Troponina I/sangre , Estudios de Casos y Controles , Cromatografía Liquida , Enfermedad Coronaria/diagnóstico , Ensayo de Inmunoadsorción Enzimática , Humanos , Isoenzimas/sangre , Espectrometría de Masas , Infarto del Miocardio/diagnósticoRESUMEN
BACKGROUND: Emicizumab has been approved for the prophylaxis of patients with hemophilia A with or without inhibitors. However, spontaneous and trauma-induced breakthrough bleeds have been reported in patients on emicizumab prophylaxis, and no laboratory assay has been validated to evaluate the hemostatic activity of emicizumab. OBJECTIVES: The thrombin generation assay (TGA) could be a surrogate marker of the hemostatic efficacy of emicizumab. The correlation between TGA and the methods used to measure emicizumab blood concentration was evaluated in this study. METHODS: TGA was modified by the use of a trigger reagent combining a very low concentration of tissue factor and activated factor (F)XI. Emicizumab quantification was performed by 3 methods: the modified 1-step FVIII assay and 2 methods based on liquid chromatography and mass spectrometry (LC-MS). RESULTS: Using tissue factor/activated FXI-triggered TGA and platelet-poor plasma, a relationship was observed between the area under the thrombin generation curve (endogenous thrombin potential [ETP]) and the clinical response of patients to emicizumab. The ultrastructure of fibrin clots was consistent with ETP results and showed that emicizumab had a hemostatic activity equivalent to 20 to 30 IU/dL of FVIII. Finally, pharmacokinetic/pharmacodynamic analyses showed no correlation between ETP and LC-MS nor with modified 1-stage FVIII assay, but a statistically significant correlation between the LC-MS methods and the time-to-peak results of the TGA. CONCLUSION: Using a modified TGA, this study showed that patients who experienced breakthrough bleeds while on emicizumab had a lower thrombin-generating capacity compared with others with good clinical response to emicizumab.
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Anticuerpos Biespecíficos , Anticuerpos Monoclonales Humanizados , Hemofilia A , Hemostasis , Trombina , Humanos , Hemofilia A/tratamiento farmacológico , Hemofilia A/sangre , Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales Humanizados/farmacocinética , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/farmacocinética , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Biespecíficos/sangre , Trombina/metabolismo , Adulto , Hemostasis/efectos de los fármacos , Masculino , Adulto Joven , Adolescente , Hemorragia/sangre , Persona de Mediana Edad , Resultado del Tratamiento , Cromatografía Liquida , Pruebas de Coagulación Sanguínea , Hemostáticos/uso terapéutico , Hemostáticos/farmacología , Hemostáticos/farmacocinética , Espectrometría de Masas , Coagulación Sanguínea/efectos de los fármacos , Niño , Índice de Severidad de la EnfermedadRESUMEN
Belatacept, a CTLA4-Ig, was designed to prevent rejection and graft loss in kidney transplant recipients. This immunotherapy showed a long-term clinical benefit mainly on renal function and better glycemic control but was also associated with a higher number of severe infectious diseases, particularly CMV disease, and lymphoproliferative disease. Therapeutic drug monitoring usually guides the benefit-risk assessment of long-term immunosuppression. In this study, an analytical method by LC-MS/MS was developed in 20 microL of plasma for the belatacept quantification. Intra- and inter-assay precision and accuracy were lower than 20% for the limit of quantification, and 15% for higher concentrations. The method was implemented in our lab and provided data about the inter-variability (N = 108) and intra-variability (N = 33) of belatacept concentrations in kidney transplant recipients with a stable renal function, after conversion from a CNI- to a belatacept-based regimen.
RESUMEN
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.
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Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Péptidos/análisis , Proteómica/métodos , Secuencias de Aminoácidos , Arginina/química , Cisteína/química , Humanos , Marcaje Isotópico , Lisina/química , Metionina/química , Datos de Secuencia Molecular , Péptidos/sangre , Proteolisis , Técnica de Dilución de Radioisótopos , Estándares de Referencia , Tripsina/químicaRESUMEN
Background: This study compared the performance of plasma infliximab and adalimumab quantification using a commercially available kit (mAbXmise kit) and mass spectrometry readout to that of two ELISA methods in patients treated for inflammatory bowel disease. Methods & results: The mAbXmise method based on liquid chromatography tandem mass spectrometry (LC-MS/MS) was linear from 2 to 100 µg/ml. It was validated according to international guidelines. Regarding cross-validation for infliximab (n = 70), the mean bias with LC-MS/MS assay was approximately threefold higher with the commercial ELISA assay compared with the in-house ELISA (-6.1 vs -1.8 µg/ml, respectively). The mean bias between the LC-MS/MS assay and in-house ELISA was -1.2 µg/ml for adalimumab (n = 35). Conclusion: The LC-MS/MS method is a powerful alternative to immunoassays to monitor concentrations of infliximab and adalimumab.
