Development of reverse phase protein microarrays for the validation of clusterin, a mid-abundant blood biomarker

<p>Abstract</p> <p>Background</p> <p>Many putative disease blood biomarkers discovered in genomic and proteomic studies await validation in large clinically annotated cohorts of patient samples. ELISA assays require large quantities of precious blood samples and are not high-throughput. The reverse phase protein microarray platform has been developed for the high-throughput quantification of protein levels in small amounts of clinical samples.</p> <p>Results</p> <p>In the present study we present the development of reverse-phase protein microarrays (RPPMs) for the measurement of clusterin, a mid-abundant blood biomarker. An experimental protocol was optimized for the printing of serum and plasma on RPPMs using epoxy coated microscope slides and a non-denaturing printing buffer. Using fluorescent-tagged secondary antibodies, we achieved the reproducible detection of clusterin in spotted serum and plasma and reached a limit of detection of 780 ng/mL. Validation studies using both spiked clusterin and clinical samples showed excellent correlations with ELISA measurements of clusterin.</p> <p>Conclusion</p> <p>Serum and plasma spotted in the reverse phase array format allow for reliable and reproducible high-throughput validation of a mid-abundant blood biomarker such as clusterin.</p

The Effect of Pre-Analytical Variability on the Measurement of MRM-MS-Based Mid- to High-Abundance Plasma Protein Biomarkers and a Panel of Cytokines

Blood sample processing and handling can have a significant impact on the stability and levels of proteins measured in biomarker studies. Such pre-analytical variability needs to be well understood in the context of the different proteomics platforms available for biomarker discovery and validation. In the present study we evaluated different types of blood collection tubes including the BD P100 tube containing protease inhibitors as well as CTAD tubes, which prevent platelet activation. We studied the effect of different processing protocols as well as delays in tube processing on the levels of 55 mid and high abundance plasma proteins using novel multiple-reaction monitoring-mass spectrometry (MRM-MS) assays as well as 27 low abundance cytokines using a commercially available multiplexed bead-based immunoassay. The use of P100 tubes containing protease inhibitors only conferred proteolytic protection for 4 cytokines and only one MRM-MS-measured peptide. Mid and high abundance proteins measured by MRM are highly stable in plasma left unprocessed for up to six hours although platelet activation can also impact the levels of these proteins. The levels of cytokines were elevated when tubes were centrifuged at cold temperature, while low levels were detected when samples were collected in CTAD tubes. Delays in centrifugation also had an impact on the levels of cytokines measured depending on the type of collection tube used. Our findings can help in the development of guidelines for blood collection and processing for proteomic biomarker studies

A micro-flow, high-pH, reversed-phase peptide fractionation and collection system for targeted and in-depth proteomics of low-abundance proteins in limiting samples

We present a method and a simple system for high-pH RP-LC peptide fractionation of small sample amounts (30–60 µg), at micro-flow rates with micro-liter fraction collection using ammonium bicarbonate as an optimized buffer for system stability and robustness. The method is applicable to targeted mass spectrometry approaches and to in-depth proteomic studies where the amount of sample is limited. Using targeted proteomics with peptide standards, we present the method's analytical parameters, and potential in increasing the detection of low-abundance proteins that are difficult to quantify with direct targeted or global LC-MS analyses. This fractionation system increased peptide signals by up to 18-fold, while maintaining high quantitative precision, with high fractionation reproducibility across varied sample sets. In real applications, it increased the detection of targeted endogenous peptides by two-fold in a 25 cell-cycle-control protein panel, and in-depth MS analyses of nuclear extracts, it allowed the detection of up to 8,896 proteins with 138,417 peptides in 24-concatenated fractions compared to 3,344 proteins with 23,093 peptides without fractionation. In a relevant biological problem of CDK4/6-inhibitors and breast cancer, the method reproduced known information and revealed novel insights, highlighting that it can be successfully applied in studies involving low-abundance proteins and limited samples. • Tested nine high-pH buffer/solvent systems to obtain a robust, effective, and reproducible micro-flow fractionation method which was devoid of commonly encountered LC clogging/pressure issues after months of use. • Peptide enrichment method to improve detection and quantitation of low-abundance proteins in targeted and in-depth proteomic studies. • Can be applied to diverse protein samples where the available amount is limited

Comparison of cytokine levels between tube types and processing protocols.

<p>Concentration of 20 cytokines in pg/ml from plasma collected in K₂EDTA tubes, P100 tubes processed with different protocols and CTAD tubes. All tubes were processed immediately after collection (T0). Brackets depict groups compared using paired t-tests and for which statistical significance * p<0.05 was found.</p

Cytokine levels at time 0.

<p>Concentration of cytokines in pg/ml from plasma collected in K₂EDTA (white bars) and P100 (black bars) tubes processed immediately after collection (T0) using protocol A. The levels of 20 cytokines detected using the Bio-Plex® Assay are shown (RANTES levels not included in the figure, concentration is off scale). Paired t-test comparisons were performed for each cytokine between tube types. * p<0.05, <b>+</b> cytokines not detected in K₂EDTA samples.</p

Percent differences in peptides measured in P100 and kEDTA tubes compared to CTAD tubes.

*<p>p<0.05.</p>**<p>p<0.001.</p