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Biological Sciences

Dynamic Range Enhancement Applied to Mass Spectrometry (DREAMS)

Sponsor: DOE Office of Biological and Environmental Research
Contact: Harold Udseth

The large variation among protein relative abundances that have potential biological significance in mammalian systems (possibly >9 orders of magnitude and even greater in some sample types such as plasma) presents a major challenge for proteomics. In many cases, the presence of highly abundant peptides can preclude detection of low-abundance peptides in the same spectrum. This issue can be effectively addressed using Dynamic Range Enhancement Applied to Mass Spectrometry (DREAMS), a recently developed approach that involves the use of data-directed ejection of the most abundant ions before ion accumulation in a 2-D ion trap external to the ICR trap.1,2 This approach eliminates the rapid filling of the external 2-D quadrupole ion trap and consequently, the ICR trap, enabling selective accumulation of lower abundance species for significantly extended time, and providing greater sensitivity and extended dynamic range.

Enhanced proteome coverage is achieved by using Dynamic Range Enhancement Applied to Mass Spectrometry (DREAMS).
Enhanced proteome coverage is achieved by using Dynamic Range Enhancement Applied to Mass Spectrometry (DREAMS). Full Image (png 31kb)

We have demonstrated the DREAMS approach in the analysis of tryptic peptides using 14N/15N labeled Deinococcus radiodurans and mouse B16 melanoma proteins and showed that the number of detected peptide pairs suitable for use in quantitative analyses is significantly increased because of the enhanced dynamic range of the measurements.3 As an example, a combined total of 17,813 unambiguous and unique 14N/15N peptide pairs with an average abundance ratio of 1.035 and a standard deviation of 0.29 was detected in a single DREAMS LC-FTICR analysis of mouse B16 melanoma proteins. Using DREAMS, the number of peptides for which quantitative information could be obtained (from the relative abundances of peptide pairs) was increased by 80%.

To determine if such a huge increase in the number of observed peptides translates into markedly better proteome coverage, we have analyzed a mixture of 14N/15N -labeled D. radiodurans cells. Using the AMT tag approach, we observed and quantified 1,244 D. radiodurans proteins (~40% of the predicted D. radiodurans proteome) as peptide pairs in a single DREAMS LC-FTICR analysis. This compares favorably with shotgun approaches; for instance, recent work by Washburn et al., using MudPIT reported an average of 869 yeast proteins (~14% of the predicted larger set of yeast proteins) observed in three analyses of 1:1, 5:1, and 10:1 mixtures of yeast grown in 14N and 15N-enriched minimal media. Further, DREAMS and nanoLC-FTICR technology were recently used in combination with SPICAT to achieve the highest proteme coverage (>2,000 Cys-containing peptide pairs) from the smallest amount of the starting protein (10,000 mammalian cells or <2 µg).

References

  1. Belov ME et al. 2001. "Dynamic range expansion applied to mass spectrometry based on data-dependent selective ion ejection in capillary liquid chromatography Fourier transform ion cyclotron resonance for enhanced proteome characterization." Analytical Chemistry 73(21):5052-5060.
  2. Harkewicz R, Belov ME, Anderson DA, Paša-Tolić L, Masselon CD, Prior DC, Udseth HR, Smith RD. 2002. "ESI-FTICR mass spectrometry employing data-dependent external ion selection and accumulation." Journal of the American Society for Mass Spectrometry 13:144-154.
  3. Paša-Tolić L et al. 1999. "High throughput proteome-wide precision measurements of protein expression using mass spectrometry." Journal of the American Chemical Society 121:7949-7950.

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