Amy VanMeter

Laser-capture microdissection

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 thorough molecular analysis, one must be able to analyze specific cell populations within the context of their heterogeneous tissue microecology. Laser-capture microdissection (LCM) is a method to procure subpopulations of tissue cells under direct microscopic visualization. LCM 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 (LOH) analysis, RNA transcript profiling, cDNA library generation, proteomics discovery and signal-pathway profiling. Herein we provide a thorough description of LCM techniques, with an emphasis on tips and troubleshooting advice derived from LCM users. The total time required to carry out this protocol is typically 1–1.5 h.

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 β Ser-108 (p < 0.005), ERK Thr-202/Tyr-204 (p < 0.003), and GSK3αβ 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.

A genomic and proteomic study of the spectrum of nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, and some of its forms are progressive. This study describes the profiling of hepatic gene expression and serum protein content in patients with different subtypes of NAFLD. Liver biopsy specimens from 98 bariatric surgery patients were classified as normal, steatosis alone, steatosis with nonspecific inflammation, and nonalcoholic steatohepatitis (NASH). Microarray hybridizations were performed in triplicate and the microarray expression levels of a selected group of genes were confirmed using real‐time quantitative reverse‐transcriptase polymerase chain reaction. Serum protein profiles of the same patients were determined by SELDI‐TOF mass spectrometry. Of 98 obese patients, 91 were diagnosed with NAFLD (12 steatosis alone, 52 steatosis with nonspecific inflammation, and 27 NASH), and 7 patients without NAFLD served as obese controls. Each group of NAFLD patients was compared with the obese controls, and 22 genes with more than twofold differences in expression levels were revealed. Proteomics analyses were performed for the same group comparisons and revealed twelve significantly different protein peaks. In conclusion, this genomic/proteomic analysis suggests differential expression of several genes and protein peaks in patients within and across the forms of NAFLD. These findings may help clarify the pathogenesis of NAFLD and identify potential targets for therapeutic intervention. (HEPATOLOGY 2005;42:665–674.)

Laser Capture Microdissection and Protein Microarray Analysis of Human Non-small Cell Lung Cancer Differential Epidermal Growth Factor Receptor (EGPR) Phosphorylation Events Associated with Mutated EGFR Compared with Wild Type

Little is known about lung carcinoma epidermal growth factor (EGF) kinase pathway signaling within the context of the tissue microenvironment. We quantitatively profiled the phosphorylation and abundance of signal pathway proteins relevant to the EGF receptor within laser capture microdissected untreated, human non-small cell lung cancer (NSCLC) (n = 25) of known epidermal growth factor receptor (EGFR) tyrosine kinase domain mutation status. We measured six phosphorylation sites on EGFR to evaluate whether EGFR mutation status in vivo was associated with the coordinated phosphorylation of specific multiple phosphorylation sites on the EGFR and downstream proteins. Reverse phase protein array quantitation of NSCLC revealed simultaneous increased phosphorylation of EGFR residues Tyr-1148 (p < 0.044) and Tyr-1068 (p < 0.026) and decreased phosphorylation of EGFR Tyr-1045 (p < 0.002), HER2 Tyr-1248 (p < 0.015), IRS-1 Ser-612 (p < 0.001), and SMAD Ser-465/467 (p < 0.011) across all classes of mutated EGFR patient samples compared with wild type. To explore which subset of correlations was influenced by ligand induction versus an intrinsic phenotype of the EGFR mutants, we profiled the time course of 115 cellular signal proteins for EGF ligand-stimulated (three dosages) NSCLC mutant and wild type cultured cell lines. EGFR mutant cell lines (H1975 L858R) displayed a pattern of EGFR Tyr-1045 and HER2 Tyr-1248 phosphorylation similar to that found in tissue. Persistence of phosphorylation for AKT Ser-473 following ligand stimulation was found for the mutant. These data suggest that a higher proportion of the EGFR mutant carcinoma cells may exhibit activation of the phosphatidylinositol 3-kinase/protein kinase B (AKT)/mammalian target of rapamycin (MTOR) pathway through Tyr-1148 and Tyr-1068 and suppression of IRS-1 Ser-612, altered heterodimerization with ERBB2, reduced response to transforming growth factor β suppression, and reduced ubiquitination/degradation of the EGFR through EGFR Tyr-1045, thus providing a survival advantage. This is the first comparison of multiple, site-specific phosphoproteins with the EGFR tyrosine kinase domain mutation status in vivo.

Reverse-phase protein microarrays: application to biomarker discovery and translational medicine

Mapping of protein signaling networks within tumors can identify new targets for therapy and provide a means to stratify patients for individualized therapy. Kinases are important drug targets, as such kinase network information could become the basis for development of therapeutic strategies for improving treatment outcome. An urgent clinical goal is to identify functionally important molecular networks associated with subpopulations of patients that may not respond to conventional combination chemotherapy. Reverse-phase protein microarrays are a technology platform designed for quantitative, multiplexed analysis of specific phosphorylated, cleaved, or total (phosphorylated and nonphosphorylated) forms of cellular proteins from a limited amount of sample. This class of microarray can be used to interrogate cellular samples, serum or body fluids. This review focuses on the application of reverse-phase protein microarrays for translational research and therapeutic drug target discovery.

