1.
Végvári, Ákos, et al.
(author)
Bioinformatic strategies for unambiguous identification of prostate specific antigen in clinical samples
2011
In: Journal of Proteomics. - : Elsevier BV. - 1874-3919. ; 75:1, s. 202-210
Journal article (peer-reviewed) abstract
Prostate specific antigen (PSA), as a widely used clinical biomarker in prostate cancer diagnostics, exists in multiple molecular forms. However, all of these forms might not be recognized in a given sample by the standard immunoassays. Therefore, we have investigated PSA isoforms separated by size using mass spectrometric analyses. The objective of these developments was to identify and specify the various forms of PSA. To optimize successful identification of different PSA forms, we have developed a bioinformatic strategy, consisting of high resolution MALDI-MS PMF and sequencing MS/MS data searches. To improve sequence-based identification, the recently introduced Proteios software environment was employed, allowing the combination of multiple database search engines in an automated manner. We could unambiguously identify PSA in clinical samples by all detectable tryptic peptides, which were found to be common in several isoforms.
2.
Jankovskaja, Skaidre, et al.
(author)
Optimization of sample preparation for transporter protein quantification in tissues by LC–MS/MS
2019
In: Journal of Pharmaceutical and Biomedical Analysis. - : Elsevier BV. - 0731-7085 .- 1873-264X. ; 164, s. 9-15
Journal article (peer-reviewed) abstract
Background: Reproducible quantification of drug transporter protein expression in tissues is important for predicting transporter mediated drug disposition. Many mass-spectrometry based transporter protein quantification methods result in high variability of the estimated transporter quantities. Therefore, we aimed to evaluate and optimize mass spectrometry-based quantification method for drug transporter proteins in tissues. Materials and methods: Plasma membrane (PM) proteins from mouse tissues were isolated by applying three extraction protocols: commercial plasma membrane extraction kit, tissue homogenization by Potter-Elvehjem homogenizer in combination with sucrose-cushion ultracentrifugation, and PM enrichment with Tween 40. Moreover, five different protein digestion protocols were applied on the same PM fraction. PM isolation and digestion protocols were evaluated by measuring the amount of transporter proteins by liquid chromatography-tandem mass spectrometry in selected reaction monitoring mode. Results: Mouse liver homogenization by Potter-Elvehjem homogenizer in combination with sucrose-cushion ultracentrifugation and PM enrichment with Tween 40 resulted in two times higher transporter protein quantity (Breast cancer resistance protein (Bcrp) 18.0 fmol/μg protein) in comparison with the PM samples isolated by extraction kit (Bcrp 9.8 fmol/μg protein). The evaluation of protein digestion protocols revealed that the most optimal protocol for PM protein digestion is with Lys-C and trypsin, in combination with trypsin enhancer and heat denaturation. Overall, quantities of Bcrp and Na+/K + ATPase proteins evaluated in mouse liver and kidney cortex by using our optimized PM isolation method, as well as, established digestion protocol were two to three times higher than previously reported and coefficient of variation (CV) for technical replicates was below 10%. Conclusion: We have established an improved transporter protein quantification methodology by optimizing PM isolation and protein digestion procedures. The optimized procedure resulted in a higher transporter protein yield and improved precision.
3.
Ahmad Tajudin, Asilah, et al.
(author)
Integrated acoustic immunoaffinity-capture (IAI) platform for detection of PSA from whole blood samples.
2013
In: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0189 .- 1473-0197. ; 13:9, s. 1790-1796
Journal article (peer-reviewed) abstract
On-chip detection of low abundant protein biomarkers is of interest to enable point-of-care diagnostics. Using a simple form of integration, we have realized an integrated microfluidic platform for the detection of prostate specific antigen (PSA), directly in anti-coagulated whole blood. We combine acoustophoresis-based separation of plasma from undiluted whole blood with a miniaturized immunoassay system in a polymer manifold, demonstrating improved assay speed on our Integrated Acoustic Immunoaffinity-capture (IAI) platform. The IAI platform separates plasma from undiluted whole blood by means of acoustophoresis and provides cell free plasma of clinical quality at a rate of 10 uL/min for an online immunoaffinity-capture of PSA on a porous silicon antibody microarray. The whole blood input (hematocrit 38-40%) rate was 50 μl min(-1) giving a plasma volume fraction yield of ≈33%. PSA was immunoaffinity-captured directly from spiked female whole blood samples at clinically significant levels of 1.7-100 ng ml(-1) within 15 min and was subsequently detected via fluorescence readout, showing a linear response over the entire range with a coefficient of variation of 13%.
4.
Ekström, Simon, et al.
