Analytical and Bioanalytical Chemistry

, Volume 404, Issue 4, pp 1103–1114 | Cite as

Comparison of data analysis parameters and MS/MS fragmentation techniques for quantitative proteome analysis using isobaric peptide termini labeling (IPTL)

  • Christian J. Koehler
  • Magnus Ø. Arntzen
  • Achim Treumann
  • Bernd Thiede
Original Paper


Isobaric peptide termini labeling (IPTL) is a quantification method which permits relative quantification using quantification points distributed throughout the whole tandem mass spectrometry (MS/MS) spectrum. It is based on the complementary derivatization of peptide termini with different isotopes resulting in isobaric peptides. Here, we use our recently developed software package IsobariQ to investigate how processing and data analysis parameters can improve IPTL data. Deisotoping provided cleaner MS/MS spectra and improved protein identification and quantification. Denoising should be used with caution because it may remove highly regulated ion pairs. An outlier detection algorithm on the ratios within every individual MS/MS spectrum was beneficial in removing false-positive quantification points. MS/MS spectra using IPTL typically contain two peptide series with complementary labels resulting in lower Mascot ion scores than non-labeled equivalent peptides. To avoid this penalty, the two chemical modifications for IPTL were specified as variables including satellite neutral losses of tetradeuterium with positive loss for the heavy isotopes and negative loss for the light isotopes. Thus, the less dominant complementary ion series were not considered for the scoring, which improved the ion scores significantly. In addition, we showed that IPTL was suitable for fragmentation by electron transfer dissociation (ETD) and higher energy collisionally activated dissociation (HCD) besides the already reported collision-induced dissociation (CID). Notably, ETD and HCD data can be identified and quantified using IsobariQ. ETD outperformed CID and HCD only for charge states ≥4+ but yielded in total fewer protein identifications and quantifications. In contrast, the high-resolution information of HCD fragmented peptides provided most identification and quantification results using the same scan speed.


CID ETD HCD Isobaric labeling IPTL IsobariQ MS/MS Quantitative proteomics 



Collision-induced dissociation


Electron transfer dissociation


Higher energy collisionally activated dissociation


Isotope-coded affinity tagging


Isotope-coded protein labeling


Isobaric peptide termini labeling


Isobaric tagging for relative and absolute quantification


Liquid chromatography


Mass spectrometry


Tandem mass spectrometry


Stable isotope labeling with amino acid in cell culture




Tandem mass tagging



We would like to thank Yue Xuan (Thermo Fisher Scientific, Bremen, Germany) and Olav Mjaavatten and Frode Berven (PROBE, Bergen, Norway) for performing the measurements on their LTQ-Orbitrap Velos Pro instruments.

Supplementary material

216_2012_5949_MOESM1_ESM.pdf (1001 kb)
ESM 1 (PDF 1001 kb)


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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Christian J. Koehler
    • 1
  • Magnus Ø. Arntzen
    • 1
  • Achim Treumann
    • 2
  • Bernd Thiede
    • 1
  1. 1.The Biotechnology Centre of OsloUniversity of OsloOsloNorway
  2. 2.NEPAFNewcastle upon TyneUK

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