Detection and quantification of proteins in clinical samples using high resolution mass spectrometry.
- Proteomics of Cellular Signaling
Quantitative proteomics has benefited from the recent development of mass spectrometers capable of high-resolution and accurate-mass (HR/AM) measurements. While targeted experiments are routinely performed on triple quadrupole instruments in selected reaction monitoring (SRM; often referred as multiple reaction monitoring, MRM) mode, the quadrupole-orbitrap mass spectrometers allow quantification in MS/MS mode, also known as parallel reaction monitoring (PRM). This technique is characterized by higher selectivity and better confidence in the assignment of the precursor and fragment ions, and thus translates into an improved analytical performance. More fundamentally, PRM introduces a change of the overall paradigm of targeted experiments, by the decoupling of the acquisition and data processing. They rely on two distinct steps, with a simplified acquisition method in conjunction with a flexible, iterative, post-acquisition data processing. This account describes in detail the different steps of a PRM experiment, which include the design of the acquisition method, the confirmation of the identity of the analytes founded upon a full MS/MS fragmentation pattern, and the quantification based on the extraction of specific fragment ions (selected post-acquisition) using tight mass tolerance. The different types of PRM experiments, defined as large-scale screening or precise targeted quantification using calibrated internal standards, together with the considerations on the selection of experimental parameters are discussed.