Is it possible iTRAQ without LC? Or can we use iTRAQ labelling directly to MALDI for protein quantification without separation of peptides using nanoLC?
Technically yes, but realistically only if you are looking at very simple protein mixtures (e.g. 2D spots) and have a MALDI-TOF/TOF available. iTRAQ quantitation needs MS/MS data (as the differentiation only comes about on fragmentation) (there is a non-isobaric form called mTRAQ though that can be used in a similar fashion as e.g. the ICPL label). Any other peptides of similar mass that you co-isolate when you do MS/MS will also contribute to the reporter ion ratio, thus your observed reporter ion ratios will actually be a weighted average across multiple precursors in the same precursor isolation window. Consequently, iTRAQ (and the related TMT label) of complex samples rather heavily relies on good upfront separation by nanoLC. For application to 2DE separated samples you can look at e.g. Schmidt et al., Proteomics. 2013 May;13(9):1417-22.
Thanks for answer Lenz. Basically, we have nanoLC and MALDI-TOF-TOF-MS-MS Ultrflxtreme of Bruker. I checked so many papers but most of the authors suggested nanaoLC followed by MS/MS. I want to standaredize iTRAQ without separation on nanoLC and go directly on MALDI. But I will keep your suggestion in mind and go naonoLC followed by MALDI. Thanks
Regards,
Robin
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