Combination of Gas-Phase Fractionation and MS3 Acquisition Modes for Relative Protein Quantification with Isobaric Tagging

Relative quantification of peptides and proteins with isobaric tags such as iTRAQ or TMT is commonly used in comparative quantitative proteomics based on tandem mass spectrometry (MS/MS). Nonetheless, isobaric tagging inherently suffers from the cofragmentation/interference phenomenon that may compromise the quality of the quantitative data. An MS3 acquisition mode has been recently proposed to address this issue. Because of the additional ion isolation and fragmentation step, the MS3 acquisition mode significantly alleviates this interference effect. However, MS3 acquisition exhibits a lower sensitivity and a higher duty cycle, both of which reduce the number of identified and quantified proteins. In the present study, we evaluated the combination of gas-phase fractionation (GPF) and MS3 acquisition modes to optimize both identification and quantification of tryptic peptides labeled with TMT using a hybrid ion trap-orbitrap (LTQ-OT) instrument. An interference model was used where TMT-labeled human plasma proteolytic digests were spiked with TMT-labeled E. coil proteolytic digests. When combined with GPF, the MS3 acquisition mode was compared with MS2 modes such as high-energy collision dissociation (HCD) and combined collision-induced dissociation (CID)/HCD. We demonstrated the benefit of using both GPF and MS3 to analyze tryptic peptides labeled with TMT in terms of quantification precision and accuracy as well as proteome coverage. We further explored parameters such as the influence of automatic gain control and additional MS3 scans. The TMT-GPF-MS3 workflow was shown to be a powerful alternative for quantitative proteomic studies that offers improved identification/quantification accuracy and enhanced proteome coverage without the need for extensive sample fractionation before MS analysis.