(author)
Integrated selective enrichment target - a microtechnology platform for matrix-assisted laser desorption/ionization-mass spectrometry applied on protein biomarkers in prostate diseases
2004
In: Electrophoresis. - : Wiley. - 0173-0835. ; 25:21-22, s. 3769-3777
Journal article (peer-reviewed) abstract
The performance of a miniaturized sample processing platform for matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), manufactured by silicon microfabrication, called integrated selective enrichment target (ISET) technology was evaluated in a biological context. The ISET serves as both sample treatment device and MALDI-MS target, and contains an array of 96 perforated nanovials, which each can be filled with 40 nL of reversed-phase beads. This methodology minimizes the number of sample transfers and the total surface area available for undesired adsorption of the analytes in order to provide high-sensitivity analysis. ISET technology was successfully applied for characterization of proteins coisolated by affinity chromatography of prostate-specific antigen (PSA) from human seminal fluid. The application of ISET sample preparation enabled multiple analyses to be performed on a limited sample volume, which resulted in the discovery that prolactin inducible protein (PIP) was coisolated from the samples.
5.
Finnskog, David, et al.
(author)
High-speed biomarker identification utilizing porous silicon nanovial arrays and MALDI-TOF mass spectrometry
2006
In: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 27:5-6, s. 1093-1103
Journal article (peer-reviewed) abstract
Speed and accuracy are crucial prerequisites in the application of proteomic methods to clinical medicine. We describe a microfluidic-based nanovial array for rapid proteolytic processing linked to MALDI-TOF MS. This microscale format consumes only minute amounts of sample, and it is compatible with rapid bioanalytical protocols and high-sensitivity readouts. Arrays of vials (300 mu m in diameter and 25 mu m deep), isotropically etched in silicon wafers were electrochemically porosified. Automated picoliter microdispensing was employed for precise fluid handling in the microarray format. Vials were prefilled with trypsin solution, which was allowed to dry. Porosified and nonporosified nanovials were compared for trypsin digestion and subsequent MS identification of three model proteins: lysozyme, alcohol dehydrogenase, and serum albumin at levels of 100 and 20 fmol. In an effort to assess the rapid digestion platform in a context of putative clinical applications, two prostate cancer biomarkers, prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2), were digested at levels of 100 fmol (PSA), 20 fmol (PSA) and 8 fmol (hK2). All biomarker digestions were completed in less than 30 s, with successful MS identification in the porous nanovial setting.
6.
Kwon, Ho Jeong, et al.
(author)
Drug Compound Characterization by Mass Spectrometry Imaging in Cancer Tissue
2015
In: Archives of Pharmacal Research. - : Springer Science and Business Media LLC. - 1976-3786 .- 0253-6269. ; 38:9, s. 1718-1727
Research review (peer-reviewed) abstract
MALDI Mass spectrometry imaging (MSI) provides a technology platform that allows the accurate visualization of unlabeled small molecules within the two-dimensional spaces of tissue samples. MSI has proven to be a powerful tool-box concept in the development of new drugs. MSI allows unlabeled drug compounds and drug metabolites to be detected and identified and quantified according to their mass-to-charge ratios (m/z) at high resolution in complex tissue environments. Such drug characterization in situ, by both spatial and temporal behaviors within tissue compartments, provide new understandings of the dynamic processes impacting drug uptake and metabolism at the local sites targeted by therapy. Further, MSI in combination with histology and immunohistochemistry, provides the added value of defining the context of cell biology present at the sites of drug localization thus providing invaluable information relating to treatment efficacy. In this report we provide mass spectrometry imaging data within various cancers such as malignant melanoma in patients administered with Vemurafenib, a protein kinase inhibitor that is targeting both the BRAF, and ERK mutated target proteins and that has shown significant efficacy in restraining disease progression. We also provide an overview of other examples of the new generation of targeted drugs, and demonstrate the data on personalized medicine drugs localization within tumor compartments within in vivo models. In these cancer models we provide detailed data on drug and target protein co-localization of YCG185 and Sunitinib. These drugs are targeting VEGFR2 within the angiogenesis mechanism. Our ability to resolve drug uptake at targeted sites of directed therapy provides important opportunities for increasing our understanding about the mode of action of drug activity within the environment of disease.
7.
Malm, Johan, et al.
(author)
Blood Plasma Reference Material – A Global Resource for Proteomic Research
2013
In: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 12:7, s. 3087-3092
Journal article (peer-reviewed) abstract
There is an ever-increasing awareness and interest within the clinical research field that has creating a large demand for blood fraction samples as well as other clinical samples. The translational research area is another field that demanding for blood samples, used widely in proteomics, genomics, as well as metabolomics. Blood samples are the global most common biological samples that find its use in a broad variety of applications in Life Science. We hereby introduce a new reference blood plasma (EDTA) that is aimed as a global resource for the Proteomics community. We have developed these reference plasma standards by defining the Control group as those with CRP levels <3mg/L and a Disease group with CRP ranges >30 mg/L. In these references we have used both newborn children 1-2 weeks, as well as youngsters 10-15 years, and middle aged 30-50 years, and elderly patients at the ages of 65+. The total number of these reference plasma pools was 80 patients in each group. We provide data on the developments and characteristics of the reference blood plasma standards, as well as what is used by the team members at the respective laboratories. The standards have been evaluated by pilot sample processing in biobanking operations, and are currently a resource that allows the Proteomic society to perform quantitative proteomic studies. By the use of high quality reference plasma samples, global initiatives, such as the Chromosome Human Proteome Project (C-HPP), will benefit as one scientific program when the entire human proteome is mapped and linked to human diseases.
