Spectronaut™ is quite versatile regarding how it quantifies features (precursors, peptides, and proteins). You can choose among the several options on the Quantification node of a settings schema (figure 1).
Figure 1. Quantification settings that can be modified as preferred
First, Spectronaut allows you to choose between MS1 (isotopic envelope) or MS2 (fragment ions) level to quantify precursors. In addition, you can choose whether you want to use the area under the curve within the XIC integration boundaries to assess quantitative values, or rather the apex peak height instead.
Then, in Spectronaut there are two levels of quantification: minor (peptide) group and major (protein) group. The peptide level will be calculated from the precursors; the protein level will be quantified based on the peptide level. You can decide which elements you prefer to be considered for each group:
- As minor (peptide) group, you can choose among: 1) stripped sequence, 2) modified sequence or 3) precursor. To know the difference between these three, please refer to our article about it here.
- As major (protein) group, you can choose between: 1) protein-group Id or 2) Gene Id
For both levels, you can choose between the mean or the sum of the lower elements to quantify each level. In addition, a Top N strategy can be applied to both peptides and proteins, allowing you to choose a minimum and a maximum number of lower level elements to be used.
Table 1 summarizes the BGS Factory Settings (default), what they imply and what are the alternatives.
Label and spike‑in workflows
Furthermore, Spectronaut is able to quantify more than one channel, i.e., is able to run label analysis in addition to label‑free ones. The supported workflows in Spectronaut are:
- Label-free: Default workflow for all single-channel experiments. Peak detection, scoring, and identification are applied as usual.
- Label: Default workflow for all 2‑channel pipelines. Peak detection and scoring will be applied to all channels. Light to heavy ratios will be reported. Post Analysis processing will be performed on the light to heavy ratio.
- Spike-in: Peak detection will be performed on only the reference (heavy) channel. Scoring and identification will be performed on the target (light) channel. The heavy channel is expected to be easily detectable and considered a peak-picking aid in this experiment. Target to reference ratios will be reported. Post Analysis processing will be performed on the target to reference ratio.
- Inverted spike-in: Similar to spike-in but the light channel is considered the reference.
Finally, there are a couple of aspects to consider about quantification. The most important one relates to the correction for interferences. Spectronaut automatically detects fragment ions that are potentially interfering with others and exclude them from the analysis.
Another aspect to consider when choosing your quantification settings is the use of the Proteotypicity Filter. By applying this filter, you will be quantifying your protein using only non‑shared (proteotypic) peptides. There are two levels of proteotypicity you can choose from: protein or protein‑group.
If you are interested in the normalization aspect of the quantification, please refer to our article about the issue here.
Created by SEZ. Last update 2018-03-14 by SEZ