Projects

Adaptive Mass Spectrometry

Intelligent data acquisition in mass spectrometry driven proteomics can enhance the accuracy and efficiency of proteomics data collection. Using methods like real-time data base searching (search a spectra in less than 10 milliseconds) we are building adaptive strategies to harness spectral data to determine quantitative disparities between cellular states.

Real-Time Spectral Library Matching for Sample Multiplexed Quantitative ProteomicsSample multiplexed quantitative proteomics assays have proved to be a highly versatile means to assay molecular phenotypes. Yet, stochastic precursor selection and precursor coisolation can dramatically reduce the efficiency of data acquisition and quantitative accuracy. To address this, intelligent data acquisition (IDA) strategies have recently been developed to improve instrument efficiency and quantitative accuracy for both discovery and targeted methods. Toward this end, we sought to develop and implement a new real-time spectral library searching (RTLS) workflow that could enable intelligent scan triggering and peak selection within milliseconds of scan acquisition. To ensure ease of use and general applicability, we built an application to read in diverse spectral libraries and file types from both empirical and predicted spectral libraries. We demonstrate that RTLS methods enable improved quantitation of multiplexed samples, particularly with consideration for quantitation from chimeric fragment spectra. We used RTLS to profile proteome responses to small molecule perturbations and were able to quantify up to 15% more significantly regulated proteins in half the gradient time compared to traditional methods. Taken together, the development of RTLS expands the IDA toolbox to improve instrument efficiency and quantitative accuracy for sample multiplexed analyses.

Full-Featured, Real-Time Database Searching Platform Enables Fast and Accurate Multiplexed Quantitative ProteomicsMultiplexed quantitative analyses of complex proteomes enable deep biological insight. While a multitude of workflows have been developed for multiplexed analyses, the most quantitatively accurate method (SPS-MS3) suffers from long acquisition duty cycles. ...

Systems Proteomics

Modern proteomics enables quantitation of up to 10,000 proteins from complex cellular matrices. At the interface of cell biology, biochemistry and proteomics, we are attempting to understand how proteins govern key cellular pathways involved in normal cellular function and disease (e.g. cancer).

Global and tissue-specific aging effects on murine proteomesMaintenance of protein homeostasis degrades with age, contributing to aging-related decline and disease. Previous studies have primarily surveyed transcriptional changes with age. To define the effects of age directly at the protein level, we performed discovery-based proteomics in 10 tissues from 20 C57BL/6J mice, representing both sexes at adult and late midlife ages (8 and 18 months). Consistent with previous studies, age-related changes in protein abundance often have no corresponding transcriptional change. Aging resulted in increases in immune protein abundance across all tissues, consistent with a global pattern of immune infiltration with age. Our protein-centric data revealed tissue-specific aging changes with potential functional consequences, including altered endoplasmic reticulum and protein trafficking in the spleen. We further observed changes in the stoichiometry of protein complexes with important roles in protein homeostasis such as the CCT/TriC complex and large ribosomal subunit. These data provide a foundation for understanding how proteins contribute to systemic aging across tissues. ### Competing Interest Statement The authors have declared no competing interest.

Informatics

The lab is actively developing programs to enable the above projects geared towards adaptive instrument control, data analysis, and data interpretation. Projects under development can be found at our GitHub page.

Improved Monoisotopic Mass Estimation for Deeper Proteome Coverage - PubMedAccurate assignment of monoisotopic peaks is essential for the identification of peptides in bottom-up proteomics. Misassignment or inaccurate attribution of peptidic ions leads to lower sensitivity and fewer total peptide identifications. In the present work, we present a performant, open-source, c …

Host-pathogen Interactions

Multidrug resistant pathogens are a continuing national health concern. To address this we are investigating how pathogen proteins and pathways that govern virulence affect host cellular state through the lens of the proteome.

Host-microbe protein interactions during bacterial infectionInterspecies protein-protein interactions are essential mediators of infection. While bacterial proteins required for host cell invasion and infection can be identified through bacterial mutant library screens, information about host target proteins and ...

Projects

Adaptive Mass Spectrometry

Systems Proteomics

Informatics

Host-pathogen Interactions

University of Washington

Genome Sciences

Projects

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