Quantification of Proteins by Label-Free LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 216

in a single experiment ( 2 - 7 ) , producing information-rich stud-
ies that could lead to the discovery of novel disease biomarkers.
Obtaining both qualitative and quantitative information also pro-
vides deeper and more comprehensive insights into the origin and
structure of biological systems by allowing global proteomic pro-
filing. Regardless of the method of choice, quantitative analysis
requires an added degree of stringency, especially when analyz-
ing complex samples taken from patients suffering from complex
diseases such as neuropsychiatric diseases, which are polygenic,
multi-symptomatic, and present with overlapping symptoms.
In order for proteomic profiling experiments to be statistically
powered, it is necessary to investigate large sample cohorts.
As discussed in more detail in Chapters 1 and 2 of this vol-
ume, there are several different platforms used for global pro-
teomic quantitative profiling. Among the most popular are gel-
based and isotopic-labeling methods, and protocols for these are
outlined in Chapters 11 , 12 , 14 , 15 and 16 of this volume; how-
ever, these come with difficulties in meeting some of the require-
ments for comprehensive and reproducible analysis ( 6 ) . These
requirements are ( 8 , 9 ) ( 1 ) the ability to detect as many proteins
as possible; ( 2 ) achieving a dynamic range that is wide enough
to detect low-abundance proteins; ( 3 ) high reproducibility and
consistency of the platform performance so that biological dif-
ferences can be sufficiently distinguished from instrumental ones,
along with successful validation of quantitative significance; and
( 4 ) the ability to profile and compare a large number of non-
pooled samples. The necessity to analyze as many discrete (non-
pooled) samples as possible cannot be overestimated. Greater
n -numbers enable researchers to apply a variety of statistical meth-
ods and increase the statistical power of the analysis that are not
possible when analyzing only a handful of samples or pooled sam-
ples. Furthermore, by analyzing samples in a discrete manner it
is possible to investigate subpopulations within a given group as
well as verify whether any demographics influence the underlying
proteomic profile.
There are several methodologies for label-free relative quan-
titation of proteins. The basic strategy is similar for all meth-
ods where the samples are analyzed sequentially and discretely
where neither proteins nor peptides are labeled. Thus the relative
quantitation relies on reproducible intensity measurements by the
MS. The label-free methods are distinguished by the MS mode
of operation, a defining factor for the analysis. There are four
types of strategies: the first two rely on acquiring data in data-
dependant analysis mode (DDA) and in the second two methods
data are acquired in MS survey mode ( see Table 13.1 ) .
A DDA experiment is typically a serial process. The cycle starts
by acquiring an MS survey scan followed by the selection of a
number of precursor ions for fragmentation (MS/MS) that may