| 1. |
- Woldmar, N., et al.
(författare)
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Proteomic analysis of brain metastatic lung adenocarcinoma reveals intertumoral heterogeneity and specific alterations associated with the timing of brain metastases
- 2023
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Ingår i: ESMO Open. - : Elsevier BV. - 2059-7029. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Brain metastases are associated with considerable negative effects on patients’ outcome in lung adenocarcinoma (LADC). Here, we investigated the proteomic landscape of primary LADCs and their corresponding brain metastases. Materials and methods: Proteomic profiling was conducted on 20 surgically resected primary and brain metastatic LADC samples via label-free shotgun proteomics. After sample processing, peptides were analyzed using an Ultimate 3000 pump coupled to a QExactive HF-X mass spectrometer. Raw data were searched using PD 2.4. Further data analyses were carried out using Perseus, RStudio and GraphPad Prism. Proteomic data were correlated with clinical and histopathological parameters and the timing of brain metastases. Mass spectrometry-based proteomic data are available via ProteomeXchange with identifier PXD027259. Results: Out of the 6821 proteins identified and quantified, 1496 proteins were differentially expressed between primary LADCs and corresponding brain metastases. Pathways associated with the immune system, cell-cell/matrix interactions and migration were predominantly activated in the primary tumors, whereas pathways related to metabolism, translation or vesicle formation were overrepresented in the metastatic tumors. When comparing fast- versus slow-progressing patients, we found 454 and 298 differentially expressed proteins in the primary tumors and brain metastases, respectively. Metabolic reprogramming and ribosomal activity were prominently up-regulated in the fast-progressing patients (versus slow-progressing individuals), whereas expression of cell-cell interaction- and immune system-related pathways was reduced in these patients and in those with multiple brain metastases. Conclusions: This is the first comprehensive proteomic analysis of paired primary tumors and brain metastases of LADC patients. Our data suggest a malfunction of cellular attachment and an increase in ribosomal activity in LADC tissue, promoting brain metastasis. The current study provides insights into the biology of LADC brain metastases and, moreover, might contribute to the development of personalized follow-up strategies in LADC.
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| 2. |
- Juhasz, P., et al.
(författare)
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ESI and MALDI LC/MS-MS approaches for large scale protein, identification and quantification : Are they equivalent?
- 2002
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Ingår i: ; , s. 55-56
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Konferensbidrag (refereegranskat)abstract
- An approach for large scale protein identification and quantification was described. The peptide mixtures from the strong cation exchange (SCX) fractionated protein digests were separated and analyzed by HPLC MS/MS. The human fibrobalst nuclei were purified from stimulated and non-stimulated cells. The quantification results with MALDI and ESI LC/MS were shown to be identical within experimental error.
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| 3. |
- Plymoth, A., et al.
(författare)
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Human bronchoalveolar lavage: biofluid analysis with special emphasis on sample preparation
- 2003
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Ingår i: Proteomics. ; 3:6
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Tidskriftsartikel (refereegranskat)abstract
- Respiratory diseases are an important health problem throughout the world. Whether caused by industrial pollutants, infections, smoking, cancer or metabolic diseases, damage to the lungs and airways often lead to morbidity or death. Bronchoalveolar lavage (BAL) obtained by fiber-optic bronchoscopy is a biofluid mirroring the expression of normally secreted pulmonary proteins and the products of activated cells and destructive processes. The characterization of the proteome within this compartment provides an opportunity to establish temporal and prognostic indicators of airway disease. The objective of this study was to develop methods of analysis of BAL samples, which achieved the highest level of annotation of the expression map of this proteome. We have optimized the process of sample preparation after investigating a variety of techniques including dialysis, ultramembrane filtration, precipitation and gel filtration. We have further studied methods to remove albumin from BAL in order to unmask proteins hidden on two-dimensional gels. In a pilot application of the method, BAL protein profiles obtained from healthy nonsmokers and smokers at risk for developing chronic obstructive pulmonary disease showed distinct differences.
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| 4. |
- Vialas, Vital, et al.
(författare)
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A multicentric study to evaluate the use of relative retention times in targeted proteomics
- 2017
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Ingår i: Journal of Proteomics. - : Elsevier BV. - 1874-3919. ; 152, s. 138-149
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Tidskriftsartikel (refereegranskat)abstract
- Despite the maturity reached by targeted proteomic strategies, reliable and standardized protocols are urgently needed to enhance reproducibility among different laboratories and analytical platforms, facilitating a more widespread use in biomedical research. To achieve this goal, the use of dimensionless relative retention times (iRT), defined on the basis of peptide standard retention times (RT), has lately emerged as a powerful tool. The robustness, reproducibility and utility of this strategy were examined for the first time in a multicentric setting, involving 28 laboratories that included 24 of the Spanish network of proteomics laboratories (ProteoRed-ISCIII). According to the results obtained in this study, dimensionless retention time values (iRTs) demonstrated to be a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups both intra- and inter-laboratories. iRT values also showed very low variability over long time periods. Furthermore, parallel quantitative analyses showed a high reproducibility despite the variety of experimental strategies used, either MRM (multiple reaction monitoring) or pseudoMRM, and the diversity of analytical platforms employed. Biological significance From the very beginning of proteomics as an analytical science there has been a growing interest in developing standardized methods and experimental procedures in order to ensure the highest quality and reproducibility of the results. In this regard, the recent (2012) introduction of the dimensionless retention time concept has been a significant advance. In our multicentric (28 laboratories) study we explore the usefulness of this concept in the context of a targeted proteomics experiment, demonstrating that dimensionless retention time values is a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups.
