A Concentration-Dependent Analysis Method for High Density Protein Microarrays

Abstract

Protein microarray technology is rapidly growing and has the potential to accelerate the discovery of targets of serum antibody responses in cancer, autoimmunity and infectious disease. Analytical tools for interpreting this high-throughput array data, however, are not well-established. We developed a concentration-dependent analysis (CDA) method which normalizes protein microarray data based on the concentration of spotted probes. We show that this analysis samples a data space that is complementary to other commonly employed analyses, and demonstrate experimental validation of 92% of hits identified by the intersection of CDA with other tools. These data support the use of CDA either as a preprocessing step for a more complete proteomic microarray data analysis or as a stand-alone analysis method.

Novel myeloma-associated antigens revealed in the context of syngeneic hematopoietic stem cell transplantation

“…20 All samples were screened using arrays from the same printing lot. The microarrays contained approximately 8000 N-terminus GST-fusion human proteins expressed in an insect cell line and spotted on nitrocellulose-coated glass slides.…”

“…Fluorescence intensities were quantified using GenePix Pro Version 5.0 software (Molecular Devices) at 635 nm, 100% power, and 600 gain. Significant Ab-protein interactions were determined using Prospector Version 5.1.0 analysis software (Invitrogen) and an algorithm 20 that incorporates the contribution of protein spot concentration to signal intensity. Signal change between pre-treatment or control plasma and post-treatment plasma was considered significant if the change in both (1) the signal magnitude (Z delta ), defined as Z post Ϫ max(0, Z pre ) and (2) the ratio (Z mult ), defined as Z post /max(1,Z pre ), were greater than a cutoff value n (n ϭ 5 for significant Ags, called "candidate Ags").…”

“…Expressed protein was immunoprecipitated using patient plasma, as described previously. 20 Immunoprecipitated proteins were subjected to SDS-PAGE on 10% Tris-HCl polyacrylamide gels (Bio-Rad), transferred to nitrocellulose membranes in Tris-glycine buffer with 20% methanol (blocked overnight in 1ϫ TBS/0.5% Tween-20), and detected using streptavidin-HRP conjugate (1:20 000 dilution; MP Biomedicals).…”

“…Significance of Ab binding to protein spots was determined using established analytical methods. 20 Candidate Ags were defined as those proteins eliciting significantly more plasma Ab binding from post-HSCT plasma compared with pre-HSCT plasma. Six proteins (C1orf116, DAPK2, PDGFRB, PIM1, PRKCB1, and RELA) met the criteria for candidate Ags (Table 2).…”

Reverse phase protein microarray technology in traumatic brain injury

“…Relative quantification is usually sufficient for this purpose. However, absolute quantification, needed for diagnostics, can be achieved by adding a certified standard to the samples printed on the slide (Breitling et al, 2004;Marina et al, 2008).…”