Current Proteomics - Volume 6, Issue 3, 2009

Inherited metabolic diseases (IMD) include a broad spectrum of biochemical alterations caused by genetic defects which affect the structure and function of proteins involved in cellular metabolic pathways. Until now, more than 500 different IMD that alter the synthesis, metabolism, transport and/or storage of biochemical compounds have been identified. Molecular and biochemical techniques can be applied to determine both the genotype and phenotype of IMD patients; they allow for accurate diagnosis and application of individual based treatment strategies. The emerging genomic, proteomic and metabolomic tools using molecular, cellular and physiological approaches provide deeper insight into the pathophysiology of metabolic diseases. Mass spectrometry is a powerful and established technology, employed in IMD research and newborn screening that leads to early diagnosis of different metabolic disorders. Over the last years, the number of different proteomic techniques applied to IMD investigation has dramatically increased. The most remarkable ones include: 2-DE and LC for protein separation; and ESI-MS, ESI-MS/MS, MALDI-MS, MALDI-MS/MS, SELDI-MS, DIOS-MS for protein/metabolite identification and analysis. Proteomics offers the ability to identify novel proteins and to study their expression patterns and function, along with the potential to discover biomarkers as diagnostic tools or for the development of new therapeutic strategies. This review describes the main proteomic techniques applied to IMD diagnosis and research.

Current methods used for the proteomic analysis of human cerebrospinal fluid (CSF) focus on depleting highly abundant proteins, to facilitate the detection of less abundant proteins. However most such methods are time-consuming and not highly effective. In the present study, we tested a newly developed hollow-fiber-membrane-based (HFM) protein separation device as a tool in preparing CSF samples for 2-D-LC-MS/MS proteomic analysis. Within 1 h, the HFM pretreatment device efficiently depleted a CSF sample for highly abundant high molecular weight proteins, thus enriching the sample for less abundant, low molecular weight proteins and peptides. Six CSF samples of three different volumes (1 mL, 2 mL and 10 mL) from three healthy subjects were pretreated with this device, and subsequent 2-D-LC-MS/MS analysis yielded 82,731 MS/MS spectra with statistically positive hits for 160 proteins. Thirty-four of these proteins, including 4 brain-specific proteins, were found in CSF for the first time. This pretreatment method may assist with the identification of new biomarkers for the diagnosis and treatment of CNS diseases.

IgA nephropathy (IgAN), the most frequent type of glomerulonephritis, is characterized at biopsy by a wide variability of functional, proteinuric and histologic features; about 30% of patients progress to end stage renal disease (ESRD). Baseline proteinuria is one of the main predictors of progression. Few studies evaluated whether some components of proteinuria improve prediction of the outcome and may identify patients for whom different therapeutic approaches are indicated (no treatment, renin-angiotensin system (RAS) inhibition, immunosuppression). The composition of proteinuria was assessed by classical methods (SDS-PAGE, immunonephelometry); recently new technologies [mainly capillary electrophoresis coupled to mass spectrometry (CE-MS)] were able to analyze the whole spectrum of urinary proteins/ polypeptides. Low molecular weight proteins in SDS-PAGE pattern predicted progression. Urinary excretion of IgG, a reliable marker of selectivity of glomerular capillary wall, predicted progression better than proteinuria/day and other markers by univariate and multivariate analysis. Fractional Excretion of IgG identified patients responsive to ACEinhibitors (ACEi) in non-crescentic IgAN and patients responsive to immunosuppression in crescentic-IgAN. CE-MS identified a specific “IgAN polypeptide pattern” significantly different from controls and from other glomerular diseases with high sensitivity and specificity. In patients treated with ACEi the analysis with 2-D PAGE coupled to nano-HPLCESI- MS/MS identified 3 proteins [kininogen, inter-α-trypsin inhibitor heavy chain H4 and transthyretin] whose excretion was significantly different in ACEi-responders vs non-responders. Baseline low kininogen excretion predicted inadeguate/ absent response to ACEi. Conclusions: urinary excretion of IgG is to date the best proteinuric predictor of progression and responsiveness to ACEi in non-crescentic IgAN and to immunosuppression in crescentic IgAN . Urinary profile assessed by proteome analysis identifies a polypeptide pattern that distinguishes IgAN from healthy controls and other glomerular diseases and some proteins whose value predicts ACEi-responsiveness. Future developments of urinary proteome characterization in larger cohorts of patients may further improve diagnosis, prognostication and therapeutic approach.

Helicobacter pylori infection induces an increase in the rate of apoptosis in gastric epithelial cells and initiates a secondary hyperproliferative response. Currently, cytotoxin-associated proteins and vacuolating cytotoxin participate in the induction of the apoptotic process in the gastric epithelium, however, the existence of other factors that participate in this process has not been ruled out. Moreover, differential protein expression by H. pylori during the apoptotic process has not been studied. The aim of this work was to study differential H. pylori protein expression during the process of apoptosis in AGS cell. AGS cells were infected with H. pylori and apoptosis was determined by measuring chromatin condensation and caspase-3 activity. In addition, bacterial and cellular protein extraction was performed during apoptosis. The H. pylori protein profile was analyzed by double-dimension electrophoresis (2-DE) and protein identification was performed by LC/NSI-MS/MS analysis. A significant increase in chromatin condensation and caspase-3 activity was seen in the AGS cells 12 h post-infection. The proteomic analysis demonstrated a significant increase in the expression of 19 proteins, from which five proteins were identified from H. pylori. Thirty-one spots were present during the pathogen-host interaction. In conclusion: H. pylori induced a significant increase in apoptosis in infected AGS cells 12 h post-infection, and the H. pylori proteins that were identified could be participating in the apoptotic event.

Cysteine endopeptidases regulate many physiological processes in the body and their impaired function may lead to several diseases. One of the methods of treating such diseases is achieved by controlling the proteolytic activity of these enzymes by using enzyme inhibition. This manuscript focuses on the mechanism of action of Papain, a cysteine endopeptidase enzyme and its inhibition using images generated with RasMol, which is one of the most popular graphics programs for the display of macromolecules like proteins and nucleic acid structures. The location of active site residues on the papain enzyme molecule is described with the RasMol images and the mode of inhibition of these active sites is described in reference with few examples of papain inhibitors. Thus, the manuscript discusses the use of RasMol program to understand the inhibition of cysteine endopeptidase enzyme papain.