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| 5. |
- Westergren-Thorsson, G., et al.
(författare)
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Proteome
- 2006
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Ingår i: Encyclopedia of Respiratory Medicine : Volume 1-4 - Volume 1-4. - 9780123708793 - 9780123708793 ; 1-4, s. 527-532
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Proteomics can be defined as the studies of protein properties on a large scale to obtain a global view of biological processes at the protein level. Essentially, proteomics requires protein separation and identification, and in many cases quantification. The cornerstones in proteomics are protein/peptide separation by gel electrophoresis and/or different chromatographic techniques, and identification by mass spectrometry followed by bioinformatic and biological interpretation of data. By combining these different separation techniques and mass spectrometry it is now possible to identify low abundant proteins with 10-1000 copies per cell. Today, substantial research efforts in proteome studies of the lung are focused on obtaining new diagnostic markers, as well as fingerprinting disease mechanisms.
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| 6. |
- Ansari, D., et al.
(författare)
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Relationship between tumour size and outcome in pancreatic ductal adenocarcinoma
- 2017
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Ingår i: British Journal of Surgery. - : Oxford University Press (OUP). - 0007-1323 .- 1365-2168. ; 104:5, s. 600-607
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Tidskriftsartikel (refereegranskat)abstract
- Background: The size of pancreatic ductal adenocarcinoma (PDAC) at diagnosis is an indicator of outcome. Previous studies have focused mostly on patients with resectable disease. The aim of this study was to investigate the relationship between tumour size and risk of metastasis and death in a large PDAC cohort, including all stages. Methods: Patients diagnosed with PDAC between 1988 and 2013 were identified from the Surveillance, Epidemiology, and End Results (SEER) database. Tumour size was defined as the maximum dimension of the tumour as provided by the registry. Metastatic spread was assessed, and survival was calculated according to size of the primary tumour using the Kaplan-Meier method. Cox proportional regression modelling was used to adjust for known confounders. Results: Some 58728 patients were included. There were 187 patients (0·3 per cent) with a tumour size of 0·5cm or less, in whom the rate of distant metastasis was 30·6 per cent. The probability of tumour dissemination was associated with tumour size at the time of diagnosis. The association between survival and tumour size was linear for patients with localized tumours, but stochastic in patients with regional and distant stages. In patients with resected tumours, increasing tumour size was associated with worse tumour-specific survival, whereas size was not associated with survival in patients with unresected tumours. In the adjusted Cox regression analysis, the death rate increased by 4·1 per cent for each additional 1-cm increase in tumour size. Conclusion: Pancreatic cancer has a high metastatic capacity even in small tumours. The prognostic impact of tumour size is restricted to patients with localized disease.
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| 7. |
- Burestedt, E., et al.
(författare)
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Optimisation and validation of an automated solid phase extraction technique coupled on-line to enzyme-based biosensor detection for the determination of phenolic compounds in surface water samples
- 1995
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Ingår i: Chromatographia. - Heidelberg : Springer Berlin/Heidelberg. - 0009-5893 .- 1612-1112. ; 41:3-4, s. 207-215
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Tidskriftsartikel (refereegranskat)abstract
- A fully integrated screening system for phenolic compounds was developed incorporating on-line solid phase extraction, fractionation and biosensor detection. Two different types of biosensors, solid graphite and carbon paste electrodes incorporating the enzyme tyrosinase, were compared and used in the screening system. Interfacing of the solid phase extraction and fractionation with the biosensor detection was given special attention since the biosensors were not compatible with the organic modifier used for desorption of phenols from the solid phase extraction step. The system was validated with conventional analytical techniques. Surface water samples from the Ebro river were spiked with 1,10, and 25μg L−1 of catechol, phenol,p-cresol, respectively. Three out of seven samples were spiked and the correct samples were identified, containing phenols equivalent to the spiked concentrations. © 1995 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH.
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| 8. |
- Cadenas, J., et al.
