List of stringently identified serum proteins and Supplementary information

Es sind Listen (Tabellen) mit 2669 Serumproteinen und ihren Peptiden, die nach einer 2-dimensionalen Chromatographie mit Hilfe der Massenspektrometrie identifiziert werden konnten. Die Listen im PDF-Format sind am 25. 08. 2008 das letzte Mal aktualisiert worden. Beispiele von Chromatogrammen der RP-LC und der 3D-Trennung und von MS-Spektren sind in den Abbildungen S1 und S2 gezeigt. Sie können als Datei herunter geladen werden. Ergänzende Abbildungen und Tabellen zu Publikationen wurden am 17. 03. 2009 und am 25. 09. 2009 aktualisiert. Alle Arbeiten wurden am Institut für Biochemie I am Universitätsklinikum in der Arbeitsgruppe Analytik durchgeführt. Die Dateien wurden erstellt von: S. Kreusch, H. Rhode, B. Colditz, H. Endmann Kontakt: [email protected] lists (tables) presented contain 2669 protein subunits and their peptides identified stringently in human serum samples. Serum was separated and fractionated by 2-dimensional chromatography. Proteins within fractions were identified by mass spectrometry using sequence tags of their peptides. Examples of RP-LC and MS spectra and of 3D separation are given in Figs. S1 and S2, respectively. Lists of proteins and peptides in PDF format may be downloaded. Last update: 25/08/08. Last update of Figures and Tables supplementary to manuscripts submitted: 17/03/2009 and 25/09/2009. Work was done by the research group Analytik of Institute of Biochemistry I, Klinikum, Friedrich-Schiller-University Jena, Germany. Team of authors: S. Kreusch, H. Rhode, B. Colditz, H. Endmann contact: [email protected]

Diagnosis of Alport syndrome-search for proteomic biomarkers in body fluids

The hereditary kidney disease Alport syndrome (AS) has become a treatable disease: intervention with angiotensin-converting enzyme (ACE)-inhibitors delays end stage renal failure by years. The efficiency of ACE inhibition depends on the onset of therapy-the earlier the better. Therefore, early diagnosis has become increasingly important. To date, robust diagnosis requires renal biopsy and/or expensive genetic analysis, which is mostly performed late after onset of the profound clinical symptoms of this progressive renal disease. Thus, disease biomarkers enabling low-invasive screening are urgently required. Fourteen potential proteomic candidate markers (proteins) identified in a previous study in sera from patients exhibiting manifest AS were evaluated in the plasma, serum, and urine collected from a cohort of 132 subjects, including patients with AS and other nephropathies and healthy controls. Quantitation was performed by immunoassays. The serum and plasma levels of none of the 14 proteins evaluated were significantly different among the three groups and therefore could not be used to discriminate between the groups. In contrast, the levels of various biomarker combinations in the urine were significantly different between AS patients and healthy controls. Importantly, some combinations had the potential to discriminate between AS and other nephropathies. These findings open a window of opportunity for the sensitive and specific early diagnosis of AS. Our results increase the potential for larger scale evaluation of an increased number of patients

Preclinical Alterations in the Serum of COL(IV)A3^–/^– Mice as Early Biomarkers of Alport Syndrome

The efficiency of the inhibition of the angiotensin converting enzyme, the most widely used therapy for the Alport syndrome, depends on the onset of the therapythe earlier the better. Hence, early progressive biomarkers are urgently required to allow for preclinical diagnosis, an early start of possible therapy as well as the monitoring of this therapy. In the present study, an improved comprehensive and precise proteomic approach has been applied to the serum of juvenile Alport-mice, nontreated and treated, and wild-type controls of various ages to search for biomarkers. With a total of 2542 stringently altered proteins, the serum composition clearly shows a dependency on age, that is, stage, and therapy. Initially, the serum constituents indicate an enhanced extracellular matrix remodeling, cell damage, and the production of particular acute phase proteins. A panel of 15 potential biomarker candidates has been identified. In later stages, renal filtration failure and systemic acute phase reaction determine the composition of the serum; an effect that is well-known for manifested human Alport syndrome. With a small number of mouse urine samples, for example, the proteomic results for gelsolin could be verified using ELISA. Once verified in man, these early biomarkers would allow for a sensitive and specific diagnosis of the Alport syndrome in children as well as facilitate the monitoring of a possible therapy

