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FIGURE 19.1
Overview of the Intact Protein Analysis System workflow for GVHD biomarkers discovery. The first step depletes the six most abundant proteins because the
plasma has a 10 12 dynamic range. A pool of 10 patients without GVHD (GVHD−) and a pool of 10 patients with GVHD (GVHD+) are compared. Intact proteins are labeled
with stable isotopes. The GVHD− pool receives the light isotope and the GVHD+ pool receives the heavy isotope. The cysteine residues are labeled with the light [ 12 C]
acrylamide isotope and the heavy [ 13 C]acrylamide isotope through a thiol alkylation shown here. The difference ( Δ ) of 3 between the isotopes allows for quantification.
The choice of acrylamide was made because the tag has to be small to avoid interference with MS/MS. There is no big change in physical-chemical characteristics of the
proteins. It allows a good yield and reproducibility and avoids by-products. It is compatible with the entire workflow. Then, the pools are mixed together for further analy-
sis. A two-dimensional protein fractionation is performed in which anion-exchange chromatography represents the first dimension of the protein separation and reversed-
phase chromatography the second dimension of separation. After digestion with trypsin, individual fractions are analyzed by a mass spectrometer. The acquired spectra
(LC-MS/MS) are automatically processed by the Computational Proteomics Analysis System for the identification of proteins, with a false discovery rate of less than 5%.
the candidate protein. The procedure is also relatively simple and highly
reproducible from performer to performer and from laboratory to labo-
ratory, limiting both inter- and intra-assay variability. Finally, because of
the urgent need for GVHD blood tests, we primarily tested proteins with
available antibodies. The main disadvantage of validation by ELISA is the
large volume of patient plasma required. Thus, we used a sequential ELISA
protocol to minimize freeze/thaw cycles and the use of precious plasma
samples [12] . In addition, we chose sequential over other available multi-
plex platforms for two reasons: (1) most antibody pairs for novel proteins
cannot easily be conjugated on beads or other materials and are both time-
consuming and expensive; and (2) individual ELISAs are more precise than
multiplex microarray or beads, secondary to the absence of cross-reactivity
[13] . Ideally, we would like to develop a multiplex platform using a limited
amount of plasma (<10 μl for 4-10 analytes) with no cross talk. Recently,
multiple-reaction monitoring has emerged as a potentially useful tech-
nique for clinical diagnostics [32] . This rapid tandem mass spectrometric
technique enables the targeted monitoring and quantification of candidate
molecules in complex samples.
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