(författare)
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Regulation of human oocyte maturation in vivo during the final maturation of follicles
- 2023
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Ingår i: Human Reproduction. - : Oxford University Press (OUP). - 0268-1161 .- 1460-2350. ; 38:4, s. 686-700
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Tidskriftsartikel (refereegranskat)abstract
- STUDY QUESTION: Which substances and signal transduction pathways are potentially active downstream to the effect of FSH and LH in the regulation of human oocyte maturation in vivo? SUMMARY ANSWER: The regulation of human oocyte maturation appears to be a multifactorial process in which several different signal transduction pathways are active. WHAT IS KNOWN ALREADY: Many studies in animal species have provided insight into the mechanisms that govern the final maturation of oocytes. Currently, these studies have identified several different mechanisms downstream to the effects of FSH and LH. Some of the identified mechanisms include the regulation of cAMP/cGMP levels in oocytes involving C-type natriuretic peptide (CNP), effects of epidermal growth factor (EGF)-related peptides such as amphiregulin (AREG) and/or epiregulin (EREG), effect of TGF-β family members including growth differentiation factor 9 (GDF9) and morphogenetic protein 15 (BMP15), activins/inhibins, follicular fluid meiosis activating sterol (FF-MAS), the growth factor midkine (MDK), and several others. However, to what extent these pathways and mechanisms are active in humans in vivo is unknown. STUDY DESIGN, SIZE, DURATION: This prospective cohort study included 50 women undergoing fertility treatment in a standard antagonist protocol at a university hospital affiliated fertility clinic in 2016-2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: We evaluated the substances and signalling pathways potentially affecting human oocyte maturation in follicular fluid (FF) and granulosa cells (GCs) collected at five time points during the final maturation of follicles. Using ELISA measurement and proteomic profiling of FF and whole genome gene expression in GC, the following substances and their signal transduction pathways were collectively evaluated: CNP, the EGF family, inhibin-A, inhibin-B, activins, FF-MAS, MDK, GDF9, and BMP15. MAIN RESULTS AND THE ROLE OF CHANCE: All the evaluated substances and signal transduction pathways are potentially active in the regulation of human oocyte maturation in vivo except for GDF9/BMP15 signalling. In particular, AREG, inhibins, and MDK were significantly upregulated during the first 12-17 h after initiating the final maturation of follicles and were measured at significantly higher concentrations than previously reported. Additionally, the genes regulating FF-MAS synthesis and metabolism were significantly controlled in favour of accumulation during the first 12-17 h. In contrast, concentrations of CNP were low and did not change during the process of final maturation of follicles, and concentrations of GDF9 and BMP15 were much lower than reported in small antral follicles, suggesting a less pronounced influence from these substances. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: Although GC and cumulus cells have many similar features, it is a limitation of the current study that information for the corresponding cumulus cells is not available. However, we seldom recovered a cumulus-oocyte complex during the follicle aspiration from 0 to 32 h. WIDER IMPLICATIONS OF THE FINDINGS: Delineating the mechanisms governing the regulation of human oocyte maturation in vivo advances the possibility of developing a platform for IVM that, as for most other mammalian species, results in healthy offspring with good efficacy. Mimicking the intrafollicular conditions during oocyte maturation in vivo in small culture droplets during IVM may enhance oocyte nuclear and cytoplasmic maturation. The primary outlook for such a method is, in the context of fertility preservation, to augment the chances of achieving biological children after a cancer treatment by subjecting oocytes from small antral follicles to IVM. Provided that aspiration of oocytes from small antral follicles in vivo can be developed with good efficacy, IVM may be applied to infertile patients on a larger scale and can provide a cheap alternative to conventional IVF treatment with ovarian stimulation. Successful IVM has the potential to change current established techniques for infertility treatment. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the University Hospital of Copenhagen, Rigshospitalet, the Independent Research Fund Denmark (grant number 0134-00448), and the Interregional EU-sponsored ReproUnion network. There are no conflicts of interest to be declared.
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| 9. |
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| 10. |
- Edfors, R., et al.
(författare)
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Use of proteomics to identify biomarkers associated with chronic kidney disease and long-term outcomes in patients with myocardial infarction
- 2020
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Ingår i: Journal of Internal Medicine. - : Wiley. - 0954-6820 .- 1365-2796. ; 288:5, s. 581-592
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Tidskriftsartikel (refereegranskat)abstract
- Background Patients with chronic kidney disease (CKD) have poor outcomes following myocardial infarction (MI). We performed an untargeted examination of 175 biomarkers to identify those with the strongest association with CKD and to examine the association of those biomarkers with long-term outcomes. Methods A total of 175 different biomarkers from MI patients enrolled in the Swedish Web-System for Enhancement and Development of Evidence-Based Care in Heart Disease Evaluated According to Recommended Therapies (SWEDEHEART) registry were analysed either by a multiple reaction monitoring mass spectrometry assay or by a multiplex assay (proximity extension assay). Random forests statistical models were used to assess the predictor importance of biomarkers, CKD and outcomes. Results A total of 1098 MI patients with a median estimated glomerular filtration rate of 85 mL min(-1)/1.73 m(2)were followed for a median of 3.2 years. The random forests analyses, without and with adjustment for differences in demography, comorbidities and severity of disease, identified six biomarkers (adrenomedullin, TNF receptor-1, adipocyte fatty acid-binding protein-4, TNF-related apoptosis-inducing ligand receptor 2, growth differentiation factor-15 and TNF receptor-2) to be strongly associated with CKD. All six biomarkers were also amongst the 15 strongest predictors for death, and four of them were amongst the strongest predictors of subsequent MI and heart failure hospitalization. Conclusion In patients with MI, a proteomic approach could identify six biomarkers that best predicted CKD. These biomarkers were also amongst the most important predictors of long-term outcomes. Thus, these biomarkers indicate underlying mechanisms that may contribute to the poor prognosis seen in patients with MI and CKD.
