Analysis of Peptides in Biological Fluids by LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 315

3. Submit searches and review data retuned by Mascot. A decoy
database should be searched to estimate the false positive
identification rate.

Relative quantification may be performed by comparing peak
areas or heights of extracted ion chromatograms of the peptide
mass across samples ( see also Fig. 19.2 ) .
1. In the 'QuanLynx' function of MassLynx, create a new
method by entering the m / z and retention time of the
peptide(s) of interest as well as internal standard peptides
( Fig. 19.2A ) .

3.5.2.Quantitative
Analysis (see Note14)

A B

523.75
1.069e+003

Angiotensin
Area:187.32

100

40 nM

%
-0

523.75
6.482e+002

Angiotensin
Area:98.57

100

20 nM

%
-0

min

523.75
9.583e+001

Angiotensin
Area:15.41

100

C

5 nM

200

187.32

%
-0

180

y = 4.7708x - 1.8165
R ² = 0.9971

min

160

140

523.75
3.760e+001

120

1 nM Angiotensin

Area:4.49

100 99

98.57

80

%
-1

60

min

40

15.0 20.0 25.0 30.0 35.0 40.0 45.0

20

15.41

523.75
9.775e+001

0

0 4.49

0 10 20 30 40 50

0 nM

99

Angioten sinspiked in urine nM

%
-1

min

15.0 20.0 25.0 30.0 35.0 40.0 45.0

Fig. 19.2 Quantification of angiotensin in urine. To demonstrate quantification of a peptide in urine, control urine
aliquots were spiked with increasing amounts of angiotensin (m/z = 523.75) and a standard curve constructed. Pep-
tide m/z values were entered in QuanLynx (A) and used to obtain extracted ion chromatograms of the peptide ions of
interest (B) whose intensities (areas or peak heights) can then be plotted to investigate linearity and dynamic range
of quantification (C) .

2. Process samples by running this method across the samples
to be compared.
3. Review XICs and correct peak integration as required
( Fig. 19.2B ) .
4. Export area values of XICs into a spreadsheet file such as
Excel.