Measuring oxidized M148 using conventional mass spectrometry methods that utilize spectral counting or extracted ion chromatograms can be lengthy and challenging in large sample sizes. In contrast, MRM is a promising technique that allows multiplexing of several targets and has been successfully applied to quantitate plasma proteins [10] and [14]. Cyclopamine supplier The addition of SIS peptides in MRM allows for absolute quantitation. Since our goal was to develop

an assay to assess the relative ratio of oxidized M148 to the native peptide rather than the absolute concentrations, the SIS peptide for the unmodified M148 was not synthesized. M148(O) SIS peptide was used to correctly identify the peaks of the in vivo M148 peaks, and optimize the transitions. The rationale for not determining the absolute concentration buy Alectinib of M148(O) in plasma was that this concentration can vary because of variations in the ApoA-I concentration. In contrast, monitoring the relative ratio of oxidized M148 to the non-oxidized peptide represents the “quality” of this peptide, is cost-effective and simple with less inherent variability. Thus, this approach

is better suited for comparing M148 oxidation ratios among different patient groups. One advantage of MRM is that different peptide variants can be selected, depending on the goal of the particular project. The M148-containing ApoA-I peptide would not normally

be selected for ApoA-I quantitation because of its susceptibility to methionine oxidation. The “ATEHLSTLSEK” ApoA-I peptide likely gives Protein kinase N1 a better estimate of total ApoA-I concentration. Several limitations of the study deserve mention. First, we have not measured the molar % oxidized of M148, as such measure would require calibration of both forms of the peptide. Second, methionines are susceptible to ex vivo oxidation that can result from inadequate or prolonged freezing, repeated thawing, or centrifugations [15]. Because an anti-oxidant solution was not immediately added before freezing the samples or after HDL isolations, this might have permitted additional oxidation. The ratios of methionine oxidation observed in our study were higher than those reported in an earlier study on diabetes where the samples were preserved in an anti-oxidant solution prior freezing [16]. In this earlier study, however, younger patients with type 1 diabetes were recruited. We have recently demonstrated that immediate freezing of samples at −80 °C without the use of an anti-oxidant solution results in low levels of ApoA-I oxidation that are stable for up to 2 years of storage [17].