Systematic optimization of targeted and multiplexed MS-based screening workflows for protein biomarkers.

Background: The capability of targeted MS-based methods to simultaneously measure multiple analytes with high selectivity and sensitivity greatly facilitates the discovery and quantitation of novel biomarkers. However, the complexity of biological samples is a major bottleneck that requires extensive sample preparation. Results: This paper reports a generic workflow to optimize surrogate peptide-based protein biomarker screening for seven human proteins in a multiplexed manner without the need for any specific affinity reagents. Each step of the sample processing and LC-MS methods is systematically assessed and optimized for better analytical performance. Conclusion: The established method is used for the screening of multiple myeloma patient samples to determine which proteins could be robustly measured and serve as potential biomarkers of the disease.

Intact mAb LC-MS for drug concentration from pre-clinical studies: bioanalytical method performance and in-life samples.

Background: Antibody biotherapeutic measurement from pharmacokinetic studies has not been traditionally based on intact molecular mass as is the case for small molecules. However, recent advancements in protein capture and mass spectrometer technology have enabled intact mass detection and quantitation for dosed biotherapeutics. A bioanalytical method validation is part of the regulatory requirement for sample analysis to determine drug concentration from in-life study samples. Results/methodology: Here, an intact protein LC-MS assay is subjected to mock bioanalytical method validation, and unknown samples are compared between intact protein LC-MS and established bioanalytical assay formats: Ligand-binding assay and peptide LC-MS/MS. Discussion/conclusion: Results are presented from the intact and traditional bioanalytical method evaluations, where the in-life sample concentrations were comparable across method types with associated data analyses presented. Furthermore, for intact protein LC-MS, modification monitoring and evaluation of data processing parameters is demonstrated.

Metabolite identification using an ion mobility enhanced data-independent acquisition strategy and automated data processing.

RATIONALE: Liquid chromatography/mass spectrometry is an essential tool for efficient and reliable quantitative and qualitative analysis and underpins much of contemporary drug metabolism and pharmacokinetics. Data-independent acquisition methods such as MSE have reduced the potential to miss metabolites, but do not formally generate quadrupole-resolved product ion spectra. The addition of ion mobility separation to these approaches, for example, in High-Definition MSE (HDMSE ) has the potential to reduce the time needed to set up an experiment and maximize the chance that all metabolites present can be resolved and characterized. We compared High-Definition Data-Dependent Acquisition (HD-DDA), MSE and HDMSE approaches using automated software processing with Mass-MetaSite and WebMetabase. METHODS: Metabolite identification was performed on incubations of glucagon-like peptide-1 (7-37) (GLP-1) and verapamil hydrochloride. The HD-DDA, MSE and HDMSE experiments were conducted on a Waters ACQUITY UPLC I-Class LC system with a VION IMS quadrupole time-of-flight (QTOF) mass spectrometer operating under UNIFI control. All acquired data were processed using MassMetaSite able to read data from UNIFI 1.9.4. WebMetabase was used to review the detected chromatographic peaks and the spectral data interpretations. RESULTS: A comparison of outcomes obtained for MSE and HDMSE data demonstrated that the same structures were proposed for metabolites of both verapamil and GLP-1. The ratio of structurally matched to mismatched product ions found by MassMetaSite was slightly greater for HDMSE than for MSE , and HD-DDA, thus improving confidence in the structures proposed through the addition of ion mobility based data acquisitions. CONCLUSIONS: HDMSE data acquisition is an effective approach for the elucidation of metabolite structures for both small molecules and peptides, with excellent accuracy and quality, requiring minimal tailoring for the compound under investigation.

Microflow UPLC and high-resolution MS as a sensitive and robust platform for quantitation of intact peptide hormones.

Aim: Recent advances in microflow ultra performance liquid chromatography (UPLC) systems offer higher sensitivity with robustness to meet the routine bioanalytical demands. Modern high-resolution mass spectrometers (HRMS) enable the development of highly selective methods with broad dynamic range. Results: The quantitative performances of tandem quadrupole MS and HRMS were comprehensively compared using seven intact peptide hormones up to 9.4 kDa. Results show comparable performance between two platforms in sensitivity, accuracy and linearity. For some peptides, HRMS provided lower background interference. The benefit of increased sensitivity using microflow UPLC was also demonstrated. Conclusion: HRMS is a versatile platform capable of both basic characterization and reliable quantitation in complex matrices. Microflow UPLC provides lower LLOQs than conventional flow systems, even with less sample volume injected.

Integration of microfluidic LC with HRMS for the analysis of analytes in biofluids: past, present and future.

Capillary LC (cLC) coupled to MS has the potential to improve detection limits, address limited sample volumes and allow multiple analyses from one sample. This is particularly attractive in areas where ultrahigh assay sensitivity, low limits of detection and small sample volumes are becoming commonplace. However, implementation of cLC-MS in the bioanalytical-drug metabolism area had been hampered by the lack of commercial instrumentation and the need for experts to operate the system. Recent advances in microfabricated devices such as chip-cube and ion-key technologies offer the potential for true implementation of cLC in the modern laboratory including the benefits of the combination of this type of separation with high-resolution MS.