Parallel reaction monitoring (PRM) and selected reaction monitoring (SRM) exhibit comparable linearity, dynamic range and precision for targeted quantitative HDL proteomics

Abstract

High-density lipoprotein (HDL), a lipid nanoparticle containing many different low abundance proteins, is an attractive target for clinical proteomics because its compositional heterogeneity is linked to its cardioprotective effects. Selected reaction monitoring (SRM) is currently the method of choice for targeted quantification of proteins in such a complex biological matrix. However, model system studies suggest that parallel reaction monitoring (PRM) is more specific than SRM because many product ions can be used to confirm the identity of a peptide. We therefore compared PRM and SRM for their abilities to quantify proteins in HDL, using 15N-labeled apolipoprotein A-I (HDL’s most abundant protein) as the internal standard. PRM and SRM exhibited comparable linearity, dynamic range, precision, and repeatability for protein quantification of HDL. Moreover, the single internal standard protein performed as well as protein-specific peptide internal standards when quantifying 3 different proteins. Importantly, PRM and SRM yielded virtually identical quantitative results for 26 proteins in HDL isolated from 44 subjects. Because PRM requires less method development than SRM and is potentially more specific, our observations indicate that PRM in concert with a single isotope-labeled protein is a promising new strategy for quantifying HDL proteins in translational studies.

Plasminogen promotes cholesterol efflux by the ABCA1 pathway

“…Proteins were quantified using spectral counts -the total number of MS/ MS spectra detected for a protein. Spectral counting of HDL proteins strongly agrees with the targeted proteomic method, selected reaction monitoring (43,(74)(75)(76)(77). Proteins considered for analysis had to be detected in 3 or more analyses with 2 or more unique peptides.…”

“…Fractions from the late-eluting peak of material with ABCA1 CEC activity were combined and then separated by cationexchange ( Figure 4, A and E) or anion-exchange (Figure 4, C and G) chromatography; each fraction was again assessed for ABCA1 CEC activity. The active fractions were analyzed with shotgun proteomics (42), and the relative concentrations of proteins (quantified as spectral counts, the number of unique peptides detected by MS/MS (43) in each fraction were correlated with ABCA1 CEC activity.…”

“…Spectral counts for each protein normalized to total spectral counts for peptides from each sample were used to calculate a spectral index. We used the index to compare relative protein composition (43).…”

Multiple apolipoprotein kinetics measured in human HDL by high-resolution/accurate mass parallel reaction monitoring

“…Nonetheless, this approach requires optimization steps to determine which fragment ions are suitable for subsequent quantifi cation studies ( 48,49 ). A recent study by Ronsein et al ( 50 ) determined that PRM and SRM are comparable in terms of sensitivity and accuracy for quantifi cation of an apoA-I protein standard curve, which spanned a femtomole to picomole range. The same study ( 50 ) used a narrow peptide isolation window (±1 Da), and the PRM scans were set to the lowest resolution setting of 17,500 K (at m/z 200), making the method comparable to MRM.…”

The High Density Lipoprotein Cholesterol Hypothesis Revisited

“…Alterations in protein cargo have been associated with pathological states, such as inflammation (28), poor response to therapy (29), autoimmune disease (30), and diabetes (31). Further improvements in quantitative techniques (32,33) and their application to clinical studies should identify panels of protein biomarkers that may be pharmacologically modifiable to reduce risk. Another important variable is HDL's ability to promote cholesterol efflux from cultured macrophages with samples derived from serum, termed cholesterol efflux capacity (CEC).…”