Gaining efficiency by parallel quantification and identification of iTRAQ-labeled peptides using HCD and decision tree guided CID/ETD on an LTQ Orbitrap.

Isobaric stable isotope labeling of peptides using iTRAQ is an important method for MS based quantitative proteomics. Traditionally, quantitative analysis of iTRAQ labeled peptides has been confined to beam-type instruments because of the weak detection capabilities of ion traps for low mass ions. Recent technical advances in fragmentation techniques on linear ion traps and the hybrid linear ion trap-orbitrap allow circumventing this limitation. Namely, PQD and HCD facilitate iTRAQ analysis on these instrument types. Here we report a method for iTRAQ-based relative quantification on the ETD enabled LTQ Orbitrap XL, which is based on parallel peptide quantification and peptide identification. iTRAQ reporter ion generation is performed by HCD, while CID and ETD provide peptide identification data in parallel in the LTQ ion trap. This approach circumvents problems accompanying iTRAQ reporter ion generation with ETD and allows quantitative, decision tree-based CID/ETD experiments. Furthermore, the use of HCD solely for iTRAQ reporter ion read out significantly reduces the number of ions needed to obtain informative spectra, which significantly reduces the analysis time. Finally, we show that integration of this method, both with existing CID and ETD methods as well as with existing iTRAQ data analysis workflows, is simple to realize. By applying our approach to the analysis of the synapse proteome from human brain biopsies, we demonstrate that it outperforms a latest generation MALDI TOF/TOF instrument, with improvements in both peptide and protein identification and quantification. Conclusively, our work shows how HCD, CID and ETD can be beneficially combined to enable iTRAQ-based quantification on an ETD-enabled LTQ Orbitrap XL.

[1]  Scott A McLuckey,et al.  Electron transfer ion/ion reactions in a three-dimensional quadrupole ion trap: reactions of doubly and triply protonated peptides with SO2*-. , 2005, Analytical chemistry.

[2]  Albert J R Heck,et al.  Triplex protein quantification based on stable isotope labeling by peptide dimethylation applied to cell and tissue lysates , 2008, Proteomics.

[3]  S. Ficarro,et al.  Optimized orbitrap HCD for quantitative analysis of phosphopeptides , 2009, Journal of the American Society for Mass Spectrometry.

[4]  M. Mann,et al.  Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics* , 2002, Molecular & Cellular Proteomics.

[5]  Madalina M. Drugan,et al.  Strong Cation Exchange-based Fractionation of Lys-N-generated Peptides Facilitates the Targeted Analysis of Post-translational Modifications* , 2009, Molecular & Cellular Proteomics.

[6]  A. Makarov,et al.  Interfacing the orbitrap mass analyzer to an electrospray ion source. , 2003, Analytical chemistry.

[7]  Ronald J. Moore,et al.  Combined pulsed-Q dissociation and electron transfer dissociation for identification and quantification of iTRAQ-labeled phosphopeptides. , 2009, Analytical chemistry.

[8]  B. Bogerts,et al.  A comparison of the synaptic proteome in human chronic schizophrenia and rat ketamine psychosis suggest that prohibitin is involved in the synaptic pathology of schizophrenia , 2008, Molecular Psychiatry.

[9]  B. V. Breukelen,et al.  Targeted SCX Based Peptide Fractionation for Optimal Sequencing by Collision Induced, and Electron Transfer Dissociation , 2008 .

[10]  M. Mann,et al.  Higher-energy C-trap dissociation for peptide modification analysis , 2007, Nature Methods.

[11]  Frank Fischer,et al.  Targeted data acquisition for improved reproducibility and robustness of proteomic mass spectrometry assays , 2010, Journal of the American Society for Mass Spectrometry.

[12]  Karl Mechtler,et al.  High precision quantitative proteomics using iTRAQ on an LTQ Orbitrap: a new mass spectrometric method combining the benefits of all. , 2009, Journal of proteome research.