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Espectrometría de Masas en Tándem , Adalimumab , Cromatografía Liquida/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Infliximab , Espectrometría de Masas en Tándem/métodosRESUMEN
Pembrolizumab is a humanized immunoglobulin G4-kappa anti-PD1 antibody used in the treatment of different solid tumors or haematological malignancies. A liquid chromatography coupled with a high resolution mass spectrometry (orbitrap technology) method was fully developed, optimized, and validated for quantitative analysis of pembrolizumab in human plasma. A mass spectrometry assay was used for the first time a full-length stable isotope-labelled pembrolizumab-like (Arginine 13C6-15N4 and Lysine 13C6-15N2) as an internal standard; the sample preparation was based on albumin depletion and trypsin digestion and, finally, one surrogate peptide was quantified in positive mode. The assay showed good linearity over the range of 1-100 µg/mL, a limit of quantification at 1 µg/mL, excellent accuracy from 4.4% to 5.1%, and also a between-day precision below 20% at the limit of quantification. In parallel, an in-house ELISA was developed with a linearity range from 2.5 to 50 µg/mL. Then, results were obtained from 70 plasma samples of cancer patients that were treated with pembrolizumab and quantified with both methods were compared using the Passing-Bablok regression analysis and Bland-Altman plotting. The LC-MS/HRMS method is easy to implement in the laboratory for use in the context of PK/PD studies, clinical trials, or therapeutic drug monitoring.
RESUMEN
BACKGROUND: Different liquid chromatography tandem mass spectrometry (LC-MS/MS) methods have been published for quantification of monoclonal antibodies (mAbs) in plasma but thus far none allowed the simultaneous quantification of several mAbs, including immune checkpoint inhibitors. We developed and validated an original multiplex LC-MS/MS method using a ready-to-use kit to simultaneously assay 7 mAbs (i.e., bevacizumab, cetuximab, ipilimumab, nivolumab, pembrolizumab, rituximab and trastuzumab) in plasma. This method was next cross-validated with respective reference methods (ELISA or LC-MS/MS). METHODS: The mAbXmise kit was used for mAb extraction and full-length stable-isotope-labeled antibodies as internal standards. The LC-MS/MS method was fully validated following current EMA guidelines. Each cross validation between reference methods and ours included 16-28 plasma samples from cancer patients. RESULTS: The method was linear from 2 to 100 µg/mL for all mAbs. Inter- and intra-assay precision was <14.6% and accuracy was 90.1-111.1%. The mean absolute bias of measured concentrations between multiplex and reference methods was 10.6% (range 3.0-19.9%). CONCLUSIONS: We developed and cross-validated a simple, accurate and precise method that allows the assay of up to 7 mAbs. Furthermore, the present method is the first to offer a simultaneous quantification of three immune checkpoint inhibitors likely to be associated in patients.
RESUMEN
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.
Asunto(s)
Nivolumab , Espectrometría de Masas en Tándem , Cromatografía Liquida , Ensayo de Inmunoadsorción Enzimática , Humanos , PlasmaRESUMEN
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.
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Anticuerpos Monoclonales/sangre , Cromatografía Liquida/métodos , Inmunoglobulina G/sangre , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina G/química , Datos de Secuencia Molecular , RatasRESUMEN
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.
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Pruebas de Química Clínica/normas , Espectrometría de Masas/normas , Proteínas/análisis , Proteómica/normas , Secuencia de Aminoácidos , Tampones (Química) , Pruebas de Química Clínica/métodos , Enterotoxinas/análisis , Enterotoxinas/química , Contenido Digestivo/química , Humanos , Concentración de Iones de Hidrógeno , Factor I del Crecimiento Similar a la Insulina/análisis , Factor I del Crecimiento Similar a la Insulina/química , Isótopos/química , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Isoformas de Proteínas , Estabilidad Proteica , Proteínas/química , Proteómica/métodos , Estándares de Referencia , Infecciones Estafilocócicas/microbiología , Troponina I/análisis , Troponina I/químicaRESUMEN
Enterotoxin A (SEA) is a staphylococcal virulence factor which is suspected to worsen septic shock prognosis. However, the presence of SEA in the blood of sepsis patients has never been demonstrated. We have developed a mass spectrometry-based assay for the targeted and absolute quantification of SEA in serum. To enhance sensitivity and specificity, we combined an immunoaffinity-based sample preparation with mass spectrometry analysis in the selected reaction monitoring (SRM) mode. Absolute quantification of SEA was performed using the PSAQ™ method (Protein Standard Absolute Quantification), which uses a full-length isotope-labeled SEA as internal standard. The lower limit of detection (LLOD) and lower limit of quantification (LLOQ) were estimated at 352pg/mL and 1057pg/mL, respectively. SEA recovery after immunocapture was determined to be 7.8±1.4%. Therefore, we assumed that less than 1femtomole of each SEA proteotypic peptide was injected on the liquid chromatography column before SRM analysis. From a 6-point titration experiment, quantification accuracy was determined to be 77% and precision at LLOQ was lower than 5%. With this sensitive PSAQ-SRM assay, we expect to contribute to decipher the pathophysiological role of SEA in severe sepsis. This article is part of a Special Issue entitled: Proteomics: The clinical link.
