Quantification of Proteins by Label-Free LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 222

Table 13.3
(A) The LC gradient for tissue extracts and cell lines. (B) The
LC gradient for depleted serum or plasma
ATi e ( in)

Flow( µ l/min) %A %B Curve

Initial 0.3 97 3 Initial

1 0.3 97 3 Linear

100 0.3 70 30 Linear

115 0.3 5 95 Linear

126 0.3 97 3 Linear

BTi e ( in) Flow( µ l/min) %A %B Curve

Initial 0.3 95 5 Initial

1 0.3 95 5 Linear

80 0.3 70 30 Linear

90 0.3 5 95 Linear

104 0.3 5 95 Linear

105 0.3 95 5 Linear

Mass accuracy is maintained throughout the analysis by the
use of a LockSpray apparatus. A reference compound (Glu-
Fibrinopeptide B, Sigma, St. Louis, MO) is continuously
infused using the LockSpray and scanned intermittently
every 30 s. During data processing, the analyte spectra are
corrected automatically based on the difference between the
detected m / z peak and the theoretical m / z peak (785.8426
[m+2H]+) of Glu-Fibrinopeptide B.

Raw data, acquired in continuum format, are processed using the
ProteinLynx Global Server software version 2.3 (also known as
Identity E ) (Waters, Milford, MA). Both quantitative and qual-
itative information are produced automatically by the software,
using the default parameters.

3.2.DataProcessing
andProtein
Identification

Intensity measurements are obtained by integration of the total
ion volume of each extracted, charge state-reduced, deiso-
toped, and mass-corrected ions across the mass spectrometric
and chromatographic volume ( Fig. 13.2 ) , as opposed to two-
dimensional integration of extracted ion chromatograms (XIC).
The algorithm calculates the observed mass and intensity mea-
surement deviation for every detected component. The chro-
matographic area associated with each component is calculated
using an integration algorithm similar to the ApexTrack peak
integration algorithm provided in the MassLynx software. If a
particular component exists in more than one charge state, the

3.2.1.Quantitative
Information