Combination of gas-phase fractionation and MS³ acquisition modes for relative protein quantification with isobaric...

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 MS(3) acquisition mode has been recently proposed to address this issue. Because of the additional ion isolation and fragmentation step, the MS(3) acquisition mode significantly alleviates this interference effect. However, MS(3) 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 MS(3) 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. coli proteolytic digests. When combined with GPF, the MS(3) acquisition mode was compared with MS(2) modes such as high-energy collision dissociation (HCD) and combined collision-induced dissociation (CID)/HCD. We demonstrated the benefit of using both GPF and MS(3) 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 MS(3) scans. The TMT-GPF-MS(3) 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.