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Análisis Químico de la Sangre/métodos , Análisis Químico de la Sangre/normas , Enterotoxinas/análisis , Enterotoxinas/sangre , Infecciones Estafilocócicas/sangre , Staphylococcus aureus/metabolismo , Secuencia de Aminoácidos , Enterotoxinas/química , Enterotoxinas/aislamiento & purificación , Enterotoxinas/metabolismo , Humanos , Inmunoquímica/métodos , Inmunoquímica/normas , Espectrometría de Masas/métodos , Espectrometría de Masas/normas , Modelos Biológicos , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/química , Proteínas/análisis , Proteínas/química , Proteómica/métodos , Proteómica/normas , Estándares de Referencia , Sensibilidad y Especificidad , Infecciones Estafilocócicas/diagnóstico , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/aislamiento & purificaciónRESUMEN
In the field of analytical chemistry, stable isotope dilution assays are extensively used in combination with liquid chromatography-mass spectrometry (LC-MS) to provide confident quantification results. Over the last decade, the principle of isotope dilution has been adopted by the proteomic community in order to accurately quantify proteins in biological samples. In these experiments, a protein's concentration is deduced from the ratio between the MS signal of a tryptic peptide and that of a stable isotope-labeled analog, which serves as an internal standard. The first isotope dilution standards introduced in proteomics were chemically synthesized peptides incorporating a stable isotope-tagged amino acid. These isotopically labeled peptide standards, which are currently widely used, are generally added to samples after protein isolation and digestion. Thus, if protein enrichment is necessary, they do not allow correction for protein losses that may occur during sample pre-fractionation, nor do they allow the tryptic digestion yield to be taken into account. To reduce these limitations we have developed the PSAQ (Protein Standard Absolute Quantification) strategy using full-length stable isotope-labeled proteins as quantification standards. These standards and the target proteins share identical biochemical properties. This allows standards to be spiked into samples at an early stage of the analytical process. Thanks to this possibility, the PSAQ method provides highly accurate quantification results, including for samples requiring extensive biochemical pre-fractionation. In this chapter, we describe the production of full-length stable isotope-labeled proteins (PSAQ standards) using cell-free expression devices. The purification and quality control of protein standards, crucial for good-quality and accurate measurements, are also detailed. Finally, application of the PSAQ method to a typical protein quantification assay is presented.
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Marcaje Isotópico/métodos , Proteínas/química , Proteómica/métodos , Aminoácidos/química , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Control de Calidad , Estándares de ReferenciaRESUMEN
For high-throughput structural studies of protein complexes of composition inferred from proteomics data, it is crucial that candidate complexes are selected accurately. Herein, we exemplify a procedure that combines a bioinformatics tool for complex selection with in vivo validation, to deliver structural results in a medium-throughout manner. We have selected a set of 20 yeast complexes, which were predicted to be feasible by either an automated bioinformatics algorithm, by manual inspection of primary data, or by literature searches. These complexes were validated with two straightforward and efficient biochemical assays, and heterologous expression technologies of complex components were then used to produce the complexes to assess their feasibility experimentally. Approximately one-half of the selected complexes were useful for structural studies, and we detail one particular success story. Our results underscore the importance of accurate target selection and validation in avoiding transient, unstable, or simply nonexistent complexes from the outset.
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Biología Computacional/métodos , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Bases de Datos de Proteínas , Proteómica , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
To comply with current proteomics guidelines, it is often necessary to analyze the same peptide samples several times. Between analyses, the sample must be stored in such a way as to conserve its intrinsic properties, without losing either peptides or signal intensity. This article describes two studies designed to define the optimal storage conditions for peptide samples between analyses. With the use of a label-free strategy, peptide conservation was compared over a 28-day period in three different recipients: standard plastic tubes, glass tubes, and low-adsorption plastic tubes. The results of this study showed that standard plastic tubes are unsuitable for peptide storage over the period studied. Glass tubes were found to perform better than standard plastic, but optimal peptide recovery was achieved using low-adsorption plastic tubes. The peptides showing poor recovery following storage were mainly hydrophobic in nature. The differences in peptide recovery between glass and low-adsorption plastic tubes were further studied using isotopically labeled proteins. This study allowed accurate comparison of peptide recovery between the two tube types within the same LC-MS run. The results of the label-free study were confirmed. Further, it was possible to demonstrate that peptide recovery in low-adsorption plastic tubes was optimal whatever the peptide concentration stored.