1. What is the difference between DDA and DIA?
Both DDA and DIA are considered discovery-based methods in bottom-up proteomics. This means that in both cases, proteins are enzymatically digested into smaller peptides, and information is extracted from the peptides to infer conclusions at 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 MS2 data for all peptides across the entire retention time range are collected. After these initial steps, the DIA method uses spectral libraries to extract information from highly abundant data and allows quantification at the MS2 level. Due to these characteristics, DIA provides unparalleled precision and quantitative accuracy, improved reproducibility, and more comprehensive peptide sampling than DDA. Compared to DIA, the mass spectrometer in DDA mode selects only certain peptides and then fragments them, ideally one at a time. While DIA is a superior acquisition method for quantitative targets, DDA is the preferred method for library generation and database searching because of its near-peptide-specific MS2 spectra. This is especially true when combined with deep fractionation. Based on a search engine algorithm applied to existing protein databases, DDA can quickly provide identification results.
2. Is there a difference between using "Blank Document" and entering a DIA peptide search?
Entering a DIA peptide search will walk you through the form you submitted. You can get all the same results or more by selecting "Blank Document" and then using Setup > Peptide Setup and Ion Transition Setup, File > Import FASTA, Optimize > Add Bait, File > Import > Results, Optimize > Reintegrate. In other words, if you know what you want to achieve, you can get the most power and flexibility by navigating the options directly in the Skyline user interface. If not, then you should find the options for entering a DIA peptide search more convenient. After you select "Blank Document", you can also bring up this form via File > Import > Peptide Search and perhaps 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 Dependent Acquisition: Classic shotgun proteomics. The mass spectrometer selects the highest intensity ions in MS1 for MS2 ionization. Advantages: Unbiased protein detection. No prior assumptions are required. Disadvantages: Since different peptides are detected in different runs, the analyzed proteins have low reproducibility and large batch effects. It also leads to a lot of data loss. There are large errors in determining the highest content protein in the test sample, especially for plasma samples with a large dynamic range of protein concentration fluctuations. 2. DIA-Data Independent Acquisition: The "sliding window" selects MS1 ions with 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 are required. A large number of proteins can be detected. Disadvantages: Too much data is acquired, resulting in a decrease in the speed of data analysis. Because too much data is collected, complex data analysis is required to obtain the intensity of individual peptides, and peptide databases are usually used to achieve this purpose.
3. Targeted MS
Any type of mass spectrometry analysis of the target peptide, usually SRM or PRM in practice, possibly with a stable isotope standard (SIS). Advantages: Fewer batch effects. SIS allows for better quantification, even absolute quantification. Good reproducibility, less missing data. Disadvantages: Targeted analysis needs to be developed. Before the experiment, it is necessary to determine which proteins to analyze. SIS is expensive.