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Sphingomyelin (ceramide-phosphocholine, CerPCho) is a common sphingolipid in mammalian cells and is composed of phosphorylcholine and ceramide as polar and hydrophobic components, respectively. In this study, a qualitative liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS/MS) analysis is proposed in which CerPCho structures were assigned based on product ion spectra corresponding to sphingosylphosphorylcholine and N-acyl moieties. From MS/MS/MS analysis of CerPCho, we observed product ion spectra of the N-acyl fatty acids as [RCO] ions as well as sphingosylphosphorylcholine. A calibration curve for CerPCho was constructed using two stable isotopically labeled CerPCho species and then used to quantify the CerPCho species in HeLa cells as a proof-of-principle study. The present study proposes an accurate method for quantifying and assigning structures to each CerPCho species in crude biologic samples by LC-ESI-MS/MS/MS analysis. [ABSTRACT FROM AUTHOR]

Methods for isobaric-tagged peptide analysis (e.g., TMT, iTRAQ), such as the synchronous precursor selection (SPS) tandem MS/MS/MS (MS 3 ) approach, enable maintenance of reporter ion accuracy and precision by reducing the ratio compression caused by coisolated precursor ions. However, the decreased throughput of the MS 3 approach necessitates careful optimization of acquisition strategies and methods to ensure maximal proteome coverage. We present a systematic analysis of acquisition parameters used to analyze isobaric-tagged peptide samples on current generation Orbitrap mass spectrometer (MS) hardware. In contrast with previously reported works, we demonstrate the limited utility of acquiring reporter ion data in the ion trap analyzer; ion trap acquisition had only a minimal increase in identification depth and reduced quantification precision. We establish that despite the significantly increased scan rate afforded through the use of higher energy collisional dissociation (HCD) in MS 3 -based ion trap isobaric tag analyses, the reduced quantification precision and reporter ion yields negate the potential benefits in proteome coverage. Lastly, using optimized parameter sets, we further demonstrate the limited utility of the ion trap detector versus the Orbitrap for reporter ion detection in an in-depth analysis of a complex proteome sample. Together, these data will serve as a valuable resource to researchers undertaking analysis on current generation Orbitrap instrumentation with isobaric tags.