Basic Design of MRM Assays for Peptide Quantification - LC-MS/MS in Proteomics: Methods and Applications - page 168

A

Discovery experiment

m/z

Q1 Q2 Q3

Precursor ion
Transmission

Fragmentation
chamber

Fragment ion
Mass analysis

Detection

MRM experiment

Time

Q1 Q2 Q3

Precursor ion
Mass filter

Fragment ion
Mass filter

Fragmentation
chamber

Detection

B

LC MS/MSMS discovery experiment
-analysis of sample composition

Selection of peptides for MRM design

Selection of fragment ions for MRM

Optimize transition selection and LC/MS parameters
through re-iterative rounds of MRM

Design of internal and external controls and number of replicates needed
Optimization of LC conditions

Perform assay

Fig. 10.1 ( A ) Outline of information-dependent analysis (IDA) mass spectrometry and
MRM mass spectrometry. In IDA experiments, the most abundant precursor ion is
selected (in Q1) and then fragmented (in Q2). This is followed by subsequent analy-
sis of all fragment ions (in Q3). The resulting MS/MS spectrum is used for identification
of the fragmented precursor ions. In an MRM experiment only pre-defined peptides (in
Q1) are selected and fragmented (in Q2). Fragment ions, which have been pre-defined,
are selectively passed through Q3 and detected. Thus in comparison, MRM is a selec-
tive workflow for mass spectrometry, which only detects a pre-defined combination of
precursor and fragment ions. ( B ) Flow diagram for MRM design.

(5) Formic acid (HCOOH)
(6) Acetonitrile (CH 3 CN)
(7) OmniSolv-grade water
(8) Trypsin
(9) Iodoacetamide (ICH 2 CONH 2 )
(10) Dithiothreitol (HSCH 2 CH(OH)CH(OH)CH 2 SH)
(11) C18 reverse phase, Reprosil AQ pur (Dr. Maisch)