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Liquid chromatography coupled with mass spectrometry (LC-MS)-based quantitative proteomics has proven to be an invaluable tool to globally characterize proteins or peptides from complex biological systems. However, the methodology employed for mass spectrometry-based protein quantification remains an area of research interest. This chapter will provide an overview of LC-MS-based label-free quantification approaches, with a focus on a technical overview of the existing experimental practices in both spectral counting and ion intensity-based strategies. As both of these strategies are heavily dependent on informatics manipulation of the acquired data to extract the relevant quantitative information, a brief overview of current software solutions will be also discussed. The final portion of this overview will include a description of best-practice standard operating protocols and experimental design considerations when employing a label-free study. These general practices have been partially developed in our own laboratory over the course of several large-scale clinical studies and their routine incorporation into all of our quantitative workflows have resulted in a quantitatively robust and reproducible label-free platform.

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