Blood serum samples are the major source for clinical proteomics approaches, which aim to identify diagnostically relevant or treatment-response related proteins. But, the presence of very high-abundance proteins and the enormous dynamic range of protein distribution hinders whole serum analysis. An innovative tool to overcome these limitations, utilizes combinatorial hexapeptide ligand libraries (ProteoMiner). Here, we demonstrate that ProteoMiner can be used for comparative and quantitative analysis of complex proteomes. We spiked serum samples with increasing amounts (3 microg to 300 microg) of whole E. coli lysate, processed it with ProteoMiner and performed quantitative analyses of 2D-gels. We found, that the concentration of the spiked bacteria proteome, reflected by the maintained proportional spot intensities, was not altered by ProteoMiner treatment. Therefore, we conclude that the ProteoMiner technology can be used for quantitative analysis of low abundant proteins in complex biological samples.
In non-alcoholic fatty liver disease (NAFLD) caused by ectopic lipid accumulation, lipotoxicity is a crucial molecular risk factor. Mechanisms to eliminate lipid overflow can prevent the liver from functional complications. This may involve increased secretion of lipids or metabolic adaptation to ß-oxidation in lipid-degrading organelles such as mitochondria and peroxisomes. In addition to dietary factors, increased plasma fatty acid levels may be due to increased triglyceride synthesis, lipolysis, as well as de novo lipid synthesis (DNL) in the liver. In the present study, we investigated the impact of fatty liver caused by elevated de novo lipid synthesis, in a transgenic mouse model with liver-specific overexpression of human sterol regulatory element-binding protein-1c (alb-SREBP-1c), on hepatic gene expression, on plasma lipids and especially on the proteome of peroxisomes by omics analyses, and we interpreted the results with knowledge-based analyses. In summary, the increased hepatic DNL is accompanied by marginal gene expression changes but massive changes in peroxisomal proteome. Furthermore, plasma phosphatidylcholine (PC) as well as lysoPC species were altered. Based on these observations, it can be speculated that the plasticity of organelles and their functionality may be directly affected by lipid overflow.
The present protocol describes a unique approach that enables the collection of cardiac transudate (CT) from the isolated, saline-perfused rat heart. After isolation and retrograde perfusion of the heart according to the Langendorff technique, the heart is inverted into an upside-down position and is mechanically stabilized by a balloon catheter inserted into the left ventricle. Then, a thin latex cap – previously cast to match the average size of the rat heart – is placed over the epicardial surface. The outlet of the latex cap is connected to silicon tubing, with the distal opening 10 cm below the base level of the heart, creating slight suction. CT continuously produced on the epicardial surface is collected in ice-cooled vials for further analysis. The rate of CT formation ranged from 17 to 147 µL/min (n = 14) in control and infarcted hearts, which represents 0.1-1% of the coronary venous effluent perfusate. Proteomic analysis and high performance liquid chromatography (HPLC) revealed that the collected CT contains a wide spectrum of proteins and purinergic metabolites.
To the Editor: Finding a way to diagnose myelodysplastic syndromes (MDS) using proteins of the peripheral blood serum instead of morphologic characteristics of bone marrow (BM) cells would be a large improvement by minimizing invasiveness of the diagnostic procedure and reviewer dependent variability. The key challenge in studying the serum proteome is its extraordinary wide dynamic range of protein concentrations spanning more than 10 orders of magnitude that makes discovery of a potential biomarker almost impossible. This makes pre-fractionation of the serum sample essential to decrease its range of protein concentrations and enrich the low-abundant proteins. We have previously demonstrated that affinity-chromatography-based combinatorial hexapeptide ligand libraries (CPLL) for serum pretreatment are a highly reproducible fractionation method enabling analysis of low abundance proteins in complex mixtures and presented an efficient and reliable workflow for deep serum proteome analysis by combining CPLL with mass spectrometric profiling of low molecular weight proteins and peptides on ProteinChips 1, 2. Here, we used this experimental set-up to analyze the deep serum proteome of 75 patients with MDS and 30 age-matched controls (Table 1). Using 56 sera as learning set (40 MDS and 16 controls), the peak detection and clustering analysis of m/z 2,000–15,000 detected 98 peptides in CM10, 80 in Q10, and 117 in IMAC30 spectra meaning 40% more peaks in an eighth of the m/z range than previous studies 3, 4. Different proteomic profiles were tested as prediction models to distinguish between MDS and controls. Their performance was calculated and the best predictive profile (peptides FC>2.5, P < 0.01) was applied to the independent validation set comprising 49 sera (35 MDS and 14 controls). Needing only one measurement per patient, less predictive peaks and no mix of different ProteinChips we observed better performances (Table 2) and area under the curve (AUC) data (Fig. 1A–C) than previous studies 3, 4. We further used our experimental set-up to classify different MDS subtypes and stages (RCMD/del(5q) = early, RAEBI/II = advanced). Using 11 IMAC30 peptides of the learning set that