Quantification of Proteins by Label-Free LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 218

The second MS survey-based approach, termed data-
independent analysis (MS E )( 13 ) , is similar to the previous except
that during MS acquisition the collision energy is alternated such
that two channels are collected. The first channel includes the
abundance measurements of the intact peptides and a second
channel for the fragmented peptides. Both are acquired at a high
sampling rate. In this type of acquisition the chromatographic
profile is reproducibly maintained throughout the run time and
thus across the sample set, allowing for the precursor ions to be
aligned in time. Once aligned the intensities of precursor ions can
be normalized and directly compared across all injections of all
samples. This method, therefore, enables relative quantitation and
identification of peptides (and thus proteins) in the same experi-
ment without the need to reanalyze samples. Furthermore, data
analysis can be performed on unidentified peptides as well as those
that were identified.
Whichever method is used, time alignment of data must be
performed to summarize the information from all samples ( see
Fig. 13.1 ) .
In this chapter we present the methodology for MS E -based
label-free proteomics since in our experience its performance is
superior to other methods described in the literature in a num-
ber of aspects ( 14 ) . It enables the reliable identification and
Fig. 13.1. A typical workflow for LC-MS E -based label-free quantitation. Samples are analyzed sequentially and randomly.
Each raw data file is processed and searched and then all data files are combined by time alignment of all detected
features.