Basic Biology of Aging at the University of Washington

Costs of Services

Proteomics (Core A)

The Protein Signatures Core at the University of Washington Nathan Shock Center uses state-of-the-art data acquisition and analysis strategies to improve the sensitivity, robustness, throughput, dynamic range, and precision of quantitative proteomics measurements.  These technologies are based on a strategy known as data independent acquisition.  Data independent acquisition (DIA) acquires comprehensive MS/MS information in a single LC-MS/MS run using a repeated cycle of wide-window MS/MS scans. The methods used are actively being improved and the software tools modified to facilitate these analyses.

A unique aspect of how our core performs these analyses is that we have decoupled the qualitative peptide detection step from the quantification (Figure 1).  The peptide detection step is performed on a pool of the samples injected multiple times to cover the entire m/z range.  This increases our sensitivity and we can then use the retention time and m/z values from the peptide detection runs to map the location of each target peptide onto the wide window acquisition data. We call the peptide detection step chromatogram library building, and we prefer this step on each batch of samples.  Quantitative analyses can still be performed on large number of samples.

Our approach has several advantages over other workflows.  First, the analysis only requires a few additional injections per experiment to assess which peptides are detectable in the proposed sample types.  Second, we only look for peptides in the wide window data that have been found in the narrow window data – minimizing the impact of multiple hypothesis testing reducing the statistical power to detect peptides.  Third, the chromatogram library is collected on the same system, using the same column, and at a similar time to the quantification samples making the mapping of peptide retention times from the library to quantification samples very accurate.  Finally, we can make use of more information in chromatogram libraries than we can in spectrum libraries, including peak shape, interference, neighboring peaks useful for detection, etc…  Data is returned to collaborators as complete data matrix with no missing data regardless of the number of samples or the complexity of the mixture.

We treat each project as a collaboration where the Nathan Shock Center is an active participant in the experiment design, data collection, and data interpretation.  Projects can involve any number of samples (N).  Currently batch size is a max of 14 samples but there is no limit to the number of batches.  Each batch has 2 controls and a chromatogram library.  The cost of a LC-MS/MS run is $100 when subsidized by the UW Nathan Shock Center.  Each batch has a minimum of two reference runs and a chromatogram library (6 runs), which costs $800.  Each quantitation sample costs $100 per sample.  Thus, the total cost per batch is $800 + (N * $100).  The cost of sample preparation for tissues or cells is $30 per sample and for fluids is $10 per sample.  Each analysis includes a consultation and full signal processing analyses.  Statistical analyses should be arranged either by a local expert or arranged separately with the UW Nathan Shock Center. 

Figure 1: Decoupling peptide detection from peptide quantification.

 

(a) In addition to collecting wide-window DIA experiments on each quantitative replicate, a pool containing peptides from every condition is measured using several staggered narrow-window DIA experiments. After deconvolution, these narrow-window experiments have 2 m/z precursor isolation, which is analogous to targeted parallel reaction monitoring (PRM) experiments, except effectively targeting every peptide between 400 and 1000 m/z. EncyclopeDIA then uses these precise coordinates for m/z, time, and intensity to detect peptides in the quantitative samples. (b) The EncyclopeDIA algorithmic workflow for searching spectrum and chromatogram libraries.

 

Metabolomics (Core B)

Targeted Metabolomic Profiling:

$100 for targeted metabolomics

$10 for sample extraction and preparation

Global Metabolomic Profiling:

$104 for global metabolomics

$14 for sample extraction and preparation

 

Invertebrate Longevity & Healthspan (Core C)

Services available:

All quoted prices are estimates for standard procedures.  Modifications to standard protocols may cost more and will be determined on a case by case basis.

General services:

  • Consultation on experimental design for invertebrate lifespan analysis (no cost)

Yeast aging:

  • Replicative lifespan analysis (microdissection): $150 per genotype, per replicate (40 mother cells)
  • Replicative lifespan analysis (microfluidics): $250 per genotype, per replicate (>100 cells per chip, with light and fluorescence microscopy)
  • Chronological lifespan analysis: $100 per genotype, 6 replicates
  • Quantitative growth rate measurements - $100 per experiment, quantitative growth rates for up to 200 wells including controls and replicates
  • Strain construction – per project basis

C. elegans aging:

  • Lifespan analysis (robot): $150 per genotype/RNAi, 3 replicates, ~30 animals per replicate
  • Amyloid beta toxicity (robot): $150 per genotype/RNAi, 3 replicates, ~30 animals per replicate
  • Healthspan (Fecundity, Developmental rate) – per project basis
  • Stress resistance (hypoxia, hyperoxia, oxidative stress, heat, etc.) – per project basis
  • Strain construction – per project basis
  • Fluorescence and light microscopy – per project basis

Shared Equipment and Resources

In collaboration with the UW Healthy Aging and Longevity (HALo) Research Institute, the Core provides access to a variety of equipment and shared resources.  These include:

  • C. elegans injection microscope and needle puller
  • COPAS Biosort Wormsorter
  • Bioscreen C MBR
  • Zeiss Lumar Dissecting Microscope
  • Dissecting and fluorescence microscopes suitable for yeast and worm work
  • A variety of yeast strains and worm RNAi clones (contact with specific requests)

 

Please note**** Pilots that use rats or mice qualify for free animals from the NIA aging rodent resource.****