Quantification of Proteins by Label-Free LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 217

Table 13.1
Four types of label-free quantitation of proteins by LC-
MS/MS

MS acquisition
mode

Method Method of quantitation

Spectral counting Data-dependant
acquisition
(DDA)

Counting the number of times a
peptide was identified

DDA-based ion
counting

Data-dependant
acquisition
(DDA)

Ion intensity of intact peptides
detected in the MS scans (sur-
vey scans)

MS survey scan-
based ion
counting

MS survey scan Ion intensity of intact peptides

MS E -based ion
counting

MS E Ion intensity of intact peptides

or may not be at the chromatographic apex. The selected pre-
cursor ions are serially isolated for an MS/MS acquisition for an
allotted period of time, or until a certain ion current is breached.
This cycle of MS and MS/MS acquisitions continues throughout
the run time.
The first label-free method, termed 'spectral counting'
approach, relies on DDA acquisition and the quantitation is
based on how many times a peptide was fragmented. The sec-
ond method relying on DDA analysis achieves quantitation based
on the measured abundance of the intact peptides in the sur-
vey scans, which are performed intermittently between MS/MS
events. Although this method produces more accurate quantita-
tion than the previous ( 10 ) it suffers from two major pitfalls. First,
in DDA analysis a survey scan is performed over long time inter-
vals; thus the sampling rate of the eluting peptides is too low to
obtain accurate quantitation. The second pitfall is that the identi-
fication is not reproducible and most proteins are typically identi-
fied with only one or two peptides ( 11 , 12 ) .
The second strategy, which relies on MS survey acquisition,
includes two approaches. The first approach requires a blinded
analysis in which the molecular weight and intensity of all pep-
tides eluting from the separation column are measured. The MS
operates in survey mode (MS scan), at a high sampling rate. The
quantitation is based on the extracted ion chromatogram (XIC) of
intact peptide intensity measurements. Identification of the pep-
tides and their corresponding proteins is performed by a separate
DDA experiment with an 'include list' (switch list) that contains
the significantly changing peptides. In this way only the selected
peptides are fragmented and thus identified and no other ones.