What are the Differences in Targeted Proteomics Techniques?

What are the distinctive features of targeted proteomics techniques? They can be broadly categorized into several types: MRM/SRM, PRM, and SWATH/DIA. Today, MtoZ Biolabs will introduce two of the more representative ones.

 

1. MRM/SRM

(1) SRM (Selective Reaction Monitoring): This technique involves selecting a single peptide ion, which, after collision-induced dissociation, generates fragment ions. Among these, only one ion is selected for detection. Due to the selection of single ions at both steps, the method is highly specific, effectively minimizing noise and interference.

 

(2) MRM (Multiple Reaction Monitoring): When multiple compounds are measured simultaneously, several SRMs are performed together. There is no strict need to differentiate between SRM and MRM; if multiple SRMs are conducted in a single experiment, it is referred to as MRM.

 

In summary, MRM/SRM pre-selects the peptides and fragment ions for analysis, allowing it to bypass the requirement of selecting only high-abundance peaks in the first stage of mass spectrometry. This ensures that low-abundance proteins are also detected.

 

A similar method to MRM/SRM is PRM (Parallel Reaction Monitoring). The key difference is that while MRM/SRM requires pre-selection of both the precursor ion and the fragment ion, PRM only requires the selection of the precursor ion. This is because PRM is performed on high-precision Orbitrap mass spectrometers (as opposed to the triple quadrupole mass spectrometers used for MRM/SRM). The high precision allows for the simultaneous and accurate measurement of multiple fragment ions. Consequently, PRM is easier to implement, as it does not require continuous optimization of instrument parameters for the fragment ions.

 

2. DIA

Techniques like MRM require pre-selection of peptides and peptide ions, which poses a challenge if we aim to discover new entities without pre-selection. In this context, the representative targeted proteomics technique to consider is DIA (data-independent acquisition), also known as a "data-independent acquisition" strategy.

 

A representative method within DIA is the SWATH technique. The core idea is to select the mass-to-charge ratio (m/z) of precursor ions within a specified range, such as 500-900 or 400-1000, using 25 Dal as a window. For example, the technique first isolates peptide segments within the 500-525 range, fragments them, and then collects peptide segments in the 525-550 range, continuing this process sequentially.

 

DIA's first-stage mass spectrometry uniformly collects peptide segments within each window range without omission and does not involve restrictive selection of precursor ions. Therefore, compared to DDA, both the accuracy and reproducibility are significantly enhanced.