An Initial Characterization of the Serum Phosphoproteome

Phosphorylation is a dynamic post-translational protein modification that is the basis of a general mechanism for maintaining and regulating protein structure and function, and of course underpins key cellular processes through signal transduction. In the last several years, many studies of large-scale profiling of phosphoproteins and mapping phosphorylation sites from cultured human cells or tissues by mass spectrometry technique have been published; however, there is little information on general (or global) phosphoproteomic characterization and description of the content of phosphoprotein analytes within the circulation. Circulating phosphoproteins and phosphopeptides could represent important disease biomarkers because of their well-known importance in cellular function, and these analytes frequently are mutated and activated in human diseases such as cancer. Here, we report an initial attempt to characterize the phosphoprotein content of serum. To accomplish this, we developed a method in which phosphopeptides are enriched from digested serum proteins and analyzed by LC-MS/MS using LTQ-Orbitrap (CID) and LTQ-ETD mass spectrometers. With this approach, we identified approximately 100 unique phosphopeptides with stringent filtering criteria and a lower than 1% false discovery rate.

The Heme Degradation Pathway is a Promising Serum Biomarker Source for the Early Detection of Alzheimer's Disease

One of the remaining challenges in Alzheimers 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.

Phosphoproteomic Analysis of Signaling Pathways in Head and Neck Squamous Cell Carcinoma Patient Samples

Molecular targeted therapy represents a promising new strategy for treating cancers because many small-molecule inhibitors targeting protein kinases have recently become available. Reverse-phase protein microarrays (RPPAs) are a useful platform for identifying dysregulated signaling pathways in tumors and can provide insight into patient-specific differences. In the present study, RPPAs were used to examine 60 protein end points (predominantly phosphoproteins) in matched tumor and nonmalignant biopsy specimens from 23 patients with head and neck squamous cell carcinoma to characterize the cancer phosphoproteome. RPPA identified 18 of 60 analytes globally elevated in tumors versus healthy tissue and 17 of 60 analytes that were decreased. The most significantly elevated analytes in tumor were checkpoint kinase (Chk) 1 serine 345 (S345), Chk 2 S33/35, eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) S65, protein kinase C (PKC) ζ/ι threonine 410/412 (T410/T412), LKB1 S334, inhibitor of kappaB alpha (IκB-α) S32, eukaryotic translation initiation factor 4E (eIF4E) S209, Smad2 S465/67, insulin receptor substrate 1 (IRS-1) S612, mitogen-activated ERK kinase 1/2 (MEK1/2) S217/221, and total PKC ι. To our knowledge, this is the first report of elevated PKC ι in head and neck squamous cell carcinoma that may have significance because PKC ι is an oncogene in several other tumor types, including lung cancer. The feasibility of using RPPA for developing theranostic tests to guide personalized therapy is discussed in the context of these data.

Functional Protein Network Activation Mapping Reveals New Potential Molecular Drug Targets for Poor Prognosis Pediatric BCP-ALL

Background In spite of leukemia therapy improvements obtained over the last decades, therapy is not yet effective in all cases. Current approaches in Acute Lymphoblastic Leukemia (ALL) research focus on identifying new molecular targets to improve outcome for patients with a dismal prognosis. In this light phosphoproteomics seems to hold great promise for the identification of proteins suitable for targeted therapy. Methodology/Principal Findings We employed Reverse Phase Protein Microarrays to identify aberrantly activated proteins in 118 pediatric B-cell precursor (BCP)-ALL patients. Signal transduction pathways were assayed for activation/expression status of 92 key signalling proteins. We observed an increased activation/expression of several pathways involved in cell proliferation in poor clinical prognosis patients. MLL-rearranged tumours revealed BCL-2 hyperphosphorylation through AMPK activation, which indicates that AMPK could provide a functional role in inhibiting apoptosis in MLL-rearranged patients, and could be considered as a new potential therapeutic target. Second, in patients with poor clinical response to prednisone we observed the up-modulation of LCK activity with respect to patients with good response. This tyrosine-kinase can be down-modulated with clinically used inhibitors, thus modulating LCK activity could be considered for further studies as a new additional therapy for prednisone-resistant patients. Further we also found an association between high levels of CYCLIN E and relapse incidence. Moreover, CYCLIN E is more expressed in early relapsed patients, who usually show an unfavourable prognosis. Conclusions/Significance We conclude that functional protein pathway activation mapping revealed specific deranged signalling networks in BCP-ALL that could be potentially modulated to produce a better clinical outcome for patients resistant to standard-of-care therapies.

Reverse Phase Protein Microarrays

Cancer is the consequence of intra- and extracellular signaling network deregulation that derives from alteration of genetic and proteomic cellular homeostasis. Mapping the individual molecular circuitry of a patients tumor cells is the starting point for rational personalized therapy.While genes and RNA encode information about cellular status, proteins are considered the engine of the cellular machine, as they are the effective elements that drive cellular functions, such as proliferation, migration, differentiation, and apoptosis. Consequently, investigations of the cellular protein network are considered a fundamental tool to understand cellular functions. In the last decades, increasing interest has been focused on the improvement of new technologies for proteomic analysis. In this context, reverse-phase protein microarrays (RPMAs) have been developed to study and analyze posttranslational modifications that are responsible for principal cell functions and activities. This innovative technology allows the investigation of protein activation as a consequence of protein-protein interaction or biochemical reactions, such as phosphorylation, glycosylation, ubiquitination, protein cleavage, and conformational alterations.Intracellular balance is carefully conserved by constant rearrangements of proteins through the activity of a series of kinases and phosphatases. Therefore, knowledge of the key cellular signaling cascades reveal information regarding the cellular processes driving a tumors growth (such as cellular survival, proliferation, invasion, and cell death) and response to treatment.Alteration to cellular homeostasis, driven by elaborate intra- and extracellular interactions, has become one of the most studied fields in the era of personalized medicine and targeted therapy. RPMA technology is a valid tool that can be applied to protein analysis of several diseases for the potential to generate protein interaction and activation maps that lead to the identification of critical nodes for individualized or combinatorial target therapy.