Optimisation of urine sample preparation for shotgun proteomics

Urine reflects the renal function and urinary and kidney systems, but it may also reflect the presence of cancer in other parts of the body. Urine also has potential for providing prognostic information during therapeutic treatments thanks to non-invasive monitoring. A quick and reproducible protein purification procedure is essential to allow data comparison between proteomic studies in urine biomarker discovery. The article describes a simple, reproducible and cheap sample preparation procedure with a maximum protein yield (400 µg) obtained from only 10 mL of urine utilising cut-off filter desalting and digestion. The reported procedure removes yellowish background coloration residues and thus prevents the errors in spectrophotometric protein concentration determination. Different extraction solvents used in the presented procedure point to the possibility of partial elimination of abundant proteins (albumin and keratin family), as well as to the improvement of the sequence coverage of proteins identified, which helps to reveal changes in the urinary proteome. With this workflow, proteins can be easily obtained on standard laboratory equipment within 3 h. Data are available via ProteomeXchange with identifier PXD019738

MALDI-TOF/MS Profiling of Whole Saliva and Gingival Crevicular Fluid in Patients with the Invisalign System and Fixed Orthodontic Appliances

The movement of teeth by orthodontic treatment with the Invisalign (IN) system and fixed orthodontic appliances (FOA) is characterized by the reconstruction of periodontal ligaments, alveolar bone, and gingiva. A reflection of these phenomena can be found in the composition of gingival crevicular fluid (GCF). A total of 90 samples from 45 participants (45 whole saliva and 45 GCF), including 15 patients with FOA, 15 patients with IN, and 15 patients with oral health, were subjected to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF/MS) analysis. Mass fingerprints were generated for each sample. Three models were tested: a quick classifier (QC), a genetic algorithm (GA), and a supervised neural network (SNN). For both groups of samples (saliva and GCF), the GA model showed the highest recognition abilities of 88.89% (saliva) and 95.56% (GCF). Differences between the treated (FOA and IN) groups and the control group in saliva and GCF samples were determined using cluster analysis. In addition, we monitored the effect of long-term orthodontic treatment (after 6 months) in the lag phase of orthodontic tooth movement. The results show increased levels of inflammatory markers (α-defensins), which may indicate an ongoing inflammatory process even after 21 days from force application

Differential Urinary Proteomic Analysis of High-Risk Cervical Intraepithelial Neoplasia

Human papillomavirus (HPV)-associated lesions and malignancies exhibit alterations in the composition and functionality of the extracellular matrix (ECM) that represent the complex molecular pathways present between infection and disease. A total of 20 urine samples were used, including from 10 patients with cervical intraepithelial neoplasia grade 3 (CIN3) and 10 healthy controls to perform the label-free quantitative analysis using the nano-HPLC and ESI-MS ion trap mass analyzer and matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF/MS) fast screening. Among 476 identified/quantified proteins, 48 were significantly changed (log2-fold change ≥1.0 or ≤−1.0, −log10 (bbinominal, p-value ≥ 1.3), of which were 40 proteins (down-regulated) and 8 proteins (up-regulated) in CIN3, in comparison to healthy controls. The biological function and key pathway enrichment of the gene set using gen set enrichment analysis (GSEA) were analyzed. The ECM-receptor interaction pathway (NES = −1.64, p = 0.026) was down-regulated by 13 proteins (HSPG2, COL6A1, COL6A3, SPP1, THBS1, TNC, DAG1, FN1, COMP, GP6, VTN, SDC1, and CD44; log2 FC range from −0.03 to −1.48) for the CIN3 group in the KEGG database. The MALDI-TOF/MS screening showed the difference of protein profiles between the control and CIN3 groups, i.e., using the scatter plot with a well-separated shape, as well as effectively distinguishing both groups (control and CIN3) using genetic algorithms (GA) with cross-validation (51.56%) and recognition capability (95.0%). Decreased levels of ECM-receptor interaction proteins may cause disturbances in the interactions of cells with the ECM and play an important role in the development and progression of cervical cancer

Proteomic and bioinformatics analysis of human saliva for the dental-risk assessment

Background: Dental caries disease is a dynamic process with a multi-factorial etiology. It is manifested by demineralization of enamel followed by damage spreading into the tooth inner structure. Successful early diagnosis could identify caries-risk and improve dental screening, providing a baseline for evaluating personalized dental treatment and prevention strategies. Methodology: Salivary proteome of the whole unstimulated saliva (WUS) samples was assessed in caries-free and caries-susceptible individuals of older adolescent age with permanent dentition using a nano-HPLC and MALDI-TOF/TOF mass spectrometry. Results: 554 proteins in the caries-free and 695 proteins in the caries-susceptible group were identified. Assessment using bioinformatics tools and Gene Ontology (GO) term enrichment analysis revealed qualitative differences between these two proteomes. Members of the caries-susceptible group exhibited a branch of cytokine binding gene products responsible for the regulation of immune and inflammatory responses to infections. Inspection of molecular functions and biological processes of caries-susceptible saliva samples revealed significant categories predominantly related to the activity of proteolytic peptidases, and the regulation of metabolic and catabolic processes of carbohydrates. Conclusions: Proteomic analysis of the whole saliva revealed information about potential risk factors associated with the development of caries-susceptibility and provides a better understanding of tooth protection mechanisms

