Current Proteomics - Volume 7, Issue 1, 2010

High-risk neuroblastoma (NB) represents a problematic tumor phenotype associated with a dreary outlook. Modern molecular achievements over the last decade have seen the increase and implementation of 'omics technologies in oncology that promises to provide for a deeper comprehension of complex tumor pathways. The emerging concept of analyzing NB-specific 'omics profiles to better understand and define the behavior of advanced-stage tumors along with providing direct and targeted therapy may ultimately translate into improved outcomes for high-risk NB. Knowledge of NB proteomics has gradually become available, but the challenge remains to integrate data obtained from different levels of biological organization. In this review, we provide an overview of the proteomics-based techniques that can be used to advance and accelerate the discovery of novel molecular biomarkers for NB. By citing specific examples, we discuss how proteomics has contributed to the early detection of advanced-stage NB and minimal residual disease. We end by contemplating the emerging technologies that are likely to have a high impact on the field of NB in the near future.

Surface enhanced laser desorbtion/ionisation time-of-flight mass spectrometry (SELDI-TOF-MS or SELDI) combines retentate chromatography and mass spectrometry in a high-throughput format. Analytes are captured using chromatographic or immunocapture surfaces, matrix applied, ionised/desorbed with a laser and mass spectra acquired. SELDI based proteomics detects peptides and small proteins (<20 kDa) and may be used to study the concentrations, modifications and interactions of specific proteins or to discover proteomic changes associated with any biological condition of interest. Over the last decade SELDI has been applied to a wide range of biological samples but most often in the search for blood-borne biomarkers of human diseases, especially cancers. As with any biomarker research many pitfalls such as insufficient sample size, insufficient quality control, overfitting and bias must be carefully avoided to provide meaningful results. Initially, very promising results were obtained using SELDI-based ‘proteomic profiling’ to detect cancer. However, nearly 10 years on from these initial reports, none of the potential biomarkers discovered by this approach have entered routine clinical practice. It is now apparent that SELDI analysis of serum predominantly detects abundant serum proteins and that ‘biomarkers’ detected in SELDI experiments most likely arise from host responses to the cancer. SELDI therefore stands at a crossroads: will some of the biomarker work to date withstand further validation and help in the development of clinical tests, will advances in the technology allow deeper proteome mining or will SELDI become obsolete as a biomarker discovery platform? We review what has been achieved by SELDI both in, and outside, of the context of biomarker discovery.

Proper cellular functioning is dependent on the timely association of thousands of proteins in discrete complexes. A major challenge in the post genomic era is the elucidation of these diverse protein complexes and networks that govern mammalian cellular function and cell fate. Identification of the individual components of these arrangements is vital to the understanding of their function in biological processes. Currently, a widely used technique for the identification of protein complexes is a combinational approach of Tandem Affinity Purification coupled with Mass Spectrometry (TAP-MS). This technique was originally developed for the study of the yeast proteome but has since been adapted for studies of the mammalian system. This review discusses the applications of TAP-MS to the mammalian proteome. Our focus includes the technical improvements made in each step of the TAP-MS pipeline including vector design, delivery of the TAP construct into cells, expression of bait proteins, purification strategies and finally the identification of the protein complexes by mass spectrometry.

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are fatal neurodegenerative disorders of humans and animals caused by conformational conversion of a normal host glycoprotein (PrPC) into an infectious isoform (PrPSc). Whereas the mechanism of PrPSc formation and its infectivity are the subject of intensive research, the exact physiological function of PrPC and the mechanism of neurotoxicity are still unknown. Since prion infections are not limited to a monofunctional event, the PrPC/PrPSc conversion, this review will focus on recent insights into the biology of the prion protein uncovered by proteomic approaches. Recent neuroproteomic studies on the protein profile modifications associated with chronic prion infection in cell systems depicted the co-occurring biochemical abnormalities which are the basis of the prion-induced neuronal death and comprise targets for curative drugs. The involvement of other pathways in prion infectivity opens new insights into understanding of the mechanism of cellular toxicity at the molecular level. This can provide further perspectives for identification of novel therapeutic targets and also allows an integrated paradigm in the prion conversion/toxicity biology.

Lung cancer is the leading cause of cancer death for both men and women in the United States, and similar trends are seen world wide. The lack of early diagnosis is one of the primary reasons for the high mortality rate. A number of biomarkers have been evaluated in lung cancer patients, however, their specificity and early stage diagnostic values are limited. Using traditional protein chemistry and proteomics tool we have demonstrated higher serum haptoglobin levels in small cell lung cancer (SCLC). Similar findings have been reported for other cancers including ovarian cancer and glioblastoma. Haptoglobin is an acute phase protein with at least six possible phenotypes. The six phenotypes, in combination with two post translational modifications, glycosylation and deamidation, lead to large numbers of possible haptoglobin isoforms. Recent studies indicate a possible correlation between specific haptoglobin glycosylation and particular disease conditions. In our current study, we have fractionated control and SCLC patient serum by 2-D gel electrophoresis to identify differentially expressed haptoglobin isoforms in SCLC serum samples.

Presently some three hundred post-translational modifications are known to occur in bacteria in vivo. Many of these modifications play critical roles in the regulation of proteins and control key biological processes. One of the most predominant modifications, N- and O-glycosylations are now known to be present in bacteria (and archaea) although they were long believed to be limited to eukaryotes. In a number of human pathogens these glycans have been found attached to the surfaces of pilin, flagellin and other surface and secreted proteins where it has been demonstrated that they play a role in the virulence of these bacteria. Mass spectrometry characterization of these glycosylation events has been the enabling key technology for these findings. This review will look at the use of mass spectrometry as a key technology for the detection and mapping of these modifications within microorganisms, with particular reference to the human pathogens, Campylobacter jejuni and Mycobacterium tuberculosis. The overall aim of this review will be to give a basic understanding of the current ‘state-of-the-art’ of the key techniques, principles and technologies, including bioinformatics tools, involved in the analysis of the glycosylation modifications.