Quantitation of Eicosanoid Pathway Proteins in Human Cerebrospinal Fluid Using a Dual Pressure Linear Ion Trap Mass Spectrometer

摘要

We have developed a dual-pressure linear ion trap-based qual/quan workflow for the absolute protein quantitation of a subset of enzymes of the eicosanoid pathway. Protein identities were verified by comparison with the internal standard using a targeted full-scan MS/MS spectrum. Selection of surrogate peptides and creating the Velos Pro~(TM) instrument method was easily achieved using Pinpoint 1.1 software. Using this qual/quan approach, quantitation accuracy was linear (median r~(2) velence 0.98) with LOQs ranging from tens of attomoles to low femtomoles of protein. Eight endogenous proteins of the eicosanoid pathway were confidently quantified at physiologically relevant concentrations in human CSF in a single LC/MS run spanning over three orders of magnitude in protein concentration. The median percent CV was 15percent for all proteins in migraine study CSF, allowing the observation of absolute protein levels in samples of migraineurs in the well and disease state.