Multiplex counts

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 Related Articles Peak Addition Tips for Quantifying Mass Spec Easy numbers Clean targeting Label-free Metabolic power Problem: Quantifying samples serially takes a long time and adds variability to the data, so Whetton wanted to measure

Mar 1, 2008
Jeffrey M. Perkel

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 different samples simultaneously (a reference sample uses the eighth tag). (The other techniques profiled here can mostly do two at a time, though PerkinElmer's ExacTag reagent can multiplex 10 samples at once.) There is a drawback, however: Sample mixing can occur only at the peptide level, after labeling, so differences in sample preparation can influence the data. With metabolic techniques, for instance, "you can mix the cell lysates or you can mix the cells, and then all the enrichment steps take place with the two samples together, so any errors engendered through the enrichment procedure are nullified," says Whetton. "That's not the case" with iTRAQ, he adds.

Equipment:
Because it relies on the production of reporter ions, iTRAQ quantitation requires a tandem mass spectrometer. Whetton's choice: an electrospray-coupled QSTAR.

Cost:
Applied Biosystems' iTRAQ reagents multiplex (4-plex) kit costs $1,250.