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Parallel Reaction Monitoring

Parallel reaction monitoring (PRM) represents a key improvement over lower resolution triple quad SRM methods, the specificity of which is limited by coanalysis of near-isobaric ions [55, 60]. Substitution of Q3 of a triple quad with a high resolution and accurate mass (HRAM) analyzer enables the detection of all target product ions in a single scan to monitor all possible transitions in parallel. PRM utilizes extracted ion chromatograms with narrow m/z windows, which increases specificity by resolving interferences to enable confident assignment of precursor to products [61]. Proof of principle of the utility of PRM to map acetylation and methylation sites was demonstrated for histones H3 and H4; novel methyl (mono-, di-, tri-), acetyl, and monomethyl/acetyl dual modifications were assigned based on the presence of diagnostic immonium ions and neutral losses of methylamine following HCD fragmentation. The data were generated with the purpose of establishing novel precursor-product pairs for SRM quantification, particularly for discriminating peptide isoforms since PRM product ion scan information enables the assignment of distinct b and y sequence ions to specific peptide PTM variants [62]. A quantification approach coupling PRM with the use of stable isotope-labeled internal standards enabled dynamic analysis and determination of alterations in specific PTM sites, including downregulation of H3 K36 trimethylation in response to vitamin C [63]. This exemplifies two types of PRM applications: screening mode, directed to defining specific peptides of interest [62], and quantification mode, which focuses on the accurate quantification of selected peptides by reference to internal standards [63].

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