Protocol for Quantitative Proteomics of Cellular Membranes and Membrane Rafts - LC-MS/MS in Proteomics: Methods and Applications - page 250

18. Depending on the number of samples to process, the in-
gel digestion procedure can take more than half a day to
complete and it may be convenient to alternatively digest
the samples overnight, at 37 ◦ C for at least the initial 2 h.
19. ESI-LC-MS/MS methods are platform dependent, can
vary significantly, and should be designed and optimised for
the specific instrument type/configuration and laboratory
conditions. However, some general considerations for MS
analysis of iTRAQ R labelled peptides of complex samples
should be considered. Quantitative reporter ions are gen-
erated from iTRAQ R labelled peptides by cleavage of an
amide bond during MS/MS, analogous to the formation
of b and y ions. Higher collision energies than standard can
generate more intense reporter ions for quantitation, e.g.
for a QToF micromass spectrometer, MS/MS data for pep-
tides are acquired using 30-50 V collision energy depend-
ing on peptide mass. For iTRAQ R analysis collision energy
is elevated by a further 5 V, similarly to analysing phospho-
peptides.

Acknowledgements

The authors would like to thank Dr. Amy Pooler and Dr. Helen
Byers for advice and assistance with reviewing the protocols.
This work was funded by the Medical Research Council and the
Department of Trade and Industry, UK.

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