The ReSpect algorithm is a robust and efficient data-fitting method used to deconvolve isotopically unresolved mass spectra of proteins, peptides, and oligonucleotides.

The ReSpect algorithm can accommodate both low-charge-state spectra and data with a low signal-to-noise ratio, and thus it does not require high-quality data to produce meaningful results. You can use it to confirm molecular masses of proteins or oligonucleotides.

The following figure shows an isotopically unresolved mass spectrum:

Isotopically unresolved mass spectrum
Isotopically unresolved mass spectrum

The ReSpect algorithm is capable of:

  • Deconvolving isotopically unresolved complex mass spectra of biomolecules.
  • Deconvolving mass spectra containing the neutral average mass of each detected molecule.
  • Determining the m/z value of every peak in an electrospray ionization (ESI) mass spectrum.
  • Evaluating all possible charge states for any particular peak, as determined by the mass ranges.

To deconvolve spectra, the ReSpect algorithm does the following:

  1. Performs an optional spectrum preconditioning method, including an automated baseline subtraction.
  2. Locates potential peaks in the spectrum by fitting a customizable peak model, which resembles a Gaussian distribution, to the spectrum.
  3. Deconvolves the spectrum using the peak model to generate a list of detected component peaks and their associated parameters, such as m/z value, signal intensity, and confidence level. The algorithm uses peak spacing patterns, which are indicative of mass, to determine the average mass of each component.
  4. Filters the peaks in the produced list by confidence level and m/z range.
  5. Deconvolves the charge states in the filtered list of peaks to identify patterns of peaks that could represent individual components with series of associated charge states.

You can specify the ReSpect algorithm parameters for an intact mass analysis experiment on the Parameters > Component Detection page of the processing method editing wizard. See Edit the Component Detection page and ReSpect deconvolution parameters.

NOTE

The ReSpect charge state deconvolution is somewhat liberal by design. In particular, it accepts a significant false-positive rate to be sure that there are no false-negative results.

For this reason, the ReSpect algorithm uses a separate quality scoring algorithm that can distinguish which components are most reliable.

For more information, see Quality score algorithm.