[13]  Carla Pasquarello,et al.  Combining low- and high-energy tandem mass spectra for optimized peptide quantification with isobaric tags. , 2010, Journal of proteomics.

[14]  K. Parker,et al.  Multiplexed Protein Quantitation in Saccharomyces cerevisiae Using Amine-reactive Isobaric Tagging Reagents*S , 2004, Molecular & Cellular Proteomics.

[15]  G. McAlister,et al.  Supplemental activation method for high-efficiency electron-transfer dissociation of doubly protonated peptide precursors. , 2007, Analytical chemistry.

[16]  Hongwei Xie,et al.  Identification of carbonylated proteins from enriched rat skeletal muscle mitochondria using affinity chromatography‐stable isotope labeling and tandem mass spectrometry , 2007, Proteomics.

[17]  S. Gygi,et al.  Quantitative analysis of complex protein mixtures using isotope-coded affinity tags , 1999, Nature Biotechnology.

[18]  Yi Zhang,et al.  Peptide and protein quantification using iTRAQ with electron transfer dissociation , 2008, Journal of the American Society for Mass Spectrometry.

[19]  G. McAlister,et al.  Decision tree–driven tandem mass spectrometry for shotgun proteomics , 2008, Nature Methods.

[20]  Christoph H Borchers,et al.  A comparison of MS/MS‐based, stable‐isotope‐labeled, quantitation performance on ESI‐quadrupole TOF and MALDI‐TOF/TOF mass spectrometers , 2009, Proteomics.

[21]  R. Aebersold,et al.  Mass spectrometry-based proteomics , 2003, Nature.

[22]  Timothy J Griffin,et al.  iTRAQ reagent-based quantitative proteomic analysis on a linear ion trap mass spectrometer. , 2007, Journal of proteome research.

[23]  A. Pandey,et al.  Comprehensive Comparison of Collision Induced Dissociation and Electron Transfer Dissociation , 2008, Analytical chemistry.

[24]  Jeroen Krijgsveld,et al.  Metabolic labeling of C. elegans and D. melanogaster for quantitative proteomics , 2003, Nature Biotechnology.

[25]  Bernhard Kuster,et al.  Robust and Sensitive iTRAQ Quantification on an LTQ Orbitrap Mass Spectrometer*S , 2008, Molecular & Cellular Proteomics.

[26]  Jeroen Krijgsveld,et al.  Mass spectrometry-based quantitative proteomics , 2004, Expert review of proteomics.

[27]  M. Mann,et al.  SILAC Mouse for Quantitative Proteomics Uncovers Kindlin-3 as an Essential Factor for Red Blood Cell Function , 2008, Cell.

[28]  Ishtiaq Rehman,et al.  iTRAQ underestimation in simple and complex mixtures: "the good, the bad and the ugly". , 2009, Journal of proteome research.

[29]  B. Blagoev,et al.  Stable isotope labeling by amino acids in cell culture (SILAC). , 2008, Methods in molecular biology.

[30]  Beatrix Ueberheide,et al.  Protein identification using sequential ion/ion reactions and tandem mass spectrometry. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[31]  F. Cross,et al.  Accurate quantitation of protein expression and site-specific phosphorylation. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[32]  Reinout Raijmakers,et al.  Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics , 2009, Nature Protocols.

[33]  Richard Unwin,et al.  Peptide quantification using 8-plex isobaric tags and electron transfer dissociation tandem mass spectrometry. , 2009, Analytical chemistry.

[34]  Ka Wan Li,et al.  Quantitative proteomics and protein network analysis of hippocampal synapses of CaMKIIalpha mutant mice. , 2007, Journal of proteome research.

[35]  Hongling Han,et al.  Electron transfer dissociation of iTRAQ labeled peptide ions. , 2008, Journal of proteome research.

[36]  J. Yates,et al.  Metabolic labeling of mammalian organisms with stable isotopes for quantitative proteomic analysis. , 2004, Analytical chemistry.