met our minimal criteria (FC>1.6, P < 0.05), we again acquired good performance data in the validation set with very high accuracy and without false positives (Table 2, Fig. 1D,E). Despite very good predictions of our proteome profiles a more elegant way to diagnose MDS from blood would be an immunoassay against an appropriate serum biomarker being less time-consuming and reaching higher inter-laboratory reproducibility. Using only one chromatographic enrichment step (H50, Bio-Rad Laboratories) before matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) fragment ion analysis (Ultraflex III, Bruker Daltonics), we identified the two peaks dominating the MDS deep serum proteome (m/z 3,818 and 3,971, >20-times higher than in controls) by SwissProt database comparison directly from the IMAC30 ProteinChip. Peptide m/z 3,971 was sequenced as DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQC, identified as 34 amino acid long N-terminal fragment of human serum albumin with its C-terminal cysteine (Cys34) sulfonated (HSA[1-34]-SO3H) while m/z 3,818 was identified as the same HSA fragment with Cys34 removed and its C-terminal glutamine amidated (HSA[1-33]-NH2). Both are part of the so-called serum fragmentome containing thousands of proteolytically derived peptides, mostly of high-abundant serum proteins, pointing toward an altered protease activity in MDS, such as previously shown for matrix metalloproteinases, their inhibitors and tryptase 5-7. In line with this are also the unusual modifications at the breakpoints before and after Cys34 as albumin is proposed to counteract oxidative stress by radical scavenging that is mainly attributed to the redox state of its free thiol at Cys34. An irreversible modification to sulfonic acid as in HSA[1-34]-SO3H causes loss of function and protein degradation and is considered a suicide function of proteins involved in antioxidation in several diseases 8. Using median split Kaplan–Meyer survival analyses, we evaluated the prognostic effect of our predictive peptides and found m/z 7,835, previously identified as stromal cell-derived factor-1 (CXCL12/SDF-1α) 9, to show highly significant differences in overall survival (P < 0.001, Fig. 2A). We confirmed this by measuring the SDF-1α concentration in sera of 56 MDS patients and 18 normal donors (Table 1) by Quantikine®ELISA (R&D Systems) revealing significantly decreased levels in MDS (P < 0.05) and significantly prolonged survival of the MDS patients with higher SDF-1α expression (P < 0.05, Fig. 2B). That may be explained by disturbed transforming growth factor-β (TGF-β)/Smad signaling in MDS 10 transcriptionally modulating SDF-1α gene expression and causing down-regulated SDF-1α production of MDS mesenchymal stromal cells of 11, 12. Furthermore, a higher expression of CXCR4, the SDF-1α receptor on hematopoietic cells, was recently identified to be a prognostic factor for overall and progression-free survival in MDS patients 13, which might be an attempt to compensate the reduced SDF-1α levels. ROC analyses of predictive deep serum proteome profiles with AUC used on the validation set distinguishing MDS patients and controls (A–C), different MDS stages (D) as well as MDS del(5q) and other MDS subtypes (E). Kaplan–Meyer plots of m/z 7,835 (A) and SDF-1α (B) categorized by median split show that survival of MDS patients with higher cluster intensities and serum concentrations (black line) is significantly longer than that of patients with lower values (gray line). The authors would like to thank Waltraud Paßlack and Sabine Lensing-Höhn for excellent technical assistance. Julia Fröbel,1,2,3* Sonja Hartwig,1 Sabine Jourdain,4 Johannes C. Fischer,5 Christoph Zilkens,3 Andrea Kündgen,2 Detlev Suckau,4 Ulrich Germing,2 Akos Czibere,2 and Stefan Lehr1 1Department of Clinical Biochemistry and Pathobiochemistry, Leibniz Center for Diabetes Research, Heinrich-Heine-University, 40225 Düsseldorf, Germany; 2Department of Hematology, Oncology and Clinical Immunology, Heinrich-Heine-University, 40225 Düsseldorf, Germany; 3Department of Orthopedic Surgery, Heinrich-Heine-University, 40225 Düsseldorf, Germany; 4Bruker Daltonics, 28359 Bremen, Germany; 5Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich-Heine-University, 40225 Düsseldorf, Germany
Besides a therapeutic target for type 2 diabetes, dipeptidyl peptidase 4 (DPP4) is an adipokine potentially upregulated in human obesity. We aimed to explore the role of adipocyte-derived DPP4 in diet-induced obesity and insulin resistance with an adipose tissue-specific knockout (AT-DPP4-KO) mouse. Wild-type and AT-DPP4-KO mice were fed for 24 wk with a high fat diet (HFD) and characterized for body weight, glucose tolerance, insulin sensitivity by hyperinsulinemic-euglycemic clamp, and body composition and hepatic fat content. Image and molecular biology analysis of inflammation, as well as adipokine secretion, was performed in AT by immunohistochemistry, Western blot, real-time-PCR, and ELISA. Incretin levels were determined by Luminex kits. Under HFD, AT-DPP4-KO displayed markedly reduced circulating DPP4 concentrations, proving AT as a relevant source. Independently of glucose-stimulated incretin hormones, AT-DPP4-KO had improved glucose tolerance and hepatic insulin sensitivity. AT-DPP4-KO displayed smaller adipocytes and increased anti-inflammatory markers. IGF binding protein 3 (IGFBP3) levels were lower in AT and serum, whereas free IGF1 was increased. The absence of adipose DPP4 triggers beneficial AT remodeling with decreased production of IGFBP3 during HFD, likely contributing to the observed, improved hepatic insulin sensitivity.
