MacCoss, Michael JNoble, William SPino, Lindsay Kristina2019-10-152019-10-152019-10-152019Pino_washington_0250E_20741.pdfhttp://hdl.handle.net/1773/44823Thesis (Ph.D.)--University of Washington, 2019The field of mass spectrometry proteomics has made great technological progress, and these techniques are now being used to address essential questions in basic biology and are increasingly being used on samples of clinical significance. In particular, the development of data independent acquisition mass spectrometry (DIA-MS) has made it possible to measure tens of thousands of peptides from a protein digest in a 1-2 hour time scale. As generating larger and larger proteomic data sets becomes easier and easier, questions about normalizing batch effects and assessing data quality have arisen in the mass spectrometry community. In the following chapters, I describe three projects that aimed to address various challenges associated with scaling up quantitative mass spectrometry experiments. I first introduce the need for reference materials for mass spectrometry proteomics. In Chapter 2, I describe a single-point external calibration strategy to calibrate signal intensity measurements to a common reference material, which places MS measurements on the same scale and harmonizes signal intensities between instruments, acquisition methods, and sites. In Chapter 3, I extend the reference material calibration approach to multi-point calibration and demonstrate the consequences of linearity in quantitative analyses. We apply this approach to yeast lysate, human cerebrospinal fluid, and formalin-fixed paraffin-embedded samples. In Chapter 4, I apply the methods developed in the previous two chapters to investigate the yeast proteome response to genetic and environmental modulators of replicative lifespan. I show that the protein-level signatures associated with replicative lifespan extension suggest a higher-level response beyond protein abundances. Lastly, I present closing remarks and future directions in Chapter 5.application/pdfen-USCC BY-NC-NDmass spectrometryproteomicsquantitative proteomicsBiochemistryGeneticsGeneticsThesis