A top-down proteomic approach reveals a salivary protein profile able to classify Parkinson's disease with respect to Alzheimer's disease patients and to healthy controls.

Parkinson's disease (PD) is a complex neurodegenerative disease with motor and non-motor symptoms. Diagnosis is complicated by lack of reliable biomarkers. To individuate peptides and/or proteins with diagnostic potential for early diagnosis, severity and discrimination from similar pathologies, the salivary proteome in 36 PD patients was investigated in comparison with 36 healthy controls (HC) and 35 Alzheimer's disease (AD) patients. A top-down platform based on HPLC-ESI-IT-MS allowed characterizing and quantifying intact peptides, small proteins and their PTMs (overall 51). The three groups showed significantly different protein profiles, PD showed the highest levels of cystatin SA and antileukoproteinase and the lowest of cystatin SN and some statherin proteoforms. HC exhibited the lowest abundance of thymosin ß4, short S100A9, cystatin A, and dimeric cystatin B. AD patients showed the highest abundance of α-defensins and short oxidized S100A9. Moreover, different proteoforms of the same protein, as S-cysteinylated and S-glutathionylated cystatin B, showed opposite trends in the two pathological groups. Statherin, cystatins SA and SN classified accurately PD from HC and AD subjects. α-defensins, histatin 1, oxidized S100A9, and P-B fragments were the best classifying factors between PD and AD patients. Interestingly statherin and thymosin ß4 correlated with defective olfactory functions in PD patients. All these outcomes highlighted implications of specific proteoforms involved in the innate-immune response and inflammation regulation at oral and systemic level, suggesting a possible panel of molecular and clinical markers suitable to recognize subjects affected by PD.

Proximity ligation assays for precise quantification of femtomolar proteins in single cells using self-priming microfluidic dPCR chip.

The quantification of low concentration proteins can facilitate the discovery of some significant biomarkers, and provide us a more profound understanding of cell heterogeneity when applied to single cell analysis. However, most state-of- art single cell protein detection platforms are bulky, expensive and complicated. Here we report a simple and low cost microfluidic dPCR (digital polymerase chain reaction) chip-based proximity ligation assay (PLA) for the quantification of low concentration proteins. First, standard hCSTB (human cystatin B) protein was used to optimize the related experimental conditions. Comparing to ordinary PLA tests, the results showed that our method achieved femtomolar limit of detection (LOD) with a linear dynamic range over three to four orders of magnitude. Then human CD147 protein, a reported biomarker for hepatoma carcinoma, was detected in single HepG2 and L02 cells. The results showed that there were wide disparities in single cell CD147 abundance for both of the two cell lines. And the average CD147 protein content in single HepG2 cells displayed 2-fold increase in comparison to that in single L02 cells. Comparing to the research findings obtained at bulk level, our method can provide more useful information for diagnosis and targeted therapy of tumors.

Expression of Cathepsin L and Its Intrinsic Inhibitors in Glomeruli of Rats With Puromycin Aminonucleoside Nephrosis.

Cathepsin L, a lysosomal cysteine proteinase, may have a key role in various biological and disease processes by intracellular and extracellular degradation of proteins. We examined the levels of cathepsin L and its intrinsic inhibitors in glomeruli of rats with puromycin aminonucleoside (PAN) nephrosis. In contrast to the weak levels of cathepsin L in normal glomeruli, on days 4 and 8, strong immunostaining was detected in almost all podocytes when proteinuria and pathological changes of the podocytes developed. Cathepsin L was reduced after day 28, but remained in a focal and segmental manner. Cystatin ß, an intracellular inhibitor, was not detected in podocytes. However, cystatin C, an extracellular inhibitor, was detected in podocytes after day 4, coincident with cathepsin L. Cystatin C levels were gradually reduced but sustained in many podocytes on day 28, while cystatin C was not detected in podocytes sustained cathepsin L. These results demonstrated that cathepsin L levels are not always accompanied by the levels of its inhibitors in podocytes of PAN nephrosis, suggesting a potential role of cathepsin L in podocyte injury, which is a critical process for the development and progression of tuft adhesion and sclerosis.

Human stefin B role in cell's response to misfolded proteins and autophagy.

Alternative functions, apart from cathepsins inhibition, are being discovered for stefin B. Here, we investigate its role in vesicular trafficking and autophagy. Astrocytes isolated from stefin B knock-out (KO) mice exhibited an increased level of protein aggregates scattered throughout the cytoplasm. Addition of stefin B monomers or small oligomers to the cell medium reverted this phenotype, as imaged by confocal microscopy. To monitor the identity of proteins embedded within aggregates in wild type (wt) and KO cells, the insoluble cell lysate fractions were isolated and analyzed by mass spectrometry. Chaperones, tubulins, dyneins, and proteosomal components were detected in the insoluble fraction of wt cells but not in KO aggregates. In contrast, the insoluble fraction of KO cells exhibited increased levels of apolipoprotein E, fibronectin, clusterin, major prion protein, and serpins H1 and I2 and some proteins of lysosomal origin, such as cathepsin D and CD63, relative to wt astrocytes. Analysis of autophagy activity demonstrated that this pathway was less functional in KO astrocytes. In addition, synthetic dosage lethality (SDL) gene interactions analysis in Saccharomyces cerevisiae expressing human stefin B suggests a role in transport of vesicles and vacuoles These activities would contribute, directly or indirectly to completion of autophagy in wt astrocytes and would account for the accumulation of protein aggregates in KO cells, since autophagy is a key pathway for the clearance of intracellular protein aggregates.

