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are not suitable for validation purposes because of time consumption, but
they remain the most efficient methods for biomarker discovery in clinical
research.
Detailed below is the IPAS proteomics workflow that we utilized. Briefly,
GVHD-negative and -positive pools of 10 patients matched for other clini-
cal characteristics were individually immunodepleted of the six most abun-
dant plasma proteins (i.e., albumin, IgG, IgA, transferrin, haptoglobin, and
anti-trypsin). Intact proteins were then labeled on cysteine residues with
acrylamide-stable isotopes. The GVHD-negative pool was labeled with the
light acrylamide isotope
12
C, whereas the GVHD-positive pool received
the heavy acrylamide isotope
13
C. The two pools were combined, and
specimens were subjected to a 2-D protein fractionation procedure that
included anion-exchange chromatography followed by reversed-phase
chromatography. The individual fractions were then digested and analyzed
on a new-generation LC-MS/MS instrument. Because protein digestion
was performed in a top-down fashion prior to MS, the term “intact” protein
analysis is used
[26]
. The acquired spectra were automatically processed by
the high-throughput Computational Proteomics Analysis System to identify
proteins in the sample, with a false discovery rate of <5%
[30]
. This process
resulted in both the reduced complexity of individual fractions subjected
to analysis and the identification of proteins with a range of concentrations
spanning 7 logs
[31]
. This technique was therefore able to detect low abun-
dance proteins and is quantitative, as each GVHD pool was labeled with
both heavy and light stable isotopes. We sequentially prioritized the list of
proteins identified by the MS/MS method described above based on the
degree of dysregulation, as indicated by at least a twofold increase in expres-
sion and known pathway networks, as well as uniqueness to the target organ
associated with a given GVHD type. In summary, the IPAS approach has the
advantages of: (i) discovering candidate biomarkers in an unbiased fashion;
(ii) being quantitative; (iii) using a top-down approach that keeps the pro-
tein intact until the last step; and (iv) being high-throughput because of
the use of liquid-phase fractionation. There are limitations however, as: (i)
IPAS is available only in specialized laboratories; (ii) it is sensitive for low
abundance proteins, but less so than antibody-based methods; and (iii) at
least in practice, follow-up is often limited to those proteins for which an
antibody is currently available.
Figure 19.1
summarizes the IPAS workflow.
454
Proteomics approach for high-throughput validation of
GVHD biomarkers
Although proteomics holds great promise for biomarker development,
gaps still remain between biomarker discovery and biomarker validation.
Indeed, validation of biomarkers has obstacles of its own. Most notewor-
thy is the paucity of affinity-capture reagents, such as high-quality antibod-
ies with the required affinities and specificities for the target, leading to a
bias in the prioritization of candidate markers. Furthermore, the number
of samples required for validation increases as the biomarker advances
through each test phase, augmenting the need for high-throughput assays.
The most applicable approach for the quantitation of individual proteins
for validation remains the sandwich enzyme-linked immunosorbent assay
(ELISA), which is highly specific and employs two antibodies specific for
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