Improving Mass Spectrometer Acquisition when using Tandem MS/MS/MS Scanning with Isobaric Tags
n this work we utilize a standard, high-complexity Human cell line tryptic digest combined with a synthetic peptide library to systematically evaluate the properties of a variety of MS operation modes and detector configurations for the optimal examination of isobaric-tagged peptide mixtures. We demonstrate that despite the ability of the IT to yield more rapid MS3 scanning, the benefit in peptide and protein identifications when examining complex mixtures is minor while the negative impact on quantification precision is significant.
Sample Processing Protocol
To test a wide variety of scan and parameter settings, a standard mixture of cell lines and synthetic peptides was prepared. The standard is a complex tryptic digest of a combined pool of 13 individual cell lines labeled with TMT 6-plex reagents (1:1:1:1:1:1, TMT126 – TMT131). To this mixture, a set of 550 synthetic peptides labeled with TMT 6-plex reagents (3:3:3:1:1:1, TMT126 – TMT131) was added.
Data Processing Protocol
Data were processed using Proteome Discoverer and R.
Hughes CS, Spicer V, Krokhin OV, Morin GB. Investigating Acquisition Performance on the Orbitrap Fusion When Using Tandem MS/MS/MS Scanning with Isobaric Tags. J Proteome Res. 2017 Apr 20 PubMed: 28418257