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.

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 patient's 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 tumor's 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.

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.

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 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 beta 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.

Identification of thioredoxin reductase 1-regulated genes using small interference RNA and cDNA microarray.

Thioredoxin reductase 1 (TrxR1) is a cytosolic enzyme that plays a central role in controlling cellular redox homeostasis. TrxR1 can transduce regulatory redox signals through NADPH-dependent reduction of thioredoxin (Trx), which is able to reduce a broad spectrum of target enzymes and regulate the activity of several transcription factors (e.g., p53 and NF-kappaB). The TrxR1/Trx system is involved in every step of cancer biology, ranging from transformation and progression to invasion, metastasis and resistance to therapy. TrxR1 was also recently identified as one key enzyme involved in cell death induced by interferon-beta (IFN-beta)/all-trans retinoic acid (ATRA) anti-cancer treatment. Our study employed small interference RNA (siRNA) and microarray techniques to investigate the effect of TrxR1 silencing on gene expression in HepG2 cells. We also investigated TrxR1-mediated cell response to IFN-beta/ATRA treatment. We identified TrxR1-dependent genes with functions related to several cellular processes such as apoptosis (SOX4), ubiquitination (Ubiquitin D, F-box protein 25), organization of cytoskeletal/extracellular matrix (Keratin 19, Fibronectin 1) and transport (Cystine/Glutamate transporter). We also investigated the effect of TrxR1 siRNA on the protein profile using surface enhanced laser desorption ionization time-of-flight (SELDI-TOF) technology. Profiles confirmed significant involvement of TrxR1 in cell response to IFN-beta/ATRA.

Development of a new reference standard for microarray experiments.

Often microarray studies require a reference to indirectly compare the samples under observation. References based on pooled RNA from different cell lines have already been described (here referred to as RNA-R), but they usually do not exhaustively represent the set of genes printed on a chip, thus requiring many adjustments during the analyses. A reference could also be generated in vitro transcribing the collection of cDNA clones printed on the microarray in use (here referred to as T3-R). Here we describe an alternative and simpler PCR-based methodology to construct a similar reference (Chip-R), and we extensively test and compare it to both RNA-R and T3-R. The use of both Chip-R and T3-R dramatically increases the number of signals on the slides and gives more reproducible results than RNA-R. Each reference preparation is also evaluated in a simple microarray experiment comparing two different RNA populations. Our results show that the introduction of a reference always interferes with the analysis. Indeed, the direct comparison is able to identify more up- or down-regulated genes than any reference-mediated analysis. However, if a reference has to be used, Chip-R and T3-R are able to guarantee more reliable results than RNA-R.