Why is high abundance removal and grading not required for DIA?
Answer: DIA is a full scan technique. The entire full scan range of the mass spectrometer is divided into several windows, and all ions in each window are selected, fragmented and detected at high speed and in cycles, so that all fragmentation information of all ions in the sample can be obtained without missing or discrepancy. Thus, DIA obtains higher coverage information while also minimizing the impact of high-abundance proteins on the mass spectrometry scan.
Why is it necessary to perform library construction first in the experimental route of DIA?
Answer: The data obtained by the full-scan approach of DIA is too complex. Each secondary spectral map has mixed information from multiple primary signals, and it is usually difficult to match theoretical libraries directly. It is usually necessary to construct the map library first, and then use the library to analyze the DIA data in an efficient and accurate way.
Why is the quantitative analysis capability of DIA close to that of targeted techniques such as PRM and MRM?
Answer: The principle of DIA quantification is similar to that of targeted techniques, both of which use secondary fragment ions for quantification.
Does DIA still need follow-up validation?
Answer: DIA is a non-targeting technique, and the results will eventually need to be validated for targeting. However, the high accuracy of DIA enables a higher articulation and consistency of subsequent target validation.
What type of experiments is DIA suitable for?
Answer: DIA is a holographic scanning mode, which can fully record the sample information with much higher quantitative accuracy, good reproducibility and few missing values. The sample is collected with retention time corrected isotope peptides, which is suitable for large sample size. The up time interval can be larger and the data can be retraced. It can be used for differential protein screening under the influence of different factors, protein expression profiling of different tissues, disease typing, etc.
What are the advantages and disadvantages of holographic scanning DIA vs. label free?
Answer: Both can be used for samples with large differences (different species, different tissue sites); protein presence/absence analysis, no isotope labeling, relatively low cost; unlimited samples, wide range of applications.
LFQ data are slightly less reproducible and have more missing values.
DIA has better reproducibility and all ion information can be collected. The disadvantage is that a database needs to be established in advance, and data analysis is more complicated than LFQ.
