Biology Reference
In-Depth Information
Chapter 15
Mass Western for Absolute Quantifi cation of Target Proteins
and Considerations About the Instrument of Choice
David Lyon , Wolfram Weckwerth , and Stefanie Wienkoop
Abstract
The Mass Western describes the absolute quantifi cation of proteins based on stable isotope labeled integral
standard peptides and liquid chromatography coupled selective reaction monitoring triple quadrupole
mass spectrometry (LC-SRM/MS). Here, we present a detailed workfl ow including tips and we discuss
advantages and disadvantages of using different types of MS for absolute quantifi cation.
Key words
Mass Western, SRM, Heavy peptide internal standard, Absolute quantifi cation, Triple
quadrupole, Orbitrap
Abbreviations
SID Stable isotope dilution
SRM Selective reaction monitoring
PTM Posttranslational modifi cation
QqQ Triple quadrupole
LC-SRM/MS Liquid chromatography coupled selective reaction monitoring triple
quadrupole mass spectrometry
HP
Heavy peptide (
15
N
13
C labeled synthetic standard)
LP
Light peptide (native peptide, no labeling)
HCD
High energy collision induced dissociation
CE
Collision energy
LDR
Linear dynamic range
LOD
Limit of detection
LOQ
Limit of quantifi cation
1
Introduction
Absolute protein quantifi cation using stable isotope dilution
(SID) in conjunction with liquid chromatography (LC) and selec-
tive reaction monitoring (SRM) triple quadrupole (QqQ) mass
