GEN-MKT-18-7897-A
Aug 22, 2017 | Biopharma, Blogs | 0 comments
The quantitation of proteins using the surrogate peptide approach can complicate nominal mass Triple Quadrupole MRM measurements due to co-extracted interference when using non-selective extraction techniques such as pellet digestion. High resolution coupled with accurate mass filtering can mitigate such interference, as reported previously for the determination of rituximab using the TripleTOF 6600 (Protein Quant Approaches). However, an additional level of selectivity can often be achieved on nominal mass systems using the orthogonal gas-phase separation approach offered by the SelexION+® Differential Mobility System technology (DMS). Interfaced between the sampling orifice and ion source, the DMS separates ions based upon differences in their migration rates under alternating low and high field waveform amplitudes (Figure 1). Ion clustering in low fields and declustering in high fields amplifies the distinction in mobility of an ion, resulting in improved resolution from interfering species of differing molecular cross-section.1-4
Applications of the DMS are often reported for cyclic peptides exhibiting poor fragmentation efficiency in order to discriminate against high matrix/chemical background noise when acquiring data using pseudo-MRM or MIM (multiple ion monitoring). In these approaches, parent ion m/z is used for both Q1/Q3, with or without a collision energy offset, respectively. For example, an LC-DMS-MIM approach was implemented by Fu et al.5 on a QTRAP 6500 for the cyclic peptide Pasireotide, achieving a 10 pg/mL detection limit in human plasma, three-fold lower than previous RIA methods and five-fold lower than LC-MRM. Researchers at Ironwood Pharmaceuticals noted similar performance for their cyclic peptide PN1944, where DMS eliminated significant interference in MIM mode.6
In the current blog, the ability of the SelexION+ system to deconvolute analyte from LOQ-limiting matrix interference is evaluated for the determination of rituximab in human plasma using a dual peptide quantitation approach monitoring the light chain (LC) and heavy chain (HC) tryptic signature peptides detailed in Table 1. In the absence of SelexION+ technology, the LOQ of the Triple Quad 6500+ MRM assay was limited by interference at the retention time of the light chain, whilst the HC was plagued by closely eluting peaks (Figure 2). Consequently, the HC peptide was precise, accurate and linear from 4.00 – 400 µg/mL, whilst quantitation of the LC peptide could only be achieved from 20 – 400 µg/mL (Table 2). The high level of interference for both peptide MRM transitions may be attributed to the lack of sample preparation selectivity when using pellet digestion.
Optimization of the DMS for the LC and HC peptides involved examination of both separation voltage (SV) and compensation voltage (CoV) as a function of temperature and chemical modifier. In these experiments, the SV was stepped and the CoV scanned from -25 V to +25 V. Exemplary ionograms of response vs. CoV at each SV step are presented in Figure 3 for the LC peptide, indicating optimal response at an SV of 3.8 kVp-p (CoV 13 V); the HC peptide CoV at the same SV was11.2 V. Of the chemical modifiers screened, the addition of 2-propanol to the carrier gas stream (1.5% mole ratio) imparted maximal differentiation in mobility between surrogate peptides and interference.
The determination of transport gas temperature was critical to DMS performance, as this parameter is inversely proportional to gas density and thus impacts collisional frequency. Further, its effect on the CoV peak position can be dramatic because of changes in both E/N ratio (electric field strength, E, normalized to the gas number density, N) and cluster ion state. For the LC of Rituximab, an increase in temperature from 150°C to 300°C increased the CoV ca. 10 V whilst concomitantly augmenting sensitivity (Figure 4).
Implementation of the optimized SelexION+ conditions detailed above resulted in the elimination of closely eluting peaks previously observed for the HC and the achievement of the 4.00 µg/mL LOQ target for the LC (Figure 5). For the dual quantitation approach, intra-day precision was < 12% with accuracies between 96% -110% for all QCs (Table 3). The DMS responses for both LC and HC SIL peptides in an extracted batch were notably stable over a 24 hr period (200 injections) with CV ca. 10% (Figure 6).
ConclusionsIn leveraging the additional stage of gas-phase selectivity offered by the SelexION+ technology, the sensitivity and specificity goals of Rituximab were realized, due largely to the elimination of the LC peptide interference which otherwise limited the overall achievable LOQ. Additional advantages of the SelexION+ technology include simplification of extraction methods (e.g. elimination of complex immunoaffinity workflows), shorter chromatographic runs and improved peak integration.The combined benefits of SelexION+ technology are particularly relevant to peptides as sensitivity is often compromised due to low bioavailability, formation of multiple-charge states, and poor to no fragmentation.
AcknowledgementsThe author would like to thank Jean-Nicholas Mess from Algorithme Pharma, an Altasciences Company, for his valuable contributions.
References
Produced by certain moulds, thriving in crops such as grain, nuts and coffee, mycotoxins have contaminated agriculture and food production industries for a long time. To intensify the challenge, mycotoxins are resilient, not easily broken down and ensuring the safety of food supply chains requires comprehensive solutions and we are here to share those solutions with you.
Electron-Activated Dissociation (EAD) is transforming the fields of metabolomics and lipidomics by providing enhanced fragmentation techniques that offer deeper insights into molecular structures. In September, Technology Networks hosted a webinar, “Enhancing Mass-Based Omics Analysis in Model Organisms,” featuring Dr. Valentina Calabrese from the Institute of Analytical Sciences at the University of Lyon. Valentina shared her insights on improving omics-based mass spectrometry analysis for toxicology studies using model organisms, particularly in metabolomics and lipidomics. This blog explores the additional functionalities EAD offers, its benefits in untargeted workflows, its incorporation into GNPS and molecular networking, and the future role it could play in these scientific domains.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has gained significant attention in the clinical laboratory due to its ability to provide best-in-class sensitivity and specificity for the detection of clinically relevant analytes across a wide range of assays. For clinical laboratories new to LC-MS/MS, integrating this technology into their daily routine operations may seem like a daunting task. Developing a clear outline and defining the requirements needed to implement LC-MS/MS into your daily operations is critical to maximize the productivity and success of your clinical laboratory.
Posted by
You must be logged in to post a comment.
Share this post with your network