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INFORMATION FOR

Targeted Proteomics

Data acquisition by mass spectrometry can be achieved by Data-Dependent Acquisition (DDA), Data Independent Acquisition (DIA), and Targeted Data Acquisition (TDA). In DDA, a defined number of precursor ions from the full scan are selected for fragmentation based on predetermined settings, such as precursor intensity and charge state. In DIA, however, the instrument acquires a full MS spectrum followed by a series of sequential MS/MS spectra of predefined isolation windows that subdivide a larger m/z region. One of the methods by which DIA data is processed uses a targeted data extraction strategy. This strategy uses the highly specific fragment ion maps in a spectral library as the basis for qualitatively and quantitatively mining DIA data sets. In contrast to DDA, in TDA, a list of precursor peptide ions are selected for fragmentation followed by detection of a few (Selected/Multiple Reaction Monitoring, SRM/MRM) or most (Parallel Reaction Monitoring, PRM) major product ions. The precursor peptide ions are measured in predefined m/z and retention time windows and they must be unique to the targeted protein.
Schematic Depictions of SRM, PRM, and DIA Analyses: In SRM a target precursor peptide ion is isolated in the Q1 quadrupole and then fragmented in q2. Specific, pre-selected product ions (typically three) are then detected and analyzed in Q3. In PRM , a target precursor peptide ion is again isolated in the Q1 quadrupole and then fragmented in q2. All fragment ions from the target peptide ion are then monitored and analyzed in the High Resolution/Accurate Mass (HR/AM) Q3 Orbitrap. In DIA all peptide ions within a defined mass-to-charge (m/z) window are isolated in Q1 and then fragmented together in q2. The highly complex mixture of all product ions are then analyzed by an Orbitrap HR/AM mass analyzer. The analysis is repeated until the full m/z range has been covered in a stepwise fashion. (Adapted from Shi et al (2016) Proteomics 16:2160-2182 with permission)

While both SRM and PRM have similar sensitivity, linearity, dynamic range, precision, and quantitative reproducibility; PRM has several advantages as compared to SRM. Since PRM does not require prior selection of transitions (i.e., precursor/product ions); it is relatively easier to build a PRM assay. In addition, with a >200 fold increase in mass resolution and an approximately 50-fold increase in mass accuracy, PRM provides greatly increased selectivlty and confidence in peptide identification. Finally, while SRM assays typically monitor three transitions/peptide, a full MS/MS spectra is acquired in PRM that enables all of the observed product ions to be used to confirm the identity of and to quantify the target peptide.


In addition to developing custom assays to quantify target biomarker proteins and their post-translational modifications, the Yale/NIDA Neuroproteomics Center also has developed rat/mouse:

  • PRM assay for 50 proteins that include 47 proteins that are enriched on going from synaptosomal to a more highly purified PSD fraction
  • DIA assay for 2134 PSD proteins that will provide a nice complement to the PRM assay
as described in more detail in Wilson et al, 2019.