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Compared to traditional label-free, the greatest advantage of DIA is its ability to efficiently measure low-abundance protein molecules in complex samples, greatly improving the reliability of quantitative analysis.
Spectral count, defined as the total number of spectra identified for a protein, has gained acceptance as a practical, label-free, semiquantitative measure of protein abundance in proteomic studies.
Disadvantages of Lable Free: pre-processing of raw data is required before extracting ion signal intensities, which is complicated; low accuracy, relying on mass spectrometry stability, and better differentiation efficiency for label-free when there is a large difference in protein abundance.
In label-free quantification, there are two commonly used quantitative schemes: (1) mass spectral peak intensities and (2) spectral counting and it has been observed that the amount of protein correlates well with peak intensities or spectral counts of peptides unique to a specific protein (Fig. 7.3).
There are various disadvantages of using MS to detect phosphorylated proteins. One limitation is its difficulty in detecting low-abundance phosphorylated proteins and rare phosphosites. The sensitivity of MS is influenced by the dynamic range of the instrument and complexity of the sample.

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Label-free quantification is a method in mass spectrometry that aims to determine the relative amount of proteins in two or more biological samples.
Spectral counting is the most straightforward label-free quantification technique, in which proteins can be quantified by the number of MS/MS events that can be attributed to them relying on the stochastic nature of MS/MS sampling.

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