title: An approach for triplex-isobaric peptide termini labeling (triplex-IPTL).
abstract: Isobaric peptide termini labeling (IPTL) is based on labeling of both peptide termini with complementary isotopic labels resulting in isobaric peptides. MS/MS analysis after IPTL derivatization produces peptide-specific fragment ions which are distributed throughout the MS/MS spectrum. Thus, several quantification points can be obtained per peptide. In this report, we present triplex-IPTL, a chemical labeling strategy for IPTL allowing the simultaneous quantification of three states within one MS run. For this purpose, dimethylation of the N-terminal amino group followed by dimethylation of lysines was used with different stable isotopes of formaldehyde and cyanoborohydride. Upon LC-MS/MS analysis, the combined samples revealed three corresponding isotopic fragment ion series reflecting quantitatively the peptide ratios. To support this multiplexing labeling strategy, we have further developed the data analysis tool IsobariQ and included multidimensional VSN normalization, statistical inference, and graphical visualization of triplex-IPTL data and clustering of protein profiling patterns. The power of the triplex-IPTL approach in combination with IsobariQ was demonstrated through temporal profiling of HeLa cells incubated with the kinesin Eg5 inhibitor S-Trityl-l-cysteine (STLC). As a result, clusters of quantified proteins were found by their ratio profiles which corresponded well to their gene ontology association in mitotic arrest and cell death, respectively.
authors: The Biotechnology Centre of Oslo, University of Oslo, P.O. Box 1125 Blindern, 0317 Oslo, Norway.
authors: Christian J
authors: CJ
authors: Koehler
authors: Magnus Ø
authors: MØ
authors: Arntzen
authors: Gustavo Antonio
authors: GA
authors: de Souza
authors: Bernd
authors: B
authors: Thiede
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D002247:Carbon Isotopes / Q000737:chemistry; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D003545:Cysteine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D003903:Deuterium / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D053719:Tandem Mass Spectrometry
chemical_list: D002247:Carbon Isotopes; D010455:Peptides; C003724:3-tritylthio-L-alanine; D003903:Deuterium; D003545:Cysteine