Irene van den Broek (Presenter)
Cedars-Sinai Medical Center
Bio: Irene van den Broek is a post-doctoral researcher at the Advanced Clinical Biosystems Research Institute at Cedars-Sinai Medical Center in Los Angeles. Her research focuses on the development and validation of clinical mass spectrometry methods for accurate quantification of proteins in complex matrices, as well as the evaluation of clinical applicability of novel methodologies and technologies.
Authorship: Irene van den Broek (1), Qin Fu (1), A. Lenore Ackerman (2), Jennifer T. Anger (2), Stuart Kushon (3), Kim Chansky (3), Kevin Millis (4), Andrew Percy (4), Tasha Agreste (4), Jennifer E. Van Eyk (1)
(1) Advanced Clinical Biosystems Research Institute, Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA, US; (2) Department of Surgery, Division of Urology, Cedars-Sinai Medical Center, Los Angeles, CA, US; (3) Neoteryx, Torrance, CA, US; (4) Cambridge Isotope Laboratories, Tewksbury, MA, US
| Short Abstract Volumetric absorptive microsampling (VAMS) allows accurate sampling of 10 µL blood from a minimally invasive finger prick and overcomes effects from hematocrit and sample heterogeneity associated with dried blood spots. We describe the first application of VAMS for mass spectrometry-based protein quantification. To achieve the required accuracy for clinical application, specific focus was placed on the realization of automated and high-throughput sample preparation as well as quality control and standardization throughout the assay pipeline. Method validation for six high abundance proteins (apoA-I, apoB, apoC-I, apoC-III, apoE, HSA) includes assessment of sampling precision, short- and long-term storage stability, extraction recovery, intra- and inter-day reproducibility, and comparison to clinical plasma samples using 15N-labeled apoA-I as internal standard. |
Long Abstract
Introduction
Volumetric absorptive microsampling (VAMS) allows accurate sampling of a fixed small volume (10 µL) of blood, while overcoming hematocrit bias and sample heterogeneity issues associated with dried blood spots [1]. Extending the applicability of VAMS from small molecules to proteins would open doors for real-time or personalized applications in (and beyond) clinical diagnostics.
Methods
We describe the integration of accurate sample collection by VAMS, using the MitraTM microsampling device, into an automated platform for robust, high-throughput, and quality controlled, protein extraction and digestion. With regard to clinical application, pre-analytical and analytical performance have been validated using a selected reaction monitoring mass spectrometry assay for six high abundance proteins (apoA-I, apoB, apoC-I, apoC-III, apoE, and human serum albumin), and stable-isotope labeled peptides or 15N-labeled apoA-I as internal standards. In addition, an exogenous protein, beta-galactosidase, and three stable-isotope labeled peptides were added just before digestion to serve as quality control for digestion efficiency among various specimens stored for different periods of time.
Results
First, we evaluated the performance of our automated digestion platform (Biomek NXp), specifically optimized for plasma [2], for blood samples. Trypsin digestion efficiency between blood and plasma was comparable, and complete cleavage was achieved within six hours for all monitored peptides, as well as for 15N-labeled apoA-I.
We then optimized the extraction of proteins from the MitraTM microsamplers by integration into our automated digestion platform. Therefore, the tips of the MitraTM microsampling devices were removed from their sampler bodies and placed in the 96 deep-well plates used for digestion. In this way, the MitraTM tips were kept in solution during the entire process of denaturation, reduction, alkylation, and digestion. The in-tip proteolytic digestion yielded better recovery in comparison to tips that were removed before digestion, and, moreover, the in-tip digestion eliminated the need for (off-line) sonication, as recommended for the extraction of small molecules from MitraTM microsamplers. With this integrated extraction and digestion procedure, average extraction recoveries, as compared to 10 μL blood volumetrically measured by a pipet (n=9), ranged between 100.3% to 111.8% for all peptides at three different hematocrit levels. In addition, the average recovery of the 15N-labeled apoA-I from the VAMS tip was 104% (average of 11 peptides, n=6 for each peptide) when compared to the response of 15N-labeled apoA-I spiked after extraction.
After development of the automated extraction and digestion workflow, we assessed the reproducibility of the total workflow as well as potential pre-analytical sources of inaccuracy, such as effects from sampling and storage. Within- and between day reproducibility of the total workflow was evaluated by the analysis of five replicates prepared on five different days (all within 20 days difference in storage time). The average within-day reproducibility ranged between 3.2-10.4 %CV, whereas total workflow reproducibility varied between 3.4-12.6 %CV for all peptides. In addition, when five untrained individuals each loaded six MitraTM microsamplers with the same source of blood, the average within-individual reproducibility ranged between 4.2-9.3 %CV.
Short- and long-term stability of peptides and proteins in MitraTM microsamplers were assessed by mimicking extreme conditions during shipment (short-term) and storage (long-term), i.e., analysis of three replicates stored at -20oC, 4oC, room temperature, and 37oC after 1.5 and 6.5 days as well as analysis of three replicates after 0, 1, 2, 3, 4 ,8, and 12 weeks storage at room temperature. Whereas certain peptides were not affected by storage time nor temperature, others showed a decrease in response after prolonged storage or by elevated temperatures.Illustratively, for quantification of apoE, two peptides were stable up to 12 weeks storage at room temperature (e.g., 91 ± 5.7% and 95 ± 8.2% of the response at t=0) whereas one peptide showed a declining response during prolonged storage time (e.g., 88 ± 11.7% after two weeks and 57 ± 11.2% after 12 weeks storage). These results emphasize the importance of careful peptide selection for accurate quantification of proteins from MitraTM microsamplers, as well as the relevance to further optimize and standardize storage conditions.
Conclusion
We demonstrated the applicability of MitraTM microsampling devices for accurate quantification of a small number of proteins with selected peptides. Ongoing experiments focus on making our integrated pipeline more widely applicable for quantification of protein biomarkers by (1) optimization of short- and long-term storage stability; (2) comparison between clinical MitraTM and plasma samples from the same patients (n=40 each) for our validated set of proteins and peptides; and (3) further evaluation of the use of 15N-labeled apoA-I as internal standard for absolute and relative quantification.
References & Acknowledgements:
References
1.Denniff, P. & Spooner, N. Volumetric absorptive microsampling: a dried sample collection technique for quantitative bioanalysis. Anal Chem 86, 8489-8495 (2014).
2.Fu, Q. et al. A highly reproducible hands free automated proteomics sample preparation workflow. submitted for publication (2016).
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