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| 11. |
- Ekstrom, S., et al.
(författare)
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Disposable polymeric high-density nanovial arrays for matrix assisted laser desorption/ionization-time of flight-mass spectrometry: II. Biological applications
- 2001
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Ingår i: Electrophoresis. - 0173-0835. ; 22:18, s. 3984-3992
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Tidskriftsartikel (refereegranskat)abstract
- A novel disposable high-density matrix assisted laser desorption/ionization (MALDI) target plate made either from polymethylmethacrylate (PMMA) or polycarbonate (PC) is presented where thousands (1200-1600) of samples can be deposited and subsequently, analyzed by MALDI-time of flight (TOF) mass spectrometry. Good reproducibility, was obtained across the plate regardless of position on the target plate with a relative standard deviation (RSD) on the peak intensity of typically 30% calculated from data generated by analysis of a 10 nM peptide mixture of angiotensin. I, II, III and bradykinin. The nanovial array format combined with microdispensing technology makes It possible to carry out in-vial, chemistry on deposited samples. This is demonstrated by the analysis of peptides from beta -casein and subsequent in-vial dephosphorylation of its phosphopeptides at 10 fmol levels by microdispensing of alkaline phosphatase, into the nanovial. The mass spectra obtained from these polymeric targets provides can also be used in high sensitivity applications as shown by peptide mass fingerprinting of human fibroblast proteins separated by two-dimensional gel, electrophoresis.
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| 12. |
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| 13. |
- Grimm, C. H., et al.
(författare)
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Selective extraction of small proteins from biological samples using a novel restricted access column with cation exchange properties
- 2000
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Ingår i: Chromatographia. - 0009-5893. ; 52:11-12, s. 703-709
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Tidskriftsartikel (refereegranskat)abstract
- The determination of proteins utilising a polymer-based restricted access support material with ion exchange properties (IERAM) is outlined. Solid phase extraction coupled on-line with a microbore reversed phase HPLC system for the quantitation of small marker proteins is demonstrated. The cation-exchange restricted access packings were characterised with respect to their adsorption and desorption kinetics. The IERAM material was also investigated by capacity, selectivity, and biocompatibility determinations when applied to the quantification of small molecular weight proteins such as cytochrome C, Lysozyme, Ribonuclease A, Myoglobin, Insulin, human serum albumin, and a Tryptic inhibitor. The integrated system was coupled to mass identity of selected proteins by MALDI-TOF mass spectrometry. The chromatographic outlet was interfaced to an "Interplate" fractionation collecter that sampled 1 μL volumes directly onto the MALDI target plate. The fully automated coupled column system was run unattended overnight and applied to protein quantitations in human plasma samples. Recovery data for a selected number of proteins varied between 90-96% (n = 10) with a limit of quantification around 2 μM with an injection volume of 100 μL. The RSD data were typically less than 8% at a 50 μM protein level (n = 7).
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| 14. |
- Hegedüs, Luca, et al.
(författare)
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The Prognostic Relevance of PMCA4 Expression in Melanoma : Gender Specificity and Implications for Immune Checkpoint Inhibition
- 2022
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 23:6
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Tidskriftsartikel (refereegranskat)abstract
- PMCA4 is a critical regulator of Ca2+ homeostasis in mammalian cells. While its biological and prognostic relevance in several cancer types has already been demonstrated, only preclinical investigations suggested a metastasis suppressor function in melanoma. Therefore, we studied the expression pattern of PMCA4 in human skin, nevus, as well as in primary and metastatic melanoma using immunohistochemistry. Furthermore, we analyzed the prognostic power of PMCA4 mRNA levels in cutaneous melanoma both at the non-metastatic stage as well as after PD-1 blockade in advanced disease. PMCA4 localizes to the plasma membrane in a differentiation dependent manner in human skin and mucosa, while nevus cells showed no plasma membrane staining. In contrast, primary cutaneous, choroidal and conjunctival melanoma cells showed specific plasma membrane localization of PMCA4 with a wide range of intensities. Analyzing the TCGA cohort, PMCA4 mRNA levels showed a gender specific prognostic impact in stage I–III melanoma. Female patients with high transcript levels had a significantly longer progression-free survival. Melanoma cell specific PMCA4 protein expression is associated with anaplasticity in melanoma lung metastasis but had no impact on survival after lung metastasectomy. Importantly, high PMCA4 transcript levels derived from RNA-seq of cutaneous melanoma are associated with significantly longer overall survival after PD-1 blockade. In summary, we demonstrated that human melanoma cells express PMCA4 and PMCA4 transcript levels carry prognostic information in a gender specific manner.