8.
Malm, Johan, et al.
(author)
Developments in biobanking workflow standardization providing sample integrity and stability
2013
In: Journal of Proteomics. - : Elsevier BV. - 1874-3919 .- 1876-7737. ; 95:SI, s. 38-45
Journal article (peer-reviewed) abstract
Recommendations and outlines for standardization in biobanking processes are presented by a research team with long-term experience in clinical studies. These processes have important bearing on the use of samples in developing assays. These measurements are useful to document states of health and disease that are beneficial for academic research, commercial healthcare, drug development industry and government regulating agencies. There is a need for increasing awareness within proteomic and genomic communities regarding the basic concepts of collecting, storing and utilizing clinical samples. Quality control and sample suitability for analysis need to be documented and validated to ensure data integrity and establish contexts for interpretation of results. Standardized methods in proteomics and genomics are required to be practiced throughout the community allowing datasets to be comparable and shared for analysis. For example, sample processing of thousands of clinical samples, performed in 384 high-density sample tube systems in a fully automated workflow, preserves sample content and is presented showing validation criteria. Large studies will be accompanied by biological and molecular information with corresponding clinical records from patients and healthy donors. These developments position biobanks of human patient samples as an increasingly recognized major asset in disease research, future drug development and within patient care. Biological significance: The current manuscript is of major relevance to the proteomic and genomic fields, as it outlines the standardization aspects of biobanking and the requirements that are needed to run future clinical studies that will benefit the patients where OMICS science will play a major role. A global view of the field is given where best practice and conventional acceptances are presented along with ongoing large-scale biobanking projects. The authors represent broadly stakeholders that cover the academic, pharma, biotech and healthcare fields with extensive experience and deliveries. This contribution will be a milestone paper to the proteomic and genomic scientists to present data in the future that will have impact to the life science area.This article is part of a Special Issue entitled: Standardization and Quality Control in Proteomics.
9.
Malm, Johan, et al.
(author)
Large Scale Biobanking of Blood – The Importance of High Density Sample Processing Procedures
2012
In: Journal of Proteomics. - : Elsevier BV. - 1874-3919. ; 76:1, s. 116-124
Journal article (peer-reviewed) abstract
We introduce a novel automated sample-processing concept that will be of mandatory importance to proteomics and future clinical research, performing patient studies from resulting blood fractions in various disease areas. Biobank storage of small sample volumes allows for high replicate numbers to be processed and aliquoted, where each sample aliquot can be used for a dedicated clinical analysis and end-point measurement. In order to preserve sample integrity and value over time, the principle of single usage is gaining recognition. We hereby present a 384-format sample tube system for the preservation and archiving of clinical patient samples that will form the basis for future proteomics studies. This high density scaling allows for reproducible aliquoting 70-µL volumes of blood fractions. Blood plasma with EDTA, Li-heparin, and citrate, as anti-coagulants, are fractioned along with the buffy coat and the erythrocyte fraction, in addition to the serum fraction. We demonstrate an automated sample handling for biobanking: samples from patients were processed and aliquoted in both 96- and 384-sample racks by liquid handling robotics and Laboratory Intelligence Management System (LIMS) overview and control. Within this study, the blood samples were analyzed by the Clinical Chemistry department at the Southern University Hospital in Malmö, using standard biomarker assays, quantifying 23 common markers used in everyday healthcare around the world. We were able to prove that the 384-format using an aluminum foil with a thin polymer film coating for sealing, is stable and can reproducibly be processed for automated biobank freezer units.
10.
Marko-Varga, György, et al.
(author)
A Multicolumn Ion Chromatographic Determination of Nitrate and Sulfate in Waters Containing Humic Substances
1986
In: International Journal of Environmental Analytical Chemistry. - : Informa UK Limited. - 0306-7319 .- 1029-0397. ; 25:1-3, s. 161-171
Journal article (peer-reviewed) abstract
A three-column ion chromatographic system for the removal of humic substances from natural waters, and subsequent on-line concentration and determination of nitrate and sulfate using non-suppressed ion chromatography is presented. Humic substances are removed using disposable adsorption columns packed with chemically bonded amine silica material. The sample is directly transfered to an ion exchange column where the anions are concentrated ca 10 times. After reversing the flow, the ions are transferred to a third column where they are separated and quantified. The detection limit is less than 1 mgL-1 of nitrate or sulfate in water containing 45 mgL-1 of humic acid.
