62 research outputs found
Protein quantification by LC-MS:A decade of progress through the pages of Bioanalysis
Over the past 10 years, there has been a remarkable increase in the use of LC-MS for the quantitative determination of proteins, and this technique can now be considered an established bioanalytical platform for the quantification of macromolecular drugs and biomarkers, next to the traditional ligand-binding assays. Many researchers have contributed to the field and helped improve both the technical possibilities of LC-MS-based workflows and our understanding of the meaning of the results that are obtained. As a tribute to Bioanalysis, which has published many important contributions, this report gives a high-level overview of the most important trends in the field of protein LC-MS, as published in this journal since its inauguration a decade ago. It describes the major technical developments with regard to sample handling, separation and MS detection of both digested and intact protein analysis. In addition, the relevance of the complex structure and in vivo behavior of proteins is discussed and the effect of protein-protein interactions, biotransformation and the occurrence of isoforms on the analytical result is addressed
Quantitative bioanalysis of proteins by digestion and LC-MS/MS:the use of multiple signature peptides
The use of multiple signature peptides for the quantification of proteins by digestion and LC-MS/MS is reviewed and evaluated here. A distinction is made based on the purpose of the use of multiple peptides: confirmation of the protein concentration, discrimination between different protein forms or species and in vivo biotransformation. Most reports that describe methods with at least two peptides use these for confirmation, but it is not always mentioned how the peptides are used and how possible differences in concentration between the peptides are handled. Differences in concentration are often reported in the case of monitoring different protein forms or in vivo biotransformation, and this offers insight into the biological fate of the protein. </p
Quantitative bioanalysis of proteins by digestion and LC-MS/MS:the use of multiple signature peptides
The use of multiple signature peptides for the quantification of proteins by digestion and LC-MS/MS is reviewed and evaluated here. A distinction is made based on the purpose of the use of multiple peptides: confirmation of the protein concentration, discrimination between different protein forms or species and in vivo biotransformation. Most reports that describe methods with at least two peptides use these for confirmation, but it is not always mentioned how the peptides are used and how possible differences in concentration between the peptides are handled. Differences in concentration are often reported in the case of monitoring different protein forms or in vivo biotransformation, and this offers insight into the biological fate of the protein. </p
Very complex internal standard response variation in LC-MS/MS bioanalysis: Root cause analysis and impact assessment
Internal standards (ISs) are essential for the development and use of reliable quantitative bioanalytical LC-MS/MS methods, because they correct for fluctuations in the analytical response that are caused by variations in experimental conditions. Sample-to-sample differences in the IS response are thus to be expected, but a large variability often is an indication of nonoptimal sample handling or analysis settings. This paper discusses a number of cases of very complex variation of IS responses that could be attributed to analytical problems such as injection errors and sample inhomogeneity, and matrix-related issues such as degradation and increased ionization efficiency. A decision tree is proposed to help find the underlying root cause for extreme IS variability
Non-Antibody-Based Binders for the Enrichment of Proteins for Analysis by Mass Spectrometry
There is often a need to isolate proteins from body fluids, such as plasma or serum, prior to further analysis with (targeted) mass spectrometry. Although immunoglobulin or antibody-based binders have been successful in this regard, they possess certain disadvantages, which stimulated the development and validation of alternative, non-antibody-based binders. These binders are based on different protein scaffolds and are often selected and optimized using phage or other display technologies. This review focuses on several non-antibody-based binders in the context of enriching proteins for subsequent liquid chromatography-mass spectrometry (LC-MS) analysis and compares them to antibodies. In addition, we give a brief introduction to approaches for the immobilization of binders. The combination of non-antibody-based binders and targeted mass spectrometry is promising in areas, like regulated bioanalysis of therapeutic proteins or the quantification of biomarkers. However, the rather limited commercial availability of these binders presents a bottleneck that needs to be addressed
Intact protein bioanalysis by liquid chromatography - High-resolution mass spectrometry
This review discusses the challenges of quantitative protein bioanalysis by LC-MS at the protein level. We will notably address the possibilities and current limitations of protein sample preparation, separation by LC, the challenge of interpreting protein ESI-MS spectra and the options for protein quantification based on extracted ion chromatograms or deconvoluted spectra. The possibilities of high-resolution mass spectrometry (HRMS) with respect to improving the signal-to-noise (S/N) ratio and the challenges of analyzing complex mass spectra will be highlighted based on examples
Monitoring of the deuterated and nondeuterated forms of levodopa and five metabolites in plasma and urine by LC-MS/MS
