Biology Reference
In-Depth Information
The Unique Nature of Af
nity Selection
In general, protein biomarkers are in general
discovered, validated, and determined routinely
by methods involving mass spectrometry (MS),
as seen in Figure 1 . A complication is that biolog-
ical extracts are frequently composed of 10 4 to
10 5 proteins. Moreover, when trypsin digestion
is being used in identi
INTRODUCTION
The discussion in this chapter focuses on
af
nity selection of proteins and peptides along
with their
cation by mass spectral
methods, similar to what is done in top-down
and bottom-up proteomics 1 ( Figure 1 ). Top-
down analyses are referred to as protein-level
identi
identi
cation, the sample
complexity problem is even worse. Mixtures of
a million or more peptides can be generated
during proteolysis of biological extracts. This
degree of complexity exceeds the analytical
limits of most MS instruments, necessitating
preliminary fractionation.
Fractionation with chromatographic and
electrophoretic systems depends on differenti-
ating between general features of analytes,
such as hydrophobicity, hydrophilicity, size,
charge, pI, metal chelating power, or reactivity.
The problem with this approach is that two
molecules can be very similar in general prop-
erties that actually differ widely in structure,
which is why fractionation methods that target
broad features
cation is
achieved with intact proteins. Bottom-up strate-
gies in contrast are designated peptide-level identi-
cation methods in which identi
fication methods based on their use of peptide
fragments derived from parent proteins. Double
selection methods combining these two strate-
gies are also discussed. The tactic in this case is
to af
rst, then
trypsin digested, and to capture several of their
signature peptide fragments in a second round
of af
nity select targeted proteins
finally to identify the
parent proteins through MS analysis of their
signature peptides. When af
nity selection, then
nity selection is
achieved with antibodies, this method is referred
to throughout this review as a mass-linked
immuno-selective analysis (MALISA).
seldom provide more than
1
protein level
affinity selection
protein level
identification
Ab on MALDI plate
MS/MS
magnetic particles
2
proteolysis
affinity chromatography
trypsin digestion
peptide level
identification
solution based or IMER
protein
mixture
4
antibody pull-down
RPC-MS/MS
peptide level
affinity selection
Ab, lectin, prot.A/G
proteolysis
magnetic particles
3
trypsin digestion
solution based or IMER
affinity chromatography
Ab, IMAC, TiO 2
FIGURE 1 Strategies for identifying proteins and peptides based on linking targeted affinity selection methods and mass
spectral (MS) analysis. The fourth route of analysis is achieved by coupling of the protein and peptide level methods.
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