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| 15. |
- Kjellström, S, et al.
(författare)
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Flow immunochemical bio-recognition detection for the determination of interleukin-10 in cell samples
- 2000
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Ingår i: Journal of Immunological Methods. - 0022-1759. ; 246:1-2, s. 30-119
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Tidskriftsartikel (refereegranskat)abstract
- On- and off-line heterogeneous non-competitive flow immunoassays for the determination of Interleukin-10 are described. The sample containing IL-10 is mixed, either on-line in a reaction coil or off-line in a test tube, with fluorescent labelled anti-IL-10 antibodies to form an antibody-antigen complex. The labelled unbound antibodies are trapped on an immobilized IL-10 column whereas the IL-10-antibody complexes are eluted and detected downstream by a fluorescence detector. The optimization of the systems was performed with respect to choice of affinity support, flow rate, carrier buffer additives, pH and antibody-antigen association. Both bio recognition assays were tested with a spiked cell medium and the IL-10 detection limits in this matrix was found to be 8 fmol using the off-line incubation mode and 40 fmol using the on-line incubation mode. The sample through-put was 26 and 40 samples per hour in the on-line and off-line incubation modes, respectively. IL-10 identification in the sample fractions was achieved using MALDI-TOF MS.
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| 16. |
- Kjellström, S, et al.
(författare)
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On-line coupling of microdialysis sampling with liquid chromatography for the determination of peptide and non-peptide leukotrienes
- 1998
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Ingår i: Journal of chromatography. A. - 0021-9673. ; 823:1-2, s. 489-496
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Tidskriftsartikel (refereegranskat)abstract
- An automated on-line sampling method was developed using microdialysis as the simultaneous sampling and sample pre-treatment technique. The extraction fraction values of microdialysis probes sampling different eicosanoids were investigated. The impact of cyclodextrins in the perfusion liquid used for sampling hydrophobic eicosanoids in biological systems was also studied. The total time for one analysis was 7.6 min allowing seven measurements per hour for monitoring kinetic changes in biological systems.
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| 17. |
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| 18. |
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| 19. |
- Malm, Johan, et al.
(författare)
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Developments in biobanking workflow standardization providing sample integrity and stability
- 2013
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Ingår i: Journal of Proteomics. - : Elsevier BV. - 1874-3919 .- 1876-7737. ; 95:SI, s. 38-45
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Tidskriftsartikel (refereegranskat)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.
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| 20. |
- Marko-Varga, György, et al.
(författare)
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Disposable polymeric high-density nanovial arrays for matrix assisted laser desorption/ionization-time of flight-mass spectrometry: I. Microstructure development and manufacturing
- 2001
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Ingår i: Electrophoresis. - 0173-0835. ; 22:18, s. 3978-3983
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Tidskriftsartikel (refereegranskat)abstract
- In order to meet the expected enormous demand for mass spectrometry (MS)through-, put as a result of the current efforts to completely map the human proteome, this paper presents a new concept for low-cost high-throughput protein identification by matrix assisted laser desorption/ionization-time of flight-(MALDI-TOF)-MS peptide mapping using disposable polymeric high-density nanovial MALDI target plates. By means of microfabrication technology precision engineered nanovial arrays are fabricated in polymer substrates such as polymethylmethacrylate (PMMA) and polycarbonate (PC). The target plate fabrication processes investigated were precision. micromilling, cold embossing and injection moulding (work in progress). Nanovial dimensions were 300, 400 or 500 mum. Typical array densities were 165 nanovials/cm(2), which corresponds to 3300 vials on a full Applied Biosystems MALDI target plate. Obtained MALDI data displayed equal mass resolution, accuracy, signal intensity for peptide standards as compared to high-density silicon nanovial arrays previously reported by our group [7], as well as conventional stainless steel or gold targets.
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| 21. |
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| 22. |
- Nilsson, C. L., et al.
(författare)
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Chromosome 19 Annotations with Disease Speciation: A First Report from the Global Research Consortium
- 2013
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 12:1, s. 134-149
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Tidskriftsartikel (refereegranskat)abstract
- A first research development progress report of the Chromosome 19 Consortium with members from Sweden, Norway, Spain, United States, China and India, a part of the Chromosome-centric Human Proteome Project (C-HPP) global initiative, is presented (http://www.c-hpp.org). From the chromosome 19 peptide-targeted library constituting 6159 peptides, a pilot study was conducted using a subset with 125 isotope-labeled peptides. We applied an annotation strategy with triple quadrupole, ESI-Qtrap, and MALDI mass spectrometry platforms, comparing the quality of data within and in between these instrumental set-ups. LC–MS conditions were outlined by multiplex assay developments, followed by MRM assay developments. SRM was applied to biobank samples, quantifying kallikrein 3 (prostate specific antigen) in plasma from prostate cancer patients. The antibody production has been initiated for more than 1200 genes from the entire chromosome 19, and the progress developments are presented. We developed a dedicated transcript microarray to serve as the mRNA identifier by screening cancer cell lines. NAPPA protein arrays were built to align with the transcript data with the Chromosome 19 NAPPA chip, dedicated to 90 proteins, as the first development delivery. We have introduced an IT-infrastructure utilizing a LIMS system that serves as the key interface for the research teams to share and explore data generated within the project. The cross-site data repository will form the basis for sample processing, including biological samples as well as patient samples from national Biobanks.