Hammasvaihteen lujuus- ja värähtelyanalyysi

In this thesis, a D1PSF60 gearbox is examined, comparing the strength and vibration qualities of lamellar cast iron and spherical cast iron as gearbox housing material, and comparing each of these housings with the welded steel Duetto design. This thesis also serves as documentation describing these qualities for developing future gearbox designs. Calculation, measurement and testing facilities in Metso Drives, Ltd. are also identified by this thesis. Three companies, Valmet Power Transmission, Ltd., Parkano Gears, Ltd. and Santasalo Gears, Ltd., merged to form Metso Drives, Ltd. These companies aimed to unify their gearbox series by unifying product features to achieve the most cost-efficient solutions. The one-stage gearbox series Duetto from Valmet Power Transmission used welded steel gearbox housing. In the Santasalo-l C-series, the housing was composed of GJL-2S0-lamellar cast iron. For a new D1PSF gearbox series, Metso Drives, Ltd. aimed to find an efficient combination. It is generally stated that lamellar cast iron possesses better vibration qualities and yields a lower noise level than steel. When testing a gearbox made of lamellar cast iron, however, its noise level was significantly higher than the same product composed of steel. In this thesis, a strength and vibration analysis was done by an I-deas program in which stresses, deformations and natural frequencies of the D1PSF60 gearbox were identified. Frequency Response Function measurement and noise and vibration measurements were also made on the D1PSF60 gearbox in the testing field. Results were compared with those generated by the I-deas program. Natural frequencies of the D1PSF60 gearbox were obtained by the I-deas program for housing materials lamellar cast iron, spherical cast iron and steel. Natural frequencies of the D1PSF60 gearbox itself were obtained using Frequency Response Function measurements. The lamellar cast iron had the highest level of noise, the second was spherical cast iron and the lowest level of noise had welded steel as a gearbox material.Tämän työn tarkoituksena oli tutkia Metso Drives Oy:n uuden D1PSF60-vaihteen lujuus- ja värähtelyominaisuuksia pallo- ja suomugrafiittivaluraudoilla sekä verrata niitä teräksiseen Duetto-vaihteeseen. Tavoitteena oli tehdä dokumentti, jossa todetaan vaihteiden ominaisuudet ja saadaan materiaalia jatkokehitykseen ja selvitetään Metso Drives Oy:n laskenta-, mittaus- ja testausmahdollisuuksia. Valmet Power Transmission, Parkano Gearsin ja Santasalon yhdistyessä Metso Drives Oy:ksi vaihdesarjoja yhdistettiin tuotepiirteitä yhdistämällä tarkoituksena saada mahdollisimman kustannustehokkaita ratkaisuja. Valmetin yksiportaisessa Duetto-vaihdesarjassa käytettiin hitsattua teräskoteloa. Santasalon 1C-sarjassa kotelon materiaalina puolestaan oli GJL-250-suomugrafiittivalurauta. Metso Drives Oy:n uudessa D1PSF-vaihdesarjassa haluttiin yhdistää näiden vaihdesarjojen ominaisuudet kustannustehokkaasti. Yleinen käsitys koneenrakennuksessa on ollut, että suomugrafiittivaluraudalla on paremmat värähtelyominaisuudet ja myös matalammat melutasot. Vaihteiden koeajossa on kuitenkin huomattu, että suomugrafiittivalurautaisen kotelon melutaso muodostui huomattavasti korkeammaksi kuin teräksisen kotelon. Tarkastelussa tehtiin D1PSF60-vaihteelle I-deasilla lujuus- ja värähtelyanalyysi, jossa selvitettiin jännitykset ja muodonmuutokset sekä ominaistaajuudet. Vaihteelle tehtiin taajuusvastefunktiomittaus sekä melu- ja tärinämittaukset koeajokentällä. Tuloksia verrattiin I-deasilla saatuihin arvoihin. Tuloksena saatiin I-deas-ohjelmistolla D1PSF60-vaihteen ominaistaajuudet suomu- ja pallografiittivaluraudalle sekä teräskotelolle. Lisäksi taajuusvastefunktiomittauksella saatiin ominaistaajuudet D1PSF60-vaihteelle. Kotelon materiaaleista meluisimmaksi osoittautui suomugrafiittivalurauta, seuraavana pallografiittimateriaali ja äänettömin kolmesta oli teräskotelo