Abstract
The parallel reaction monitoring (PRM) assay has emerged as an alternative method of targeted quantification. The PRM assay is performed in a high resolution and high mass accuracy mode on a mass spectrometer. This review presents the features that make PRM a highly specific and selective method for targeted quantification using quadrupole-Orbitrap hybrid instruments. In addition, this review discusses the label-based and label-free methods of quantification that can be performed with the targeted approach.
Highlights
In discovery-based proteomics experiments, proteins that are specific to disease and treatment conditions, or are potential biomarker candidates, are identified in an unbiased manner
Targeted experiments are primarily performed on a triple-quadrupole (QQQ) and hybrid quadrupole-linear ion trap (QTrap) mass spectrometers using a data acquisition method known as Selected Reaction Monitoring (SRM) [1,2]
The spectrum on the upper panel is from the peptide when Peptide Retention Time Calibration (PRTC) was run by data-dependent acquisition (DDA) mode; the spectrum on the lower panel is from the peptide when one of the concentrations of PRTC (40 fmol spiked into 100 ng HeLa digest) was run by Parallel Reaction Monitoring (PRM) mode
Summary
In discovery-based proteomics experiments, proteins that are specific to disease and treatment conditions, or are potential biomarker candidates, are identified in an unbiased manner. Targeted experiments are primarily performed on a triple-quadrupole (QQQ) and hybrid quadrupole-linear ion trap (QTrap) mass spectrometers using a data acquisition method known as Selected Reaction Monitoring (SRM) [1,2]. An advantage of the q-OT mass spectrometer is that both discovery and targeted experiments can be performed on the same instrument This makes it easier to transfer instrumental parameters (e.g., collision energy, quadrupole isolation window, automatic gain control, retention time etc.) between the two data acquisition methods. PPRRMM iinn VVaalliiddaattiioonn ooff PPrrootteeiinn RReellaattiivvee AAbbuunnddaannccee. Sweredoski et al [28] have used the PRM assay in combination with the middle-down electron transfer dissociation (ETD) approach to quantify multiply charged peptides that are larger than 5 kDa. When analyzing large multiply charged peptides by data-dependent acquisition method (middle-down proteomics), multiple charge states of the same peptide are resampled, precluding the selection and fragmentation of other proteoforms. Sweredoski et al [28] have used histone H3 fractions from untreated and DMSO-treated Murine ErythroLeukemia (MEL) cells and identified combinatorial PTMs on 254 histone H3 N-terminal fragments
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