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| 23. |
- Nilsson, C. L., et al.
(författare)
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Use of ENCODE Resources to Characterize Novel Proteoforms and Missing Proteins in the Human Proteome
- 2015
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 14:2, s. 603-608
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Tidskriftsartikel (refereegranskat)abstract
- We describe the utility of integrated strategies that employ both translation of ENCODE data and major proteomic technology pillars to improve the identification of the "missing proteins", novel proteoforms, and PTMs. On one hand, databases in combination with bioinformatic tools are efficiently utilized to establish microarray-based transcript analysis and supply rapid protein identifications in clinical samples. On the other hand, sequence libraries are the foundation of targeted protein identification and quantification using mass spectrometric and immunoaffinity techniques. The results from combining proteoENCODEdb searches with experimental mass spectral data indicate that some alternative splicing forms detected at the transcript level are in fact translated to proteins. Our results provide a step toward the directives of the C-HPP initiative and related biomedical research.
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| 24. |
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| 25. |
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| 26. |
- Plymoth, Amelie, et al.
(författare)
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Rapid proteome analysis of bronchoalveolar lavage samples of lifelong smokers and never-smokers by micro-scale liquid chromatography and mass spectrometry
- 2006
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Ingår i: Clin Chem.. - : Oxford University Press (OUP). ; 52:4, s. 671-679
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The aim of this study was to determine whether relative qualitative and quantitative differences in protein expression could be related to smoke exposure or smoke-induced airway inflammation. We therefore explored and characterized the protein components found in bronchoalveolar lavage (BAL) fluid sampled from either lifelong smokers or never-smokers. METHODS: BAL fluid samples obtained by bronchoscopy from 60-year-old healthy never-smokers (n = 18) and asymptomatic smokers (n = 30) were analyzed in either pooled or individual form. Initial global proteomic analysis used shotgun digestion approaches on unfractionated BAL fluid samples (after minimal sample preparation) and separation of peptides by gradient (90-min) liquid chromatography (LC) coupled with on-line linear ion trap quadropole mass spectrometry (LTQ MS) for identification and analysis. RESULTS: LTQ MS identified 481 high- to low-abundance proteins. Relative differences in patterns of BAL fluid proteins in smokers compared with never-smokers were observed in pooled and individual samples as well as by 2-dimensional gel analysis. Gene ontology categorization of all annotated proteins showed a wide spectrum of molecular functions and biological processes. CONCLUSIONS: The described method provides comprehensive qualitative proteomic analysis of BAL fluid protein expression from never-smokers and from smokers at risk of developing chronic obstructive pulmonary disease. Many of the proteins identified had not been detected in previous studies of BAL fluid; thus, the use of LC-tandem MS with LTQ may provide new information regarding potentially important patterns of protein expression associated with lifelong smoking.
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| 27. |
- Ressine, Anton, et al.
(författare)
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Macro/Nano-Structured Silicon as Solid Support for Antibody Arrays: Surface Design, Reproducibility, and Assay Characteristics Enabling Discovery of Kallikrein Gene Products for Prostate Disease Diagnostics
- 2005
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Ingår i: Nanobiotechnology. - 1551-1286. ; 1:1, s. 93-104
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Tidskriftsartikel (refereegranskat)abstract
- To facilitate high-throughput biomarker discovery and high-density protein-chip array analyses of complex biological samples, a novel macro- and nanoporous silicon surface for protein microarrays was developed. The surface offers three-dimensional surface enlarging properties and spot confinement, enabling both high sensitivity bioassays and design of high density arrays. Reproducible manufacturing of the protein chip surface was accomplished as demonstrated by the low imprecision when standard IgG bioassays were performed at 100 pM antigen level on a series of protein chips scanned at widely different locations within a silicon wafer, as well as between different wafers from two different manufacturers. The relative standard deviation (RSD) of fluorescence spot intensity within an array on a chip was less than 20%. Mean spot intensity RSD was 19% for all 25 microarray chips in the study. Within-manufacturer-lot RSDs in chips from either manufacturer were <15% of mean spot intensity. The detection limit and dynamic range of the novel protein chip surface were examined to evaluate whether they match criteria required in a search for novel biomarkers such as for prostate cancer. Monoclonal IgG against prostate-specific antigen (PSA) was arrayed on the porous silicon chips. These were subsequently incubated in serum samples containing widely different levels of fluorescence-labeled PSA. Detection of PSA in serum at concentrations from 0.7 ng/mL (26 pM) up to 104-fold higher levels verified assay characteristics required in the search for prostate biomarkers (e.g., kallikrein gene products) at clinically relevant levels.
