MSACL 2018 US Abstract

Topic: Proteomics

Everything You Wanted to Know (sort of) about High Resolution Q-TOF Quantitation of Peptides But Were Afraid to Ask

Michael Lassman (Presenter)
Merck & Co

Bio: Michael Lassman started his career at Merck in 2005 in Diabetes Discovery Research. There, he built a mass spectrometry lab to support a broad range of discovery efforts focused on biomarkers. Michael has held positions with similar responsibilities within the Atherosclerosis and In Vivo Pharmacology groups at Merck and in 2011 assumed his current position within Clinical Development. Michael is a Sr. Principal Scientist and the Director of the Mass Spectrometry laboratory in Merck's Translational Biomarkers group. His group is tasked with developing and implementing biomarker assays to support early and late phase clinical studies within all disease areas at Merck. He has led multiple collaborations with academic partners and CROs and maintains relationships with leading technology companies to develop innovative products. In 2016 Michael added the role of biomarker lead for all o

Authorship: Megan Wang, Michael E Lassman
Merck & Co

Short Abstract

The application of high resolution mass spectrometry (HR-MS) has drawn considerable attention by researchers interested in protein and peptide quantitation. HR-MS could replace traditional QQQ, requiring reduced method development time for targeted quantitation of peptides. Perhaps the paucity of published HR-MS quantitation assays is a direct result of uncertainty regarding how and even if HR-MS can be used for reliable quantitation of peptides. In this study, using oxyntomodulin as a test molecule, we evaluate the effects of different instrument acquisition and processing methods on the sensitivity of a Q-TOF instrument, as defined by accuracy and precision.

Long Abstract

Introduction

The application of high resolution mass spectrometry (HR-MS) has drawn considerable attention by researchers interested in protein and peptide quantitation. HR-MS could replace traditional QQQ, requiring reduced method development time for targeted quantitation of peptides. Furthermore, HR-MS presents an advantage for peptides that are not amenable to fragmentation, high molecular weight peptides and post-translationally modified peptides. Perhaps the paucity of published HR-MS quantitation assays is a direct result of uncertainty regarding how and even if HR-MS can be used for quantitation of peptides. Oxyntomodulin is a peptide hormone containing 37 amino acids and has been the focus of both clinical and preclinical pharmaceutical research due to its prominent effects on weight loss and energy balance. In this study, using oxyntomodulin as a test molecule, we evaluate the effects of different instrument acquisition and processing methods on the sensitivity of a Q-TOF instrument, as defined by accuracy and precision.

Methods

Data was acquired on a Waters Xevo G2-XS with an ionKey nanospray source at a flow rate of 4 µL/min. The Acquity UHPLC consisted of a binary solvent manager and an autosampler. Chromatographic separation was achieved by gradient elution on a Waters Acquity HSS, 1.7 µm, 150 µm x 50 mm column. For each assay, a standard curve was created from 60 pM to 3.3 pM by serial dilution. A stable-isotope labeled internal standard was added to each sample at a constant final concentration of 40 pM. Technical replicates of 6 injections of each sample and regression was carried out. Data analysis was performed with TargetLynx.

Results

HR-MS was used to accurately quantify OXM in plasma at 12.1, 39.8 and 52.7 pM, with CVs varying from 10% to 16%; however it was not able to quantify plasma concentrations below 12.1 pM.

In comparison, using identical sample volumes (0.5 ml plasma), our group previously observed a LOQ of 0.78 pM using selected ion monitoring with a triple quadrupole mass spectrometer (Lee et al., 2016, Clin. Chem. 62:227) using identical sample preparation and chromatography. Another group demonstrated a LOQ of 1.8 pM with full scan HR-MS with an Orbitrap mass spectrometer at 70,000 resolution (Cox et al., 2016, Bioanalysis 8:1579) using different sample preparation and chromatography.

Conclusions & Discussion

Based on the data we collected with the Waters Xevo G2-XS, we identified the following optimal acquisition and processing parameters in terms of LOQ based on accuracy and precision:

The use of profile rather than centroid resulting in modest improvement.

The use of Target Enhancement lowering the LOQ < 2x.

An extraction window size of 2.5x FWHM.

Summing isotopes with greater than 50% of base peak intensity.


References & Acknowledgements:


Financial Disclosure

DescriptionY/NSource
Grantsno
SalaryyesMerck & Co
Board Memberno
Stockyes Merck & Co
Expensesno

IP Royalty: no

Planning to mention or discuss specific products or technology of the company(ies) listed above:

no