Label-free mass spectrometric profiling of urinary proteins and metabolites from paediatric idiopathic nephrotic syndrome

Idiopathic nephrotic syndrome (INS) is caused by renal diseases that increase the permeability of the glomerular filtration barrier without evidence of a specific systemic cause. The aim of the present work was to reveal inherent molecular features of INS in children using combined urinary proteomics and metabolomics profiling. In this study, label-free mass spectrometric analysis of urinary proteins and small molecule metabolites was carried out in 12 patients with INS versus 12 sex- and age-matched control subjects with normal renal function. Integration and biological interpretation of obtained results were carried out by Ingenuity IPA software. Validation of obtained proteomics data was carried out by Western blot method. Proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD000765. This study indicates for the first time that paediatric INS is associated with up-regulation of afamin, hydroxyphenylacetate and uridine, and concomitant down-regulation in glutamine and phenylalanine levels, and many of these molecular species were previously shown to be involved in oxidative stress. Further studies in larger patient population are underway to investigate the role of oxidative stress in renal injury in paediatric INS.     

Capillary electrophoresis for clinical problem solving: analysis of urinary diagnostic metabolites and serum proteins

Many clinical laboratories employ gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) to detect abnormal compounds occurinng in urine and serum due to disease. The methods, particularly GC-MS, often require laborious sample pre-treatment, and separation times may exceed an hour. We describe the use of capillary electrophoresis (CE) equipped a with a diode-array detector in an attempt to improve the efficiency of an analytical system routinely used for diagnosis of human metabolic disease. It was found that urine samples could be injected directly onto the CE instrument without any pre-treatment, and over 50 metabolites were separated in 15 min. Identification of abnormal metabolites was based on migration times and characteristic diode-array spectra. The method readily diagnosed adenolysuccinase deficiency, 5-oxoprolinuria, propionic acidemia and disorders having orotic acid as diagnostic metabolite (e.g. the HHH-syndrome). The results show that CE may become a useful additional tool for diagnosis of metabolic disease. In a different project CE was used to study sera from the Janus-bank. This large serum bank comprises samples collected at intervals from nearly 300 000 blood donors. As the sera are stored at −25°C and not at a lower temperature, a major concern has been the stability of the specimens. GC-MS, 2D-protein electrophoresis, certain immunological assays and enzyme measurements have previously been used to evaluate the stability of the sera. We can now also show that the protein profile, as determined by CE, is remarkably stable even after 22 years of storage. The results moreover confirmed that the CE-method and traditional gel electrophoresis gave almost identical results, except for small amounts of fibrinogen which did not show up on the CE-pattern.     

Sodium dodecyl sulphate electrophoresis of urinary proteins

The analysis of urinary proteins and their identification are discussed, particularly in regard to the technique of sodium dodecyl sulphate electrophoresis in polyacrylamide gradient gels. Urine collection, storage and preparation are evaluated, especially in regard to problems connected with concentration and dialysis of such samples. The instrumental approach to sodium dodecyl sulphate polyacrylamide gel electrophoresis represented by the Phast System appears to be particularly valuable in routine clinical analysis of urine specimens, since no sample pretreatment is required. The following types of proteinurias are evaluated: (a) orthostatic proteinurias; (b) post-renal proteinurias; (c) Bence—Jones proteinuria; (d) lower and upper urinary tract infection (cystitis and pyelonephritis) and (e) diabetes mellitus proteinurias.     

Studies on urinary gamma-glutamyl transpeptidase in nephrotic syndrome patients

(1) Variations in the levels of GGT were measured in urine specimens taken in the early morning in control and in 20 consecutive adult patients with uncomplicated nephrotic syndrome. (2) The urinary GGT activity was increased in all cases of nephrotic syndrome patients investigated with different etiology. (3) A significant correlation was found between urinary GGT activity and serum albumin (r = 0.727) but not with serum cholesterol (r = 0.129). (4) These findings suggest that enhanced excretion of urinary GGT may be stimulated by decreased albumin concentration or oncotic pressure but does not appear to be due to leakage from plasma. (5) A systematic study on urinary GGT showed that GGT activity was decreased to the upper limit of normal control values in nephrotic syndrome patients after remission.     

Two-dimensional electrophoresis of horse serum proteins: genetic polymorphism of ceruloplasmin and two other serum proteins

Two-dimensional agarose gel (pH 8.6)-horizontal polyacrylamide gel (pH 9.0) electrophoresis of horse serum proteins revealed genetic polymorphism of ceruloplasmin (Cp) and two unidentified serum proteins tentatively designated serum protein 1 (SP1) and serum protein 2 (SP2). Family data were consistent with the hypothesis that the observed Cp and SP1 phenotypes were each controlled by two co-dominant, autosornal alleles. The three common SP2 phenotypes were shown to be controlled by two codominant, autosomal alleles. Population data and limited family data indicated the occurrence of two additional SP2 alleles. Altogether more than 600 horses representing 13 different breeds were typed for Cp, SP1 and SP2, and allele frequency estimates were calculated. SP2 was highly polymorphic in all breeds studied whereas SP1 and Cp showed quite low degrees of polymorphism. SP1 polymorphism was observed in seven breeds while Cp polymorphism was observed only in the Icelandic toelter horse breed.