Biology Reference
In-Depth Information
Chapter 16
Selected Reaction Monitoring Mass Spectrometry:
A Methodology Overview
H. Alexander Ebhardt
Abstract
Moving past the discovery phase of proteomics, the term targeted proteomics combines multiple approaches
investigating a certain set of proteins in more detail. One such targeted proteomics approach is the combi-
nation of liquid chromatography and selected or multiple reaction monitoring mass spectrometry (SRM,
MRM). SRM-MS requires prior knowledge of the fragmentation pattern of peptides, as the presence of the
analyte in a sample is determined by measuring the
m/z
values of predefi ned precursor and fragment ions.
Using scheduled SRM-MS, many analytes can robustly be monitored allowing for high-throughput sample
analysis of the same set of proteins over many conditions. In this chapter, fundaments of SRM-MS are
explained as well as an optimized SRM pipeline from assay generation to data analyzed.
Key words
Selected reaction monitoring (SRM), Multiple reaction monitoring (MRM), Targeted
mass spectrometry
1
Introduction
SRM-MS is a mass spectrometry method typically performed on
triple-quadrupole instruments taking full advantage of the large
dynamic range of quadrupoles. The fi rst quadrupole is used to
defi ne the precursor ion mass (Q1) with a specifi ed window, e.g.,
m/z
± 0.35. All precursor ions passing through the fi rst window are
fragmented in the second quadrupole. The third quadrupole (Q3)
acts again as a fi lter with a specifi c window, e.g.,
m/z
± 0.35, to
allow for specifi c fragment ion to pass through toward the detec-
tor. These dual fi lters provide a higher sensitivity for monitoring
specifi c analytes as supposed to conventional shotgun experiments.
The values of Q1, a particular precursor ion, and Q3, a specifi c
fragment ion, are often referred to as
transitions
. The intensity of
the transition is recorded by a detector (electron multiplier tube)
resulting in an ion chromatograms as a function of time and transi-
tion measured. Per precursor ion, typically fi ve transitions are
