A multiple-reaction-monitoring mass spectrometric method for simultaneous quantitative analysis of five plasma apolipoproteins

A multiplexed targeted proteomic assay using a mTRAQ-MRM/MS-based approach was developed and assessed to systemat- ically quantify the relative expressions of five candidate plasma apolipoproteins that have been previously shown to be dysreg- ulated in neuropsychiatric disorders and cognitive dysfunction： apolipoprotein H （APOH）, apolipoprotein J （APOJ）, apolipo- protein A4 （APOA4）, apolipoprotein E （APOE）, and apolipoprotein D （APOD）. The peptides and transitions of each APO were carefully selected according to the tandem MS signals acquired on a TripleTOFTM 5600, followed by optimization of the declustering potential and collision energy voltages for transitions on a QTRAP 5500. Our results showed that the collision en- ergies of mTRAQ-labeled peptides were approximately 15%-20% higher than corresponding non-labeled peptides. Through optimized transitions and parameters, we analyzed the relative abundances of the five APOs in human plasma with and without depletion of high abundant proteins. The results indicated that the MRM signals of four target APOs were significantly in- creased after depletion, while the MRM signal of one APO, APOD, was decreased. Furthermore, the relative abundances of the five target APOs in healthy human plasma were stable, and the ranking of these proteins according to their MS responses changed slightly. Therefore, we deduced that the rank order of the MS signals for these target proteins can be developed as a diagnostic signature for diseased plasma.