To compare pharmacokinetics, metabolism and excretion of levodopa and a triply deuterated form, which is being developed as an improved treatment for Parkinson's disease, methods were needed for quantification of the deuterated and nondeuterated forms of levodopa and five metabolites in human plasma and urine. Results: The natural heavy isotopes in the nondeuterated compounds caused an absolute contribution of up to 100% in the response of the deuterated compounds. Similarly, heavy isotopes in the deuterated analytes contributed to the response of the internal standards, but this did not affect the reliability of the results. Conclusion: Deuterated and nondeuterated analytes can be quantified together by LC-MS/MS, but overestimation of the concentrations of the deuterated molecules may be unavoidable and a careful interpretation of the concentration data is essential
Pertuzumab Charge Variant Analysis and Complementarity-Determining Region Stability Assessment to Deamidation
Pertuzumab is a monoclonal antibody used for the treatment of HER2-positive breast cancer in combination with trastuzumab. Charge variants of trastuzumab have been extensively described in the literature; however, little is known about the charge heterogeneity of pertuzumab. Here, changes in the ion-exchange profile of pertuzumab were evaluated by pH gradient cation-exchange chromatography after stressing it for up to 3 weeks at physiological and elevated pH and 37 °C. Isolated charge variants arising under stress conditions were characterized by peptide mapping. The results of peptide mapping showed that deamidation in the Fc domain and N-terminal pyroglutamate formation in the heavy chain are the main contributors to charge heterogeneity. The heavy chain CDR2, which is the only CDR containing asparagine residues, was quite resistant to deamidation under stress conditions according to peptide mapping results. Using surface plasmon resonance, it was shown that the affinity of pertuzumab for the HER2 target receptor does not change under stress conditions. Peptide mapping analysis of clinical samples showed an average of 2-3% deamidation in the heavy chain CDR2, 20-25% deamidation in the Fc domain, and 10-15% N-terminal pyroglutamate formation in the heavy chain. These findings suggest that in vitro stress studies are able to predict in vivo modifications. </p
Enrichment and Liquid Chromatography-Mass Spectrometry Analysis of Trastuzumab and Pertuzumab Using Affimer Reagents
[Image: see text] Trastuzumab and pertuzumab are monoclonal antibodies used in the treatment of human epidermal growth factor receptor-2 (HER2)-positive breast cancer. Therapeutic proteins may undergo chemical modifications that may affect the results of bioanalytical assays, as well as their therapeutic efficacy. Modifications may arise during production and storage, as well as after administration to patients. Studying in vivo biotransformation of monoclonal, therapeutic antibodies requires their enrichment from plasma to discriminate them from endogenous antibodies, as well as from other plasma proteins. To this end, we screened Affimer reagents for selectivity toward trastuzumab or pertuzumab. Affimer reagents are alternative binding proteins possessing two variable binding loops that are based on the human protease inhibitor stefin A or phytocystatin protein scaffolds. Affimer reagents were selected from an extensive library by phage display. The four best-performing binders for each therapeutic antibody were prioritized using a microtiter plate-based approach combined with liquid chromatography–mass spectrometry (LC–MS) in the selected reaction monitoring (SRM) mode. These Affimer reagents were immobilized via engineered 6-His or Cys tags to Ni(2+)- or maleimide beads, respectively. Recovery values of 70% and higher were obtained for both trastuzumab and pertuzumab when spiked at 100, 150, and 200 μg/mL concentrations in human plasma followed by trypsin digestion in the presence of 0.5% sodium deoxycholate and 10 mM dithiothreitol (DTT). Notably, the maleimide beads showed undetectable unspecific binding to endogenous immunoglobulin G (IgGs) or other plasma proteins when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enrichment method was applied to samples from stress tests of the antibodies at 37 °C to mimic in vivo conditions
Pertuzumab Charge Variant Analysis and Complementarity-Determining Region Stability Assessment to Deamidation
Pertuzumab is a monoclonal antibody used for the treatment of HER2-positive breast cancer in combination with trastuzumab. Charge variants of trastuzumab have been extensively described in the literature; however, little is known about the charge heterogeneity of pertuzumab. Here, changes in the ion-exchange profile of pertuzumab were evaluated by pH gradient cation-exchange chromatography after stressing it for up to 3 weeks at physiological and elevated pH and 37 °C. Isolated charge variants arising under stress conditions were characterized by peptide mapping. The results of peptide mapping showed that deamidation in the Fc domain and N-terminal pyroglutamate formation in the heavy chain are the main contributors to charge heterogeneity. The heavy chain CDR2, which is the only CDR containing asparagine residues, was quite resistant to deamidation under stress conditions according to peptide mapping results. Using surface plasmon resonance, it was shown that the affinity of pertuzumab for the HER2 target receptor does not change under stress conditions. Peptide mapping analysis of clinical samples showed an average of 2-3% deamidation in the heavy chain CDR2, 20-25% deamidation in the Fc domain, and 10-15% N-terminal pyroglutamate formation in the heavy chain. These findings suggest that in vitro stress studies are able to predict in vivo modifications. </p
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