Comparison of Proteomic Profiling Technologies for Discovery of Biomarkers

A comparison of proteomic profiling technologies for discovery of biomarkers is shown in Table 2.2.

Verification for Interlaboratory Reproducibility of Protein Biomarkers

Discovery proteomics often results in a list of tens or even hundreds of potential biomarkers, but because analyzing each biomarker can take up to several weeks and the sample numbers are low, the false discovery rates tend to run high. This is not

Table 2.2 Comparison of proteomic profiling technologies for discovery of biomarkers

Approaches

Ability to detect isoforms

Precision in relative quantitation

Ability to detect low abundance proteins

High speed, low cost and simplicity

Optical methods (e.g., ELISA, SPR)

No

Yes

Yes

Yes

MudPIT

Yes

No

No

No

ProteinChip®

Yes

No

No

Yes

ICAT

Yes

Yes

No

No

ITRAQ™ (Applied Biosystems)

Yes

Yes

No

No

Isonostics™ (Target Discovery Inc)

Yes

Yes

Yes

Yes

Modified from Hall and Schneider (2004)

Abbreviations: SPR surface plasma resonance, MudPIT multidimensional protein identification technology, ELISA enzyme-linked immunosorbent assay, ICAT isotope-coded affinity tag

necessarily because of technical variability but rather is the consequence of biological variability in the samples.

Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Such technology has been limited by the availability of well- characterized antibodies, a well-known problem in protein research. Developing high-quality immunoassays also is costly and time-consuming. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated.

A multilaboratory study has assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRM-based assays, conducted by NCI-CPTAC (Addona et al. 2011). Using common materials and standardized protocols, it was shown that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low microgram per milliliter protein concentrations in unfractionated plasma. The study has provided data and benchmarks against which individual laboratories can compare their performance and evaluate new technologies for biomarker verification in plasma. Such methods, combined with protein- and peptide-enrichment strategies, are able to hit target values for limits of quantitation that are in the very bottom of the nanogram per milliliter range for proteins in blood, where many biomarkers of clinical utility are located.

 
Source
< Prev   CONTENTS   Source   Next >