Byos has three default released glycan analysis workflows that are quickly adaptable to a multitude of experimental conditions.
Analysts can easily configure any of these default workflows for the experiment, with options such as different trace types, customizable glycan tables and labeling options, positive and negative modes, and the ability to restrict associations by retention time. For more information on these settings, please see the application note titled, “Detached Glycan Analysis in Byos”, accessible on our website.
Here, we cover advanced settings to overcome data complexity associated with some Detached Glycan analysis.
Glycans often undergo in-source decay during the electrospray ionization process, leading to fragmented, complex spectra. Byos can automatically detect putative glycan fragments in order to remove the time-consuming manual interpretation associated with released glycans.
The following are advanced commands for in-source decay and duplicate assignments:
RemoveISD, if enabled (set to 1), excludes in-source dissociation candidates from auto-assignment. A lower m/z glycan may be treated as a subcomposition of another candidate glycan for the same trace peak. This option is disabled (set to 0) by default. When disabled, in-source dissociation candidates will be treated no differently from other candidates during auto-assignment. A glycan is either the same composition or a supercomposition of the other candidate glycans for the same peak.
RemoveDuplicates ensures that the candidates will only be assigned to the most abundant trace peak during auto-assignment. This option is enabled (set to 1) by default. If disabled (set to 0), candidates may be assigned to more than one trace peak.
Enabling these advanced commands allows the analyst to quickly visualize and validate candidates that are not due to ISD.
What's New: Scan Summing and Noise Reduction
MS1 scan summing, similar to that which is available in Intact Mass, is available in all chromatogram workflows to remove signals with poor correlation. Scan summing makes it easier to find and assign low level glycans and peptides in noisy MS1 spectra.
To enable ensure scan summing, add the following advanced command during project creation:
EnableMS1Sum = true
When scan summing is enabled (set to true), the displayed MS1 will be the sum of all scans between the two pink bars that mark the peak boundaries. When disabled, the displayed MS1 scan will be a single scan taken at the point indicated by the blue bar (usually the peak apex).
TimeCorrFilterValue reduces noisy background in MS1 spectra. The time correlation filter takes advantage of the built-in time correlation function responsible for coloring the MS1 peaks. This is according to the mathematical correlation coefficient of the MS1 intensity over the time-intensity profile of the trace peak as a function of m/z. The correlation result in the color coding you see in the MS1 spectrum, where the well correlated peaks are colored green (correlation coefficients above 0.8), less well correlated peaks are colored magenta (correlation coefficients between 0.4 and 0.8), and poorly correlated peaks are colored gray (correlation coefficients below 0.4). The threshold setting allows you to remove any of the poorly correlated ions from the MS1 spectrum.
The analyst has control over the amount of filtering of their data. We suggest to first try with TimeCorrFilterValue=0.1 to reduce noise levels without over filtering the data. Increasing this value to 0.4 removes most of the gray, poorly correlated peaks. Further increasing to 0.6 removes the poorly correlated pink colored peaks.