? TR&D 1 The overarching goal of the Resource is to dramatically improve on the existing proteomics paradigm by achieving orders-of-magnitude gains in both measurement sensitivity and throughput, as well as to address important measurement `blind spots' and shortcomings of current methods. At present, a minimum of thousands of cells are generally required for in-depth coverage of proteins in a biological sample, precluding many important applications involving extremely small samples, rare cell types or spatially resolved measurements. We have recently developed a Nanodroplet Processing in One-pot for Trace Samples (nanoPOTS) technology which, when coupled with ultrasensitive MS-based measurements, enables effective analysis of as few as 10 mammalian cells. The technology efforts of TR&D 1 will extend this robotic/microfluidic nanoPOTS platform to provide the `up-front' processing required to efficiently handle ultra-small samples (extending to single cells) and deliver these samples optimally to the Structures for Lossless Ion Manipulations (SLIM)-based ion mobility-mass spectrometry (IM-MS) platforms to be further advanced under TR&D 2. The robotic platform will enable samples to be transferred using nanoelectrospray ionization to the SLIM IM-MS platforms with high ionization and utilization efficiencies. The nanoPOTS platform and workflow will be optimized to enable broad proteome coverage for sample sizes in the range of 1-1000 cells so as to enable broad proteome coverage. The efforts will also establish seamless, automated integration of nanoPOTS with the widely used cell isolation technologies of fluorescence activated cell sorting (FACS) and laser-capture microdissection (LCM), enabling ultra-rare cell analysis and high-resolution proteome mapping of clinical tissues. We will also extend nanoPOTS processing to address the inherently small sample sizes and sensitivity challenges associated with functional proteomics measurements, including activity-based proteomics and measurements of key post-translational modifications such as phosphorylation, using a combination of direct surface functionalization, magnetic bead-based workflows within the nanowells and microcolumn fractionation to enrich and process functional protein subpopulations.
We aim to reduce required sample sizes for such functional measurements by more than 100-fold. The efficient processing and greatly reduced sample losses enabled by the nanoPOTS platform will complement the large gains in throughput and sensitivity afforded by TR&D 2 and its fast and high-resolution gas-phase separations and manipulations in SLIM. The workflows to be developed will enable both online and offline separations (e.g., liquid chromatography) with SLIM IM-MS using nanoPOTS-prepared samples. The development of this platform will occur in close collaboration with the TR&D 2 and 3 efforts and is key to most of the driving biological projects.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Biotechnology Resource Grants (P41)
Project #
2P41GM103493-16
Application #
9571628
Study Section
Special Emphasis Panel (ZRG1)
Project Start
Project End
Budget Start
2018-06-01
Budget End
2019-05-31
Support Year
16
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Battelle Pacific Northwest Laboratories
Department
Type
DUNS #
032987476
City
Richland
State
WA
Country
United States
Zip Code
99352
Bilbao, Aivett; Gibbons, Bryson C; Slysz, Gordon W et al. (2018) An algorithm to correct saturated mass spectrometry ion abundances for enhanced quantitation and mass accuracy in omic studies. Int J Mass Spectrom 427:91-99
Dou, Maowei; Chouinard, Christopher D; Zhu, Ying et al. (2018) Nanowell-mediated multidimensional separations combining nanoLC with SLIM IM-MS for rapid, high-peak-capacity proteomic analyses. Anal Bioanal Chem :
Chouinard, Christopher D; Nagy, Gabe; Webb, Ian K et al. (2018) Rapid Ion Mobility Separations of Bile Acid Isomers Using Cyclodextrin Adducts and Structures for Lossless Ion Manipulations. Anal Chem 90:11086-11091
Nagy, Gabe; Chouinard, Christopher D; Attah, Isaac K et al. (2018) Distinguishing enantiomeric amino acids with chiral cyclodextrin adducts and structures for lossless ion manipulations. Electrophoresis 39:3148-3155
Yu, Lei; Petyuk, Vladislav A; Gaiteri, Chris et al. (2018) Targeted brain proteomics uncover multiple pathways to Alzheimer's dementia. Ann Neurol 84:78-88
Wells, Alan; Wiley, H Steven (2018) A systems perspective of heterocellular signaling. Essays Biochem 62:607-617
Kedia, Komal; Wendler, Jason P; Baker, Erin S et al. (2018) Application of multiplexed ion mobility spectrometry towards the identification of host protein signatures of treatment effect in pulmonary tuberculosis. Tuberculosis (Edinb) 112:52-61
Kramer, Philip A; Duan, Jicheng; Gaffrey, Matthew J et al. (2018) Fatiguing contractions increase protein S-glutathionylation occupancy in mouse skeletal muscle. Redox Biol 17:367-376
Zhu, Ying; Piehowski, Paul D; Zhao, Rui et al. (2018) Nanodroplet processing platform for deep and quantitative proteome profiling of 10-100 mammalian cells. Nat Commun 9:882
Sigdel, Tara K; Mercer, Neil; Nandoe, Sharvin et al. (2018) Urinary Virome Perturbations in Kidney Transplantation. Front Med (Lausanne) 5:72

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