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| 28. |
- Ressine, A., et al.
(författare)
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Reproducible porous silicon protein microarrays -chip manufacturing and application to clinical biomarkers
- 2005
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Ingår i: Micro Total Analysis Systems - Proceedings of MicroTAS 2005 Conference : 9th International Conference on Miniaturized Systems for Chemistry and Life Sciences - 9th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - 0974361119 - 9780974361116 ; 1, s. 826-828
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Konferensbidrag (refereegranskat)abstract
- Full 3" wafer scale fabrication of porous silicon chips (pore chip protein arrays -PCPA) is reported. The PCPA is developed for the analysis of protein molecules based on the fluorescence and MALDI-TOF MS detection. The surface porosification process allows the creation of chips with different surface geometries to control the physical properties of the matrix in a highly reproducible way.
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| 29. |
- Rezeli, M., et al.
(författare)
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Quantification of total apolipoprotein E and its specific isoforms in cerebrospinal fluid and blood in Alzheimer's disease and other neurodegenerative diseases
- 2015
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Ingår i: EuPA Open Proteomics. - : Elsevier. - 2212-9685. ; 8, s. 137-143
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Tidskriftsartikel (refereegranskat)abstract
- A targeted mass spectrometric assay was developed for identification and quantification of apoE isoforms (apoE2, E3 and E4), and it was utilized for screening of samples from AD patients (. n=. 39) and patients with other neurodegenerative disorders (. n=. 38). The assay showed good linearity with LOQ corresponds to total apoE concentration of 0.8 and 40. ng/mL in CSF and plasma/serum, respectively. We identified apoE phenotypes with 100% accuracy in clinical samples. We found strong association between genotypes of the individuals and their apoE levels in blood; ε4 allele carriers had significantly lower apoE levels in blood than non-carriers. © 2015.
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| 30. |
- Wan, Guihong, et al.
(författare)
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Development and validation of time-to-event models to predict metastatic recurrence of localized cutaneous melanoma
- 2024
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Ingår i: Journal of the American Academy of Dermatology. - 0190-9622. ; 90:2, s. 288-298
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Tidskriftsartikel (refereegranskat)abstract
- Background: The recent expansion of immunotherapy for stage IIB/IIC melanoma highlights a growing clinical need to identify patients at high risk of metastatic recurrence and, therefore, most likely to benefit from this therapeutic modality. Objective: To develop time-to-event risk prediction models for melanoma metastatic recurrence. Methods: Patients diagnosed with stage I/II primary cutaneous melanoma between 2000 and 2020 at Mass General Brigham and Dana-Farber Cancer Institute were included. Melanoma recurrence date and type were determined by chart review. Thirty clinicopathologic factors were extracted from electronic health records. Three types of time-to-event machine-learning models were evaluated internally and externally in the distant versus locoregional/nonrecurrence prediction. Results: This study included 954 melanomas (155 distant, 163 locoregional, and 636 1:2 matched nonrecurrences). Distant recurrences were associated with worse survival compared to locoregional/nonrecurrences (HR: 6.21, P < .001) and to locoregional recurrences only (HR: 5.79, P < .001). The Gradient Boosting Survival model achieved the best performance (concordance index: 0.816; time-dependent AUC: 0.842; Brier score: 0.103) in the external validation. Limitations: Retrospective nature and cohort from one geography. Conclusions: These results suggest that time-to-event machine-learning models can reliably predict the metastatic recurrence from localized melanoma and help identify high-risk patients who are most likely to benefit from immunotherapy.
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| 31. |
- Wang, X. D., et al.
(författare)
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Association of chromosome 19 to lung cancer genotypes and phenotypes
- 2015
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Ingår i: Cancer and Metastasis Reviews. - : Springer Science and Business Media LLC. - 0167-7659 .- 1573-7233. ; 34:2, s. 217-226
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Forskningsöversikt (refereegranskat)abstract
- The Chromosome 19 Consortium, a part of the Chromosome-Centric Human Proteome Project (C-HPP, ), is tasked with the understanding chromosome 19 functions at the gene and protein levels, as well as their roles in lung oncogenesis. Comparative genomic hybridization (CGH) studies revealed chromosome aberration in lung cancer subtypes, including ADC, SCC, LCC, and SCLC. The most common abnormality is 19p loss and 19q gain. Sixty-four aberrant genes identified in previous genomic studies and their encoded protein functions were further validated in the neXtProt database (). Among those, the loss of tumor suppressor genes STK11, MUM1, KISS1R (19p13.3), and BRG1 (19p13.13) is associated with lung oncogenesis or remote metastasis. Gene aberrations include translocation t(15, 19) (q13, p13.1) fusion oncogene BRD4-NUT, DNA repair genes (ERCC1, ERCC2, XRCC1), TGF beta 1 pathway activation genes (TGFB1, LTBP4), Dyrk1B, and potential oncogenesis protector genes such as NFkB pathway inhibition genes (NFKBIB, PPP1R13L) and EGLN2. In conclusion, neXtProt is an effective resource for the validation of gene aberrations identified in genomic studies. It promises to enhance our understanding of lung cancer oncogenesis.