Ratio of Urinary Proteins to Albumin Excretion Shifts Substantially during Progression of the Podocytopathy Alport Syndrome, and Spot Urine Is a Reliable Method to Detect These Pathologic Changes

The urinary albumin- and protein-to-creatinine ratios (UACR and UPCR, respectively) are key endpoints in most clinical trials assessing risk of progression of chronic kidney disease (CKD). For the first time, the current study compares the UACR versus the UPCR head-to-head at early stages of CKD, taking use of the hereditary podocytopathy Alport syndrome (AS) as a model disease for any CKD. Urine samples originated from the prospective randomized, controlled EARLY PRO-TECT Alport trial (NCT01485978). Urine samples from 47 children with confirmed diagnoses of AS at very early stages of CKD were divided according to the current stage of AS: stage 0 (UACR 300 mg/g). The range of estimated glomerular filtration rate was 75–187.6 mL/min. The mean age was 10.4 ± 4.5 years. In children at stage 0, proteinuria in spot urine, confirmed in 24 h urine, was almost ten times higher than albuminuria (106.4 ± 42.2 vs. 12.5 ± 9.7; p p p = 0.36). In 17 children, UACRs and UPCRs were measured simultaneously in 24 h urine and spot urine in the same study visit. Interestingly, the UACR (and UPCR) in 24 h urine vs. in spot urine varied by less than 10% (266.8 ± 426.4 vs. 291.2 ± 530.2). In conclusion, our study provides the first evidence that in patients with normal glomerular filtration rate (GFR) and low amounts of albuminuria, especially in children with podocytopathies such as AS, measuring the UACR and UPCR in spot urine is a reliable and convenient alternative to 24 h urine collection. Our study advocates both the UACR and the UPCR as relevant diagnostic biomarkers in future clinical trials in children with glomerular diseases because the UPCR seems to be a very significant parameter at very early stages of podocytopathies. The German Federal Ministry of Education and Research funded this trial (01KG1104)

Neurofilament markers for ALS correlate with extent of upper and lower motor neuron disease

OBJECTIVE: To determine the diagnostic performance and prognostic value of phosphorylated neurofilament heavy chain (pNfH) and neurofilament light chain (NfL) in CSF as possible biomarkers for amyotrophic lateral sclerosis (ALS) at the diagnostic phase. METHODS: We measured CSF pNfH and NfL concentrations in 220 patients with ALS, 316 neurologic disease controls (DC), and 50 genuine disease mimics (DM) to determine and assess the accuracy of the diagnostic cutoff value for pNfH and NfL and to correlate with other clinical parameters. RESULTS: pNfH was most specific for motor neuron disease (specificity 88.2% [confidence interval (CI) 83.0%-92.3%]). pNfH had the best performance to differentially diagnose patients with ALS from DM with a sensitivity of 90.7% (CI 84.9%-94.8%), a specificity of 88.0% (CI 75.7%-95.5%) and a likelihood ratio of 7.6 (CI 3.6-16.0) at a cutoff of 768 pg/mL. CSF pNfH and NfL levels were significantly lower in slow disease progressors, however, with a poor prognostic performance with respect to the disease progression rate. CSF pNfH and NfL levels increased significantly as function of the number of regions with both upper and lower motor involvement. CONCLUSIONS: In particular, CSF pNfH concentrations show an added value as diagnostic biomarkers for ALS, whereas the prognostic value of pNfH and NfL warrants further investigation. Both pNfH and NfL correlated with the extent of motor neuron degeneration. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that elevated concentrations of CSF pNfH and NfL can accurately identify patients with ALS.status: publishe