1. What is the difference between DDA and DIA?
Both DDA and DIA are considered discovery-based approaches in bottom-up proteomics. This means that in both cases the protein is enzymatically digested into smaller peptides and information is extracted from the peptides to infer conclusions on the protein level. In DIA, almost all peptides are fragmented together and then analyzed using mass spectrometry. This technique produces complex fragmentation (MS2) spectra but collects MS2 data for all peptides over the entire retention time range. After these initial steps, the DIA method uses spectral libraries to extract information from the highly rich data and allows quantification at the MS2 level. Due to these properties, DIA provides unparalleled precision and quantitative accuracy, improved reproducibility, and more comprehensive peptide sampling than DDA. In contrast to DIA, a mass spectrometer in DDA mode only selects certain peptides and then fragments them, ideally one at a time. While DIA is the superior acquisition method for quantitative targets, DDA is the method of choice for library generation and database searching because of its nearly peptide-specific MS2 spectra. This is especially true when used in conjunction with deep fractionation. Based on search engine algorithms applied to existing protein databases, DDA can quickly provide identification results.
2. What is the difference between using "Blank Document" and entering DIA peptide search?
Entering a DIA peptide search will guide you step-by-step through the submitted form. You can get all the same or more by selecting "Blank Document" and then using Settings > Peptide Settings and Transition Settings, File > Import FASTA, Optimize > Add Bait, File > Import > Results, Optimize > Reintegrate the result of. In other words, if you know what you want to achieve, you can gain maximum power and flexibility by navigating options directly within the Skyline user interface. If not, you may find it more convenient to enter the DIA peptide search option. After you select "Blank Document" you can also bring up this form via File > Import > Peptide Search and possibly make some preliminary adjustments to the missing parts of the peptide search form.
3. What are the differences in data acquisition methods between DDA proteomics, DIA proteomics and targeted proteomics? What are their advantages and limitations?
1. DDA-data dependency collection:
Classic shotgun proteomics. The mass spectrometer selects the highest intensity ions in MS1 for MS2 ionization.
Advantages: Unbiased protein detection. No prior assumptions required.
Disadvantages: Since different peptides are detected in different runs, the reproducibility of the analyzed proteins is low and the batch effect is large. Also results in a lot of data loss. There is a large error in determining the most abundant protein in a test sample, especially for plasma samples where the dynamic range of protein concentration fluctuates widely.
2. DIA-data independent collection:
The "sliding window" selects MS1 ions in a certain mass-to-charge ratio range for subsequent MS2 analysis, thereby achieving scanning analysis of all ions.
Advantages: Unbiased protein detection. No prior assumptions required. A large number of proteins can be detected.
Disadvantages: Too much data is acquired, resulting in a slowdown in analyzing the data. Because so much data is collected, complex data analysis is required to obtain the intensity of individual peptides, often using peptide databases for this purpose.
3. Targeted MS:
Perform any type of mass spectrometric analysis on the target peptide, usually an SRM or PRM in fact, possibly with a stable isotope standard (SIS).
Advantages: Less batch effect. SIS enables better quantification, even absolute quantification. Good reproducibility and few missing data.
Disadvantages: Requires development of targeted assays. Before the experiment, you need to decide which proteins you want to analyze. SIS is expensive.