Limits for resolving isobaric tandem mass tag reporter ions using phase constrained spectrum deconvolution

Christian D Kelstrup; Konstantin Aizikov; Tanveer S Batth; Arne Kreutzman; Dmitry Grinfeld; Oliver Lange; Daniel Mourad; Alexander A. Makarov; Jesper V. Olsen

doi:10.1021/acs.jproteome.8b00381

Limits for resolving isobaric tandem mass tag reporter ions using phase constrained spectrum deconvolution

Christian D Kelstrup, Konstantin Aizikov, Tanveer S Batth, Arne Kreutzman, Dmitry Grinfeld, Oliver Lange, Daniel Mourad, Alexander A. Makarov, Jesper V. Olsen

6 Citations (Scopus)

Abstract

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.

Original language	English
Journal	Journal of Proteome Research
Volume	17
Issue number	11
Pages (from-to)	4008-4016
Number of pages	9
ISSN	1535-3893
DOIs	https://doi.org/10.1021/acs.jproteome.8b00381
Publication status	Published - 2 Nov 2018

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@article{d0269f95ea404916b363314b2b6f094e,

title = "Limits for resolving isobaric tandem mass tag reporter ions using phase constrained spectrum deconvolution",

abstract = "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.",

author = "Kelstrup, {Christian D} and Konstantin Aizikov and Batth, {Tanveer S} and Arne Kreutzman and Dmitry Grinfeld and Oliver Lange and Daniel Mourad and Makarov, {Alexander A.} and Olsen, {Jesper V.}",

year = "2018",

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doi = "10.1021/acs.jproteome.8b00381",

language = "English",

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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 -

Limits for resolving isobaric tandem mass tag reporter ions using phase constrained spectrum deconvolution

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