Top-down HPLC-ESI-MS detection of S-glutathionylated and S-cysteinylated derivatives of cystatin B and its 1-53 and 54-98 fragments in whole saliva of human preterm newborns.

Analysis by a HPLC-ESI-MS top-down proteomic platform of specimens of human preterm newborn whole saliva evidenced high relative amounts of cystatin B and its S-glutathionylated,S-cysteinylated, and S-S 2-mer (on Cys(3)) derivatives, decreasing as a function of postconceptional age (PCA). The percentage of S-unmodified cystatin B was higher than the S-modified isoforms in the early PCA period, differently from adults where cystatin B was detectable only as S-modified derivatives. The percentage of S-modified derivatives increased as a function of PCA, reaching at the normal term of delivery values similar to those determined in at-term newborns, babies, and adults. Moreover, in the early PCA period, high relative amounts of the 1-53 and 54-98 cystatin B fragments were detected, decreasing as a function of PCA and disappearing at the normal term of delivery. In agreement with intact cystatin B, fragment 1-53 was detectable as S-unmodified and S-modified derivatives, and their percentages changed accordingly with the percentages of intact proteins, suggesting that the fragmentation process could be subsequent to and independent from the S-modification of the protein. This study highlights specific enzymatic activity in the oral cavity of preterm newborns not present in at-term newborns and adults, which can be a clue to specialized pathways occurring during fetal oral development.

Saliva electrophoretic protein profiles in infants: changes with age and impact of teeth eruption and diet transition.

OBJECTIVE: The objective of this study was to describe the changes in salivary protein profiles in infants between the ages of 3 and 6 months, and to evaluate the impact of teeth eruption and introduction of solid foods on such profiles. DESIGN: 73 infants were followed longitudinally at 3 and 6 months of age. Their whole saliva proteins were separated by SDS-PAGE electrophoresis and semi-quantified by image analysis. Amylase activity was also measured on a sub-sample of the population (n=42 infants). Bands which abundance was significantly different between the two ages according to paired comparisons were identified by mass spectrometry techniques. RESULTS: Out of 21 bands, 13 were significantly different between 3 and 6 months of age. Two short variants of amylase increased in abundance with age, as did amylase activity. Other changes possibly translated developmental physiological events, for example maturation of the adaptive immune system. The balance between S-type cystatins and cystatins A and B was modified, in favour of S-type cystatins at 6 months of age. Teeth eruption resulted in an increase in albumin abundance, whilst introduction of solid foods was associated with higher levels of ß-2 microglobulin and S-type cystatins. CONCLUSIONS: Salivary profiles were modified substantially between the ages of 3 and 6 months. Both teeth eruption and diet had an impact on abundance changes for some proteins, revealing dynamic interactions between saliva proteome, oral physiology and diet.

Cystatin B as a tissue and urinary biomarker of bladder cancer recurrence and disease progression.

PURPOSE: Using proteomic techniques, we sought to identify novel protein biomarkers in tissue and urine from patients with transitional cell carcinoma (TCC). EXPERIMENTAL DESIGN: Urinary and tissue proteomes were analyzed and differentially expressed proteins were identified by mass spectrometry. One of the proteins, cystatin B, was further analyzed in TCC tissue by immunohistochemistry and in urine by semiquantitative Western blot analysis. RESULTS: Cystatin B tissue staining intensity significantly increased concordantly with TCC grade (P = 0.0008). Elevated urinary cystatin B levels correlated with increasing tumor grade (P = 0.062) and stage (P = 0.0047). Patients with elevated levels of cystatin B had a shorter mean +/- SE time to disease recurrence (12 +/- 1.82 months) compared with patients who had low levels (28.8 +/- 2.26 months; P = 0.0047). Similarly, patients with elevated cystatin B levels had a shorter time to grade/stage progression compared with patients with low urinary cystatin B (P = 0.0007). By multivariate Cox regression analysis, an elevated cystatin B level was the most significant variable predicting disease recurrence (hazard ratio, 3.8; 95% confidence interval, 1.5-9.5; P = 0.0049) and grade/stage progression (hazard ratio, 10.4; 95% confidence interval, 1.6-201.5; P = 0.0104). CONCLUSIONS: Cystatin B is elevated in tissue and urine of bladder cancer patients. Cystatin B urine levels are positively correlated with tumor grade, stage, and shorter time to disease recurrence and progression. Consequently, cystatin B may be useful as a novel predictive biomarker in TCC of the bladder.