Clean targeting

User:
John Yates III, professor of chemical physiology, The Scripps Research Institute, La Jolla, Calif.

Project:
Identifying insulin-signaling targets in Caenorhabditis elegans

Problem:
Yates needed a rapid and accurate way to measure the change in abundance of three specific targets over time and growth temperature.

Solution:
Yates uses single-reaction monitoring (SRM). "You can do this with other methods but it takes longer, and SRM also provides the most accurate measures," he says. SRM measures the abundance of a given parent ion based on the abundance of its specific collision-induced fragmentation products. Such transitions of parent ion to fragment are unique. For example, several peptides might weigh 2,326.6 Da, but only one will produce a fragment ion of 1,242.3 Da. Then, quantify by comparing the number of diagnostic fragmentation ions to a labeled reference protein extract.

SRM's precision, Yates says, stems from its two levels of filtration. Only one selected ion in a mixture can pass into the collision cell, and then only one fragmentation ion is counted. As a result, "you get excellent signal-to-noise because you filter out all the background." But, SRM cannot be used for exploratory work. "We weren't fishing, we were looking for something very specific," he says.

Equipment:
SRM requires a tandem mass spec, but just about any will do. The triple quadrupole offers the best linear dynamic range and sensitivity. Yates didn't have a triple-quad then, so he used an electrospray-coupled Thermo LTQ hybrid triple-quadrupole linear ion trap instead.

Cost:
Spectra 9-N growth medium from Spectra Stable Isotopes, for labeling reference extracts, costs $135 per L.