Lung Cancer Serum Biomarker Discovery Using Label-Free Liquid Chromatography- Tandem Mass Spectrometry Xuemei Zeng, PhD, Brian L. Hood, PhD, Ting Zhao,

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Lung Cancer Serum Biomarker Discovery Using Label-Free Liquid Chromatography- Tandem Mass Spectrometry Xuemei Zeng, PhD, Brian L. Hood, PhD, Ting Zhao, PhD, Thomas P. Conrads, PhD, Mai Sun, MS, Vanathi Gopalakrishnan, PhD, Himanshu Grover, BS, Roger S. Day, ScD, Joel L. Weissfeld, MD, MPH, David O. Wilson, MD, MPH, Jill M. Siegfried, PhD, William L. Bigbee, PhD Journal of Thoracic Oncology Volume 6, Issue 4, Pages 725-734 (April 2011) DOI: 10.1097/JTO.0b013e31820c312e Copyright © 2011 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 1 Effectiveness of MARS14 depletion of high-abundance serum proteins. Pie charts show the proportions of serum protein mass (A) or spectral counts (B) contributed by high-abundance proteins subjected to MARS14 depletion. The observed spectral counts of lower abundance proteins, which constitute less than 7% of the total serum protein mass, accounted for 93% of the total spectral counts observed in the immunodepleted samples, demonstrating the overall effectiveness of MARS14 depletion. Journal of Thoracic Oncology 2011 6, 725-734DOI: (10.1097/JTO.0b013e31820c312e) Copyright © 2011 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 2 Hierarchical clustering based on spectral counting of the selected significant proteins (p ≤ 0.01). Both sample distance (column distance) and protein feature distance (row distance) were based on Pearson's correlation, and the linkage of both clusters was based on average linkage. The values for spectral counts were standardized for each protein. Green and red colors in the clustering represent under- and overexpression, respectively. The selected features are able to separate case pools (pools P01–P16) from the controls (pools P17–P32) with only two exceptions (pools P15 and P18). P01–P09, adenocarcinoma pools (red labels); P11–P16, squamous cell carcinoma pools (purple); P17–P24, clinical control pools (blue); and P25–P32, healthy control pools (green). Journal of Thoracic Oncology 2011 6, 725-734DOI: (10.1097/JTO.0b013e31820c312e) Copyright © 2011 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 3 MRM analysis and quantitation of clusterin (CLU), serum amyloid A (SAA), alpha-1-acid glycoprotein 1 (AAG1), and alpha-1-acid glycoprotein 2 (AAG2). Spectral counting data indicated that the SAA, AAG1, and AAG2 were differentially abundant between the case and control pools, whereas CLU abundance was independent of case/control pool status. Scatterplot distributions of the logarithm (base 10) of MRM peak areas (average of triplicate MRM assays) according to case/control pool status are shown. Journal of Thoracic Oncology 2011 6, 725-734DOI: (10.1097/JTO.0b013e31820c312e) Copyright © 2011 International Association for the Study of Lung Cancer Terms and Conditions