Biology Reference
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abundance proteins such as cytokines; and (iv) high-throughput. The dis-
advantages are that there are only a restricted number of antibodies on the
array (thus, introduction of bias) and high cross-reactivity between antibod-
ies and nontarget proteins
[13]
.
MS-based approaches
The majority of nonantibody proteomics strategies are based on MS, which
has become a powerful tool for both characterizing and assessing both
qualitative and quantitative changes in complex protein mixtures
[14]
. Two
types of MS techniques in clinical proteomics have been used: (i) pattern
profiling; and (ii) detailed characterization of proteins. Pattern profiling
compares polypeptide spectra obtained by matrix-assisted laser desorp-
tion/ionization time of flight (MALDI-TOF) MS, which is used to show
which patients suffer from a particular disease without the identification
of individual profile components. A variant of MALDI-TOF MS is surface-
enhanced laser desorption/ionization (SELDI-TOF) MS, which combines
MALDI-TOF with selective sample fractionation on modified surfaces
placed directly on the sample target
[15]
. These MS profiling methods do not
require an in-depth analysis and, thus, are relativ ely high-throughput. They
are consequently less suitable for in-depth discovery approaches. Further-
more, because the factors influencing the final oligopeptide profiles of body
fluid samples are so complex, MS profiling has not yet met the standards
required in clinical practice. This technique has been applied in aGVHD
research to screen biomarker candidates in both serum
[16]
and saliva
[17]
.
Other approaches rely on separation of protein samples followed by MS.
The most frequently employed gel-based techniques for protein separation
are two-dimensional (2-D) polyacrylamide gel electrophoresis
[18]
and 2-D
differential gel electrophoresis (DIGE)
[19]
. Three-dimensional separation
of proteins, differentially labeled with fluorescent dyes [Cy3 (green) and Cy5
(red)] according to their charge, hydrophobicity, and molecular mass, have
been applied to aGVHD diagnoses
[20]
and heart ischemic insult
[21]
.
453
Despite the utility of gel-based techniques, gel-free separation methods,
such as liquid chromatography (LC)
[22,23]
and capillary electrophoresis
(CE)
[24]
, have provided better separation because they overcome several
limitations of gel separation, such as time consumption; poor separation of
proteins with low molecular weight (MW), high MW, or an extreme isoelec-
tric point; and difficult quantification of mixed spots. Gel-free techniques
also offer the prospect of an easy workflow with a direct connection with
the mass spectrometer. MS is the final step in the analytical procedure and
enables both the reliable identification of proteins and the determination of
their isoforms and post-translational modifications. MS allows unambigu-
ous quantification, particularly when tandem MS (i.e., MS/MS) is employed
[25]
, and has been used most recently for quantification with either label-
free methods or isotopically labeled tags
[26-28]
. In addition, new instru-
mentation, such as the ultra-high-resolution linear ion trap Orbitrap mass
spectrometer (Orbitrap Elite), facilitates top-down LC-MS/MS and versa-
tile peptide fragmentation modes
[29]
, increasing the number of proteins
identified by the Intact Protein Analysis System (IPAS) workflow by at least
twofold (personal communication). The mass spectra are then matched to
a sequence database to identify proteins
[30]
. At present, these approaches
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