Analysis of Peptides in Biological Fluids by LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 314

% solvent B (balance
with solvent A) Time

0 1

30 35

31 80

40 80

40.1 1

4. Flow rate should be set at 400 nl/min ( see Note 11 ).
5. The column should be equilibrated between runs at 1% B
for at least 10 min.
6. Voltages at the tune page of the tandem mass spectrometer
should be tuned for maximal sensitivity and resolution.
7. Data-dependent acquisition (DDA) experiments involve
performing MS/MS on ions that meet predefined criteria.
Typical settings are chosen to select multiply charged ions
for MS/MS that produce at least 50 ion counts/s in a 0.5 s
survey scan. This is then followed by three MS/MS scans of
the three most intense peptide ions for 1 s each.

3.5.DataAnalysis

Depending on the purpose of the study, qualitative or quanti-
tative analyses (or both) may be performed. A qualitative anal-
ysis may just involve obtaining identities of as many peptides as
possible using LC-MS/MS data; this is accomplished by match-
ing MS/MS spectra to theoretical fragmentation of peptides in
protein databases using search engines such as Mascot, Protein
Prospector and Sequest. Relative quantification of peptides may
be performed when the aim of the study is to compare polypep-
tide amounts across several samples. This workflow involves com-
paring peak areas or heights of extracted ion chromatograms of
peptides across the samples to be evaluated. Absolute quantifica-
tion of peptides in urine by LC-MS is also possible. This workflow
ideally needs the construction of standard curves and isotopically
labelled internal standards.

1. Load LC-MS/MS raw data folders into Mascot Distiller in
order to obtain peak lists of MS/MS spectra.
2. Load peak lists into Mascot. Settings should be chosen to
match the performance of the mass spectrometer and the
particulars of the experimental workflow and origin of the
sample. Appropriate mass windows, database, modifications
and enzyme should be chosen ( see Note 13 ).

3.5.1.Qualitative
Analysis (see Note12)