TY - JOUR
T1 - Limits for resolving isobaric tandem mass tag reporter ions using phase constrained spectrum deconvolution
AU - Kelstrup, Christian D
AU - Aizikov, Konstantin
AU - Batth, Tanveer S
AU - Kreutzman, Arne
AU - Grinfeld, Dmitry
AU - Lange, Oliver
AU - Mourad, Daniel
AU - Makarov, Alexander A.
AU - Olsen, Jesper V.
PY - 2018/11/2
Y1 - 2018/11/2
N2 - A popular method for peptide quantification relies on isobaric labeling such as tandem mass tags (TMT) which enables multiplexed proteome analyses. Quantification is achieved by reporter ions generated by fragmentation in a tandem mass spectrometer. However, with higher degrees of multiplexing, the smaller mass differences between the reporter ions increase the mass resolving power requirements. This contrasts with faster peptide sequencing capabilities enabled by lowered mass resolution on Orbitrap instruments. It is therefore important to determine the mass resolution limits for highly multiplexed quantification when maximizing proteome depth. Here we defined the lower boundaries for resolving TMT reporter ions with 0.0063 Da mass differences using an ultra-high-field Orbitrap mass spectrometer. We found the optimal method depends on the relative ratio between closely spaced reporter ions and that 64 ms transient acquisition time provided sufficient resolving power for separating TMT reporter ions with absolute ratio changes up to 16-fold. Furthermore, a 32 ms transient processed with phase-constrained spectrum deconvolution provides >50% more identifications with >99% quantified, but with a slight loss in quantification precision and accuracy. These findings should guide decisions on what Orbitrap resolution settings to use in future proteomics experiments relying on isobaric TMT reporter ion quantification.
AB - A popular method for peptide quantification relies on isobaric labeling such as tandem mass tags (TMT) which enables multiplexed proteome analyses. Quantification is achieved by reporter ions generated by fragmentation in a tandem mass spectrometer. However, with higher degrees of multiplexing, the smaller mass differences between the reporter ions increase the mass resolving power requirements. This contrasts with faster peptide sequencing capabilities enabled by lowered mass resolution on Orbitrap instruments. It is therefore important to determine the mass resolution limits for highly multiplexed quantification when maximizing proteome depth. Here we defined the lower boundaries for resolving TMT reporter ions with 0.0063 Da mass differences using an ultra-high-field Orbitrap mass spectrometer. We found the optimal method depends on the relative ratio between closely spaced reporter ions and that 64 ms transient acquisition time provided sufficient resolving power for separating TMT reporter ions with absolute ratio changes up to 16-fold. Furthermore, a 32 ms transient processed with phase-constrained spectrum deconvolution provides >50% more identifications with >99% quantified, but with a slight loss in quantification precision and accuracy. These findings should guide decisions on what Orbitrap resolution settings to use in future proteomics experiments relying on isobaric TMT reporter ion quantification.
U2 - 10.1021/acs.jproteome.8b00381
DO - 10.1021/acs.jproteome.8b00381
M3 - Journal article
C2 - 30220210
SN - 1535-3893
VL - 17
SP - 4008
EP - 4016
JO - Journal of Proteome Research
JF - Journal of Proteome Research
IS - 11
ER -