User:
Anthony Whetton, professor of cancer cell biology, University of Manchester, UK
Project:
Measuring proteomic changes induced by a panel of oncogenic tyrosine kinases in cultured cells
Problem:
Quantifying samples serially takes a long time and adds variability to the data, so Whetton wanted to measure protein abundance reliably in one run.
Solution:
Whetton uses Applied Biosystems iTRAQ (isobaric tag for relative and absolute quantification) reagents to quantify as many as eight samples at once. Isobaric means that each tag has the same mass (about 145 Da). For each set of tags, that mass is distributed unequally on either side of a molecular fault line. In the mass spec collision cell, each tag breaks to produce a diagnostic reporter ion of between 113 and 121 Da, whose abundance reflects the abundance of the original protein. The peptide itself fragments too, providing sequence data.
An 8-plex iTRAQ kit can quantify seven ...
