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To uncover mechanisms of nonalcoholic steatohepatitis (NASH) associated hepatocarcinogenesis, we compared the proteomes of human NASH-associated liver biopsies, resected hepatocellular carcinomas (HCCs) and HCCs of HCV+ patients with normal liver tissue of patients with gastrointestinal tumor metastasis, in formalin-fixed paraffin-embedded samples obtained after surgery in our hospital during the period from 2006 to 2011. In addition, proteome analysis of liver tumors in male STAM NASH-model mice was performed. Similar changes in the proteome spectrum such as overexpression of enzymes involved in lipid, cholesterol and bile acid biosynthesis and examples associated with suppression of fatty acid oxidation and catabolism, alcohol metabolism, mitochondrial function as well as low expression levels of cytokeratins 8 and 18 were observed in both human NASH biopsies and NASH HCCs, but not HCV+ HCCs. Alterations in downstream protein expression pointed to significant activation of transforming growth factor β, SMAD family member 3, β-catenin, Nrf2, SREBP-LXRα and nuclear receptor-interacting protein 1 (NRIP1), and inhibition of PPARs and p53 in human NASH biopsies and/or HCCs, suggesting their involvement in accumulation of lipids, development of fibrosis, oxidative stress, cell proliferation and suppression of apoptosis in NASH hepatocarcinogenesis. In STAM mice, PPARs inhibition was not obvious, while expression of cytokeratins 8 and 18 was elevated, indicative of essential differences between human and mouse NASH pathogenesis.

Type 2 diabetes mellitus (T2DM) is one of the most widely spread metabolic diseases. Because of its asymptomatic onset and slow development, early diagnosis and adequate glycaemic control are the prerequisites for successful T2DM therapy. In this context, individual amino acid residues might be sensitive indicators of alterations in blood glycation levels. Moreover, due to a large variation in the half-life times of plasma proteins, a generalized biomarker, based on multiple glycation sites, might provide comprehensive control of the glycemic status across any desired time span. Therefore, here, we address the patterns of glycation sites in highly-abundant blood plasma proteins of T2DM patients and corresponding age- and gender-matched controls by comprehensive liquid chromatography-mass spectrometry (LC-MS). The analysis revealed 42 lysyl residues, significantly upregulated under hyperglycemic conditions. Thereby, for 32 glycation sites, biomarker behavior was demonstrated here for the first time. The differentially glycated lysines represented nine plasma proteins with half-lives from 2 to 21 days, giving access to an integrated biomarker based on multiple protein-specific Amadori peptides. The validation of this biomarker relied on linear discriminant analysis (LDA) with random sub-sampling of the training set and leave-one-out cross-validation (LOOCV), which resulted in an accuracy, specificity, and sensitivity of 92%, 100%, and 85%, respectively.

Background Whole-proteome distributions of protein isoelectric point (pI) values in different organisms are bi- or trimodal with some variations. It was suggested that the observed multimodality of the proteome-wide pI distributions is associated with subcellular localization-specific differences in the local pI distributions. However, the factors responsible for variation of the intracellular localization-specific pI profiles have not been investigated in detail. Results In this work, we explored proteome-wide pI distributions of 32,138 human proteins predicted to reside in 10 subcellular compartments, as well as the pI distributions of experimentally observed lysosomal and Golgi proteins. The distributions were found to differ significantly, although all of them adhered to the major recurrent bimodal pattern. Grossly, acid-biased and alkaline-biased patterns with various minor statistical features were observed at different subcellular locations. Bioinformatics analysis revealed the existence of strong statistically significant correlations between protein pI and subcellular localization. Most markedly, protein pI was found to correlate positively with nuclear and mitochondrial locations and negatively with cytoskeletal, cytoplasmic, lysosomal and peroxisomal environment. Further analysis demonstrated that subcellular compartment-specific pI distributions are greatly influenced by local pH and organelle membrane charge. Multiple nonlinear regression analysis identified a polynomial function of the two variables that best fitted the mean pI values of the localization-specific pI distributions. A high coefficient of determination calculated for this regression ( R 2 = 0.98) suggests that local pH and organelle membrane charge are the major factors responsible for variation of the intracellular localization-specific pI profiles. Conclusions Our study demonstrates that strong correlations exist between protein pI and subcellular localization. The specific pI distributions at different subcellular locations are defined by local environment. Predominantly, it is the local pH and membrane charge that shape the organelle-specific protein pI patterns. These findings expand our understanding of spatial organization of the human proteome. Electronic supplementary material The online version of this article (10.1186/s12860-019-0221-4) contains supplementary material, which is available to authorized users.

Type 2 diabetes mellitus (T2DM) is a complex group of disorders, characterized by hyperglycemia, insulin resistance and insulin deficiency. In human blood, hyperglycemia ultimately results in the enhancement of glycation -a posttranslational modification formed by the interaction of protein amino groups with glucose. The resulting fructosamines (Amadori compounds) readily undergo further degradation resulting in advanced glycation end products (AGEs), known to be pro-inflammatory in humans. These compounds are highly heterogeneous and characteristic of advanced stages of the disease, whereas fructosamines are recognized markers of early diabetes stages (HbA 1C , glycated albumin). Recently, individual plasma protein glycation sites were proposed as promising T2DM biomarkers sensitive to short-term fluctuations of plasma glucose. However, corresponding absolute quantification strategies, applicable in regular clinical practice, are still not established. Therefore, here we propose a new analytical approach aiming at reproducible and precise quantification of multiple glycated peptides in human plasma tryptic digests. Thereby, the standard peptides comprised a 13 C, 15 N-labeled lysyl residue, a dabsyl moiety for determination of standard amounts, and a cleavable linker. Known amounts of these peptides were spiked to plasma samples prior to tryptic digestion, quantification relying on stable isotope dilution. The method was demonstrated to be applicable for quantification of individual glycated sites in T2DM patients and non-diabetic controls.