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Improved isobaric tandem mass tag quantification by ion mobility mass spectrometry.

ABSTRACT: Isobaric tandem mass tags are an attractive alternative to mass difference tags and label-free approaches for quantitative proteomics due to the high degree of multiplexing that can be performed with their implementation. A drawback of tandem mass tags are that the co-isolation and co-fragmentation of labeled peptide precursors can result in chimeric tandem mass (MS/MS) spectra that can underestimate the fold-change expression of each peptide. Ion mobility (IM) separations coupled to quadrupole time-of-flight (Q-TOF) instruments have the potential to mitigate MS/MS spectra chimeracy since IM-MS has the ability to separate ions based on charge, m/z, and collision cross section (CCS).Two complex protein mixtures, labeled with DiLeu isobaric tandem mass tags in opposite ratios, were mixed together and analyzed by data-dependent LC/IM-MS/MS. The accuracy of reporters from interfering pairs was compared with and without IM separation.IM separation was able to mitigate isobaric interference from differentially charged interfering ion pairs, as well as pairs of the same charge. Of the eight example precursors shown, only one had reporters that remained compressed below the significance threshold after IM separation.The results of this investigation demonstrate proof-of-principle that IM separation of tagged precursors prior to MS/MS fragmentation can help mitigate quantitative inaccuracies caused by isobaric interference. Future improvements of the method would include software for automated correction and use of higher resolution IM instrumentations.

SUBMITTER: Sturm RM

PROVIDER: S-EPMC4000571 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Improved isobaric tandem mass tag quantification by ion mobility mass spectrometry.

Sturm Robert M RM Lietz Christopher B CB Li Lingjun L

Rapid communications in mass spectrometry : RCM 20140501 9

<h4>Rationale</h4>Isobaric tandem mass tags are an attractive alternative to mass difference tags and label-free approaches for quantitative proteomics due to the high degree of multiplexing that can be performed with their implementation. A drawback of tandem mass tags are that the co-isolation and co-fragmentation of labeled peptide precursors can result in chimeric tandem mass (MS/MS) spectra that can underestimate the fold-change expression of each peptide. Ion mobility (IM) separations coup ...[more]

PMID: 24677527

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Project description:In this report, we explored the benefits of cyclic ion mobility (cIM) mass spectrometry in the analysis of isomeric post-transcriptional modifications of RNA. Standard methyl-cytidine samples were initially utilized to test the ability to correctly distinguish different structures sharing the same elemental composition and thus molecular mass. Analyzed individually, the analytes displayed characteristic arrival times (tD ) determined by the different positions of the modifying methyl groups onto the common cytidine scaffold. Analyzed in mixture, the widths of the respective signals resulted in significant overlap that initially prevented their resolution on the tD scale. The separation of the four isomers was achieved by increasing the number of passes through the cIM device, which enabled to fully differentiate the characteristic ion mobility behaviors associated with very subtle structural variations. The placement of the cIM device between the mass-selective quadrupole and the time-of-flight analyzer allowed us to perform gas-phase activation of each of these ion populations, which had been first isolated according to a common mass-to-charge ratio and then separated on the basis of different ion mobility behaviors. The observed fragmentation patterns confirmed the structures of the various isomers thus substantiating the benefits of complementing unique tD information with specific fragmentation data to reach more stringent analyte identification. These capabilities were further tested by analyzing natural mono-nucleotide mixtures obtained by exonuclease digestion of total RNA extracts. In particular, the combination of cIM separation and post-mobility dissociation allowed us to establish the composition of methyl-cytidine and methyl-adenine components present in the entire transcriptome of HeLa cells. For this reason, we expect that this technique will benefit not only epitranscriptomic studies requiring the determination of identity and expression levels of RNA modifications, but also metabolomics investigations involving the analysis of natural extracts that may possibly contain subsets of isomeric/isobaric species.

Dataset Information

Improved isobaric tandem mass tag quantification by ion mobility mass spectrometry.

Publications

Improved isobaric tandem mass tag quantification by ion mobility mass spectrometry.

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