Nicholas Dupuis (Presenter)
Bio: Nicholas Dupuis is a Senior Scientist at Biodesix, Inc., a leader in development of MALDI-TOF clinical diagnostic assays. Prior to joining Biodesix, Nicholas completed his graduate work with Dr. Bowers at UCSB in the area of ion mobility mass spectrometry instrument development and applications to protein structure analysis. He followed this with a NIST-NRC Post-doctoral Fellowship in JILA at the University of Colorado. His research interests lie in the area of developing new methods to enhance measurement precision in biological matrices to enable development of clinically relevant diagnostic tests.
Authorship: Nicholas F. Dupuis (1), Maximillian Steers (1), Alex A. Nickel (1), Mark W. Duncan (2), Gary Kruppa (3), Christoph H. Borchers (3), and Gary A. Pestano (1)
1) Biodesix, Inc., 2970 Wilderness Place, Ste 100, Boulder, CO, 2) Division of Endocrinology, Metabolism & Diabetes, Department of Medicine Anschutz Medical Campus, University of Colorado, Aurora, CO (3) MRM Proteomics, Inc. 4464 Markham Street, Ste #2108, Victoria, BC, Canada
Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) is a rapid, reproducible, and sensitive tool for measuring protein profiles in biological fluids. Profiling has many potential clinical applications, including diagnosis, predicting disease aggressiveness, and assessing differential therapeutic benefit for cancer patients. In this study we: 1) incorporate multiple internal standards to allow quantitative comparisons between samples and 2) use a proportional-integral-derivative (PID) control algorithm to optimize spectral quality during acquisition. This approach expands the potential applications of MALDI profiling, markedly enhances spectral quality, and reduces reliance on operator intervention during acquisition.
Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) is a rapid, reproducible, and sensitive analytical tool which is finding increasing application in clinical chemistry. For example, profiling approaches compare features within a spectrum and use this information to predict prognosis and/or response to therapy (e.g., 1-2). By contrast, most clinical tests quantify one or a few target analytes within a biological fluid and report values in units of concentration, which are then compared to a reference range/interval. However, valid inter-sample comparisons and precise absolute quantitation with MALDI-TOF MS require the inclusion of an internal standard. Here, we combined elements of both approaches - profiling and quantitation - to generate data that not only allow precise, within spectrum, relative protein abundances to be determined, but also allow us to precisely determine and compare analyte levels across samples. Together, this multi-dimensional information enhances the potential utility of MALDI-TOF MS in the discovery of new clinically relevant biomarker signatures.
METHODS and RESULTS
In MALDI-TOF MS the desorption and ionization processes can contribute substantial variability of signal intensities across samples. To correct for this, and to allow reliable across sample comparisons, we made multiple improvements to the MALDI-TOF acquisition workflow and data acquisition strategy. First we adopted thin-layer (thin film) sample preparation to optimize the homogeneity of the sample surface characterized by smaller crystals. Second, we added internal standard peptides (ACTH fragment 18-39 and Insulin chain B) to normalize the spectra. And, third, we used a proportional-integral-derivative (PID) control algorithm to actively adjust and optimize laser power to enhance the quality of spectral acquisition. All three of these improvements were coupled with a multiple raster acquisition strategy with off-line spectrum filtering and averaging.
The optimized PID workflow was then evaluated on 20 individual diseased samples [10 non-small cell lung cancer (NSCLC), 5 hepatocellular carcinoma (HCC) and 5 cirrhotic] and compared with a more traditional acquisition approach using constant laser power and total-ion-current (TIC) normalization. The inclusion of PID acquisition control and internal normalization standards helped reduce the measurand CVs for a supplemented protein from 27.9% (using traditional methods) to 15% across the diseased sample set. Additionally, six diverse NSCLC samples with biomarker protein concentrations spanning a large dynamic range were evaluated. With these cumulative improvements peak areas (expressed as a ratio of peak/internal standard intensities) the CV fell from over 40% to 16% across the sample set and within each sample the run-to-run reproducibility was good giving normalized peak area CVs of about 7.7%. The MALDI-TOF MS methods were then compared with an orthogonal MRM protein quantitation method with stable-isotope standards (SIS) for absolute quantitation and the two results were found to be linear with an R2 value of >0.998 over >2 orders of magnitude.
The addition of internal standards for spectrum normalization, as well as PID acquisition control, enables generation of high quality MALDI-TOF serum profiles from a complex biofluid (e.g., serum). These spectra not only allow measurement of relative protein abundances but also place the abundances on a linear quantitative scale to enhance inter-sample comparisons. By providing both types of biological information (profiles and quantitation), these improved methods may enable more effective biomarker discovery for clinical applications.
References & Acknowledgements:
1. Taguchi et. al. (2007) Mass Spectrometry to Classify Non–Small-Cell Lung Cancer Patients for Clinical Outcome After Treatment With Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors: A Multicohort Cross-Institutional Study, JNCI 99:838-846.
2. Gregorc et. al. (2014) Predictive value of a proteomic signature in patients with non-small-cell lung cancer treated with second-line erlotinib or chemotherapy (PROSE): a biomarker-stratified, randomised phase 3 trial, The Lancet Oncology 15:713-721
IP Royalty: no
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