Selected Reaction Monitoring Applied to Quantitative Proteomics - LC-MS/MS in Proteomics: Methods and Applications - page 160

method. Information can be extracted from either pub-
lic repositories such as Peptide-Atlas or MRM-Atlas ( 9 )
or in-house discovery results. They are directly imported
in the software package used to design the method (e.g.,
Pinpoint). Existing MS/MS spectral libraries are queried
to extract fragment ions to generate the SRM transitions,
usually the most intense assigned fragment ions.
e. If the proteins have not been reported before, the pep-
tide selection is performed in silico using the protein
sequences. Precursor and fragment ion m / z are calcu-
lated, and the elution time is predicted using hydropho-
bicity indexes ( 7 ) .
2) For each peptide, a series of transitions are defined, to ensure
a high degree of selectivity. If the MS/MS spectra of the
peptides were previously reported, five to eight of the most
intense y ions are selected from the reference spectrum ( see
Note 4) . If no MS/MS information is available, the m / z
values of the fragments are calculated, and y fragments with
m / z values above the precursor ion are favored.
3) The collision energy (CE) in an initial study is calculated for
each peptide based on a generic formula of CE (V)

= 2+

precursor ion m / z .
4) This initial transition list is exported as a csv file and
uploaded into the instrument method.

0.034 ×

3.5.LC-MSAnalysis
inSRMMode

The instrument settings for an SRM experiment are described in
Section 3.1 .
1) Import the SRM method, which includes the Q1 and Q3
m / z values and the collision energy of each transition.
2) By default, the start time and the stop time of each transition
are set to 0 and the end of the gradient time, respectively.
Specific elution time windows are active in t-SRM experi-
ments (see below).
3) The SRM is performed using the HPLC, MS settings, and
the specific SRM method.

3.6.DataAnalysis
andEvaluation

1) The raw data files are processed with the instrument soft-
ware (e.g., Pinpoint and Xcalibur) and the results are
exported in a tabular format (e.g., Excel sheet).
2) The peptide identity is confirmed by verifying the co-elution
of multiple fragment ions of each analyte and their intensities
are compared with a reference MS/MS spectrum.
3) If isotopically labeled reference peptides are used, the co-
elution of the reference and the endogenous peptides
is a strong evidence of the identity, together with their