Proteomics in graft-versushost disease and graftversus-leukemia - Immune Biology of Allogeneic Hematopoietic Stem Cell Transplantation - page 446

Current proteomics technologies for biomarker
discovery

Clinical proteomics can be defined as the identification and validation of
disease biomarkers with the objective to improve the current state of the
art in clinical practice. The classical paradigm that DNA determines the
fate of the cell is currently being questioned, as complex regulatory pro-
cesses at the level of both transcription and translation are better appreci-
ated. In addition, advances in engineering have allowed for increased data
throughput, enabling the study of complete sets of molecules (“-omics”)
with exponential speed, accuracy, and cost-effectiveness. Thus, the analysis
of the entire spectrum of molecular and cellular organization is now pos-
sible, enabling researchers to gain insight into the mechanism of disease,
with fewer a priori assumptions. However, from genes (~20,000) to proteins,
there are two more levels of complexity: the transcriptome (~100,000 RNA
transcripts) and the proteome (~1,000,000 proteins). Here, we focus on the
use of proteomics for the molecular diagnosis of GVHD post-HSCT, since
proteins are more proximal than other cellular metabolites to the ongoing
pathophysiology of this disease. Indeed, studies using genomics, transcrip-
tomics, and gene polymorphisms incompletely correlate with the expres-
sion of functionally active proteins, which more accurately reflect cellular
cross talk, such that it is likely that proteins will provide the most ideal dis-
ease biomarkers [10] .

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Proteomics technologies are currently used in two related areas: biomarker
discovery and the elucidation of pathologic processes to identify novel
therapeutic targets. The first could lead to new proteins that provide new
insights into the biology of GVHD. Both non-mass spectrometry (MS)- and
MS-based proteomic approaches have been employed to search for poten-
tial GVHD biomarkers. Several studies have examined specific proteins,
whereas other large-scale studies have investigated qualitative and quan-
titative differences in the complete protein profiles among samples from
patients with and without GVHD.

Antibody-based approaches

Immunoassays are sensitive, analytical tests that harness the unique proper-
ties of antibodies. They proved to be one of the most productive technologi-
cal contributions to medicine and fundamental life science research in the
20th century. The unique characteristics of antibodies are derived from their
three important properties: (i) their ability to bind to an extremely wide range
of natural and man-made chemicals, biomolecules, and cells, as antibody-
binding sites are derived from a huge number of potential combinations of
amino acid sequences; (ii) their exceptional binding specificity that enables
the measurement of picomolar (10 −12 ) amounts of proteins in blood samples;
and (iii) the strength of binding between an antibody and its target that makes
the test accurate and precise, even at low concentrations [11] . To screen for
aGVHD biomarkers, antibody microarrays dotted with hundreds of antibod-
ies have been employed, allowing hundreds of proteins in complex biological
matrices to be measured [12] . In summary, the advantages of immunoassays
are that they are (i) suited to the characterization of complex protein mix-
tures, such as human plasma; (ii) quantitative; (iii) highly sensitive for low