Serum low-molecular-weight protein fractionation for biomarker discovery.

Protein biomarkers provide the key diagnostic information for the detection of disease, risk of disease progression, and a patient's likely response to drug therapy. Potential biomarkers exist in biofluids, such as serum, urine, and cerebrospinal fluid. Unfortunately, discovering and validating protein biomarkers are hindered by the presence of high-molecular-weight proteins, such as serum albumin and immunoglobulins, which comprise 90% of the proteins present in these samples. High-abundance, high-molecular-weight proteins mask the low-molecular-weight (LMW) proteins and peptides using conventional protein detection methods. Candidate biomarkers are believed to exist in very low concentrations and comprise less than 1% of serum proteins, and may be highly labile as well. Therefore, it is imperative to isolate and enrich LMW proteins from complex mixtures for biomarker discovery. This chapter describes a continuous -elution electrophoresis method, based on molecular weight sieving, to isolate specific molecular weight fractions for mass spectrometric, western blotting, or protein array analysis.

The heme degradation pathway is a promising serum biomarker source for the early detection of Alzheimer's disease.

One of the remaining challenges in Alzheimer's disease (AD) research is the establishment of biomarkers for early disease detection. As part of a prospective study spanning a period of five years, we have collected serial serum samples from cognitively normal, mild cognitively impaired (MCI), and mild AD participants, including same patient samples before and after cognitive decline. Using mass spectrometry we identified several promising leads for biomarker development, such as prosaposin, phospholipase D1, biliverdin reductase B, and S100 calcium binding protein A7. Selected candidate markers were verified using reverse phase protein microarray assays. Of 15 protein/protein abundance ratios that were significantly altered in sera from subjects with mild AD compared to Normal or MCI subjects, 14 were composed of ratios containing heme oxygenase-1, biliverdin reductase A, or biliverdin reductase B. Moreover, an increase in the protein abundance ratio of matrix metallopeptidase 9/biliverdin reductase differentiated stable MCI subjects from MCI subjects progressing into mild AD before the onset of cognitive decline. These findings strongly implicate the heme degradation pathway as a promising source of protein biomarkers for the early detection of AD.

A portrait of tissue phosphoprotein stability in the clinical tissue procurement process.

Little is known about the preanalytical fluctuations of phosphoproteins during tissue procurement for molecular profiling. This information is crucial to establish guidelines for the reliable measurement of these analytes. To develop phosphoprotein profiles of tissue subjected to the trauma of excision, we measured the fidelity of 53 signal pathway phosphoproteins over time in tissue specimens procured in a community clinical practice. This information provides strategies for potential surrogate markers of stability and the design of phosphoprotein preservative/fixation solutions. Eleven different specimen collection time course experiments revealed augmentation (+/-20% from the time 0 sample) of signal pathway phosphoprotein levels as well as decreases over time independent of tissue type, post-translational modification, and protein subcellular location (tissues included breast, colon, lung, ovary, and uterus (endometrium/myometrium) and metastatic melanoma). Comparison across tissue specimens showed an >20% decrease of protein kinase B (AKT) Ser-473 (p < 0.002) and myristoylated alanine-rich C-kinase substrate protein Ser-152/156 (p < 0.0001) within the first 90-min postexcision. Proteins in apoptotic (cleaved caspase-3 Asp-175 (p < 0.001)), proliferation/survival/hypoxia (IRS-1 Ser-612 (p < 0.0003), AMP-activated protein kinase beta Ser-108 (p < 0.005), ERK Thr-202/Tyr-204 (p < 0.003), and GSK3alphabeta Ser-21/9 (p < 0.01)), and transcription factor pathways (STAT1 Tyr-701 (p < 0.005) and cAMP response element-binding protein Ser-133 (p < 0.01)) showed >20% increases within 90-min postprocurement. Endothelial nitric-oxide synthase Ser-1177 did not change over the time period evaluated with breast or leiomyoma tissue. Treatment with phosphatase or kinase inhibitors alone revealed that tissue kinase pathways are active ex vivo. Combinations of kinase and phosphatase inhibitors appeared to stabilize proteins that exhibited increases in the presence of phosphatase inhibitors alone (ATF-2 Thr-71, SAPK/JNK Thr-183/Tyr-185, STAT1 Tyr-701, JAK1 Tyr-1022/1023, and PAK1/PAK2 Ser-199/204/192/197). This time course study 1) establishes the dynamic nature of specific phosphoproteins in excised tissue, 2) demonstrates augmented phosphorylation in the presence of phosphatase inhibitors, 3) shows that kinase inhibitors block the upsurge in phosphorylation of phosphoproteins, 4) provides a rational strategy for room temperature preservation of proteins, and 5) constitutes a foundation for developing evidence-based tissue procurement guidelines.

Laser capture microdissection technology.

Deciphering the cellular and molecular interactions that drive disease within the tissue microenvironment holds promise for discovering drug targets of the future. In order to recapitulate the in vivo interactions through molecular analysis, one must be able to analyze specific cell populations within the context of their heterogeneous tissue microecology. Laser capture microdissection is a method to procure subpopulations of tissue cells under direct microscopic visualization. Laser capture microdissection technology can harvest the cells of interest directly or can isolate specific cells by cutting away unwanted cells to give histologically pure enriched cell populations. A variety of downstream applications exist: DNA genotyping and loss-of-heterozygosity analysis, RNA transcript profiling, cDNA library generation, mass spectrometry proteomics discovery and signal pathway profiling.