Urinary Protein-Biomarkers Reliably Indicate Very Early Kidney Damage in Children With Alport Syndrome Independently of Albuminuria and Inflammation.

Introduction: Alport syndrome (AS) is a hereditary type IV collagen disease. It starts shortly after birth, without clinical symptoms, and progresses to end-stage kidney disease early in life. The earlier therapy starts, the more effectively end-stage kidney disease can be delayed. Clearly then, to ensure preemptive therapy, early diagnosis is an essential prerequisite. Methods: To provide early diagnosis, we searched for protein biomarkers (BMs) by mass spectrometry in dogs with AS stage 0. At this very early stage, we identified 74 candidate BMs. Of these, using commercial enzyme-linked immunosorbent assays (ELISAs), we evaluated 27 in dogs and 28 in children, 50 with AS and 104 healthy controls. Results: Most BMs from blood appeared as fractions of multiple variants of the same protein, as shown by their chromatographic distribution before mass spectrometry. Blood samples showed only minor differences because ELISAs rarely detect disease-specific variants. However, in urine , several proteins, individually or in combination, were promising indicators of very early and preclinical kidney injury. The BMs with the highest sensitivity and specificity were collagen type XIII, hyaluronan binding protein 2 (HABP2), and complement C4 binding protein (C4BP). Conclusion: We generated very strong candidate BMs by our approach of first examining preclinical AS in dogs and then validating these BMs in children at early stages of disease. These BMs might serve for screening purposes for AS before the onset of kidney damage and therefore allow preemptive therapy.

A next generation setup for pre-fractionation of non-denatured proteins reveals diverse albumin proteoforms each carrying several post-translational modifications.

Proteomic biomarker search requires the greatest analytical reproducibility and detailed information on altered proteoforms. Our protein pre-fractionation applies orthogonal native chromatography and conserves important features of protein variants such as native molecular weight, charge and major glycans. Moreover, we maximized reproducibility of sample pre-fractionation and preparation before mass spectrometry by parallelization and automation. In blood plasma and cerebrospinal fluid (CSF), most proteins, including candidate biomarkers, distribute into a multitude of chromatographic clusters. Plasma albumin, for example, divides into 15-17 clusters. As an example of our technique, we analyzed these albumin clusters from healthy volunteers and from dogs and identified cluster-typical modification patterns. Renal disease further modifies these patterns. In human CSF, we found only a subset of proteoforms with fewer modifications than in plasma. We infer from this example that our method can be used to identify and characterize distinct proteoforms and, optionally, enrich them, thereby yielding the characteristics of proteoform-selective biomarkers.

Proteomic sample preparation by microdialysis: easy, speedy, and nonselective.

Microdialysis tools have been developed for parallelized medium exchange designated for sample volumes from 10 to 100 µl, compatible with the microplate format, and guaranteeing maximum recoveries without selectivity. These tools are applicable to both protein and peptide analysis. Moreover, they may be used for binding studies as well as for reconcentration and as unique sample containers for complex operating sequences allowing contemporaneous processing and high throughput.

UV measurements in microplates suitable for high-throughput protein determination.

An UV spectrophotometric method for protein determination using microplates is described. Using the SPECTRAmax PLUS reader, the UVStar 96- and 384-well microplates and a 96 or 384 parallel channel liquid handling technique, large-scale determinations can be performed with intraassay precision better than 3% CV (coefficient of variation) in the range from 1 to 8000 microg of protein/ml, measuring at 205, 215, and 280 nm and using different volume-dependent light-path lengths. Since the absorbance coefficient at 205 nm is found to be 30 ml/(mgxcm) for eight different proteins with a CV of 5.6% only with the Path Check option of the reader, protein concentration can be determined without any individual calibration. Samples in the volume range of 60-250 microl can be analyzed without time-consuming and expensive treatment and without sample loss. Using a special 96 or 384 parallel dialyzing device, low molecular weight substances which interfere with the analysis by their UV absorbance, such as buffers and detergents, can effectively be removed. Application examples for serum protein separation are also shown in the presence of the strongly UV absorbing detergent Triton X-100.