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| 32. |
- Yaropolov, A I, et al.
(författare)
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An amperometric biosensor based on laccase immobilized in polymer matrices for determining phenolic compounds
- 2005
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Ingår i: Journal of Analytical Chemistry. - : Springer Science and Business Media LLC. - 1608-3199 .- 1061-9348. ; 60:6, s. 553-557
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Tidskriftsartikel (refereegranskat)abstract
- An amperometric enzyme electrode based on laccase for determining phenolic compounds is proposed. The following three types of polymer materials were used for enzyme immobilization on the surface of a glassy-carbon electrode: positively charged cetyl ethyl poly (ethyleneimine) (CEPEI) and negatively charged commercial Nafion and Eastman AQ 29D polymers. The advantages and disadvantages of each of the above polymers for enzyme immobilization are discussed. The detection limits of the model phenolic compounds hydroquinone and pyrocatechol in a buffer solution on laccase immobilization in a Nation membrane were 3.5 x 10(-8) and 5.0 x 10(-8) M, respectively, at a signal-to-noise ratio of 3. Electrodes with laccase immobilized in Nation and Eastman AQ 29D membranes exhibited the shortest response time. The operating stability and the stability in storage can be significantly improved by the additional incorporation of gelatin in the polymer matrices. Gelatin prevents enzyme inactivation as a result of enzyme modification by the free-radical oxidation products of phenolic compounds.
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| 33. |
- Önnerfjord, P., et al.
(författare)
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Development of fluorescence based flow immunoassays utilising restricted access columns
- 2000
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Ingår i: Chromatographia. - 0009-5893. ; 51:3-4, s. 199-204
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Tidskriftsartikel (refereegranskat)abstract
- The development of high-speed flow immunoassay techniques is described. The principles are based on heterogeneous flow immunoassay interactions. High sample throughput can be used for screening small analytes in a number of biological matrices originating from samples of water from environmentally polluted areas, or biological fluids such as urine and plasma. The immunochemical detection principle is based on chromatographic separation of the immunocomplex formed (AbAg or AbAg*) and the free antigen (Ag) by a restricted access (RA) column, utilising size-exclusion and reversed phase mechanisms. A fluorescein-labelled analyte (Ag*) was used in the competitive assay format with fluorescence detection. Sample throughput was 80 h-1 and detection limits 1.4 nM (300pgml-1) for atrazine and 2.3 nM (500 pg ml-1) for the sum of triazines. Analyses could be performed at a sample throughput of 400 6 h-1 shift. Basic immunoaffinity interactions of a number of immunoreagents, using fluorescence polarisation were studied and outlined both for triazines and for 2,4-D. Structural variations in tracer synthesis confirmed that this is an important part in the design and optimisation of flow immuno methodologies.
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| 34. |
- Önnerfjord, P., et al.
(författare)
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Fluorescence polarisation for immunoreagent characterisation
- 1998
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Ingår i: Journal of Immunological Methods. - 0022-1759. ; 213:1, s. 31-39
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies were characterised using fluorescence polarisation, a homogeneous assay technique in which all reagents are in solution. Kinetic studies on the association and dissociation of the immunocomplex were performed. A competitive assay was used and the sensitivities, operational linearities, as well as the specificities of the immunoassays were experimentally determined for various antibody preparations with specificity for triazines. Detection limits for atrazine in water samples were determined to be within the range of 0.08-0.4 ng ml-1 using a 5-min incubation time and a 0.5-ml sample volume.
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| 35. |
- Önnerfjord, P., et al.
(författare)
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On-line solid-phase extraction in liquid chromatography using restricted access pre-columns for the analysis of s-triazines in humic-containing waters
- 1996
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 737:1, s. 35-45
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Tidskriftsartikel (refereegranskat)abstract
- A coupled-column liquid chromatographic system using solid phase extraction pre-columns for the seven chlorotriazines, atrazine, desethylatrazine, desisopropylatrazine (DIA), propazine, simazine, desethylterbuthylazine and terbuthylazine, using photodiode-array detection (PDAD) was developed. Restricted access (RA), was investigated as a pre- column material, making use of non-adsorptive size exclusion of macromolecules and simultaneous dynamic portioning of the analytes, for the pre-concentration in environmental analysis. The RA phase was characterised and shown to solve problems with difficult matrix effects in humic-containing surface water samples. Other pre-column materials, e.g. reversed-phase C18, mono-functional C18, extraction disk mixture of C18 and styrene- divinylbenzene (PS-DVB) and LiChrolut EN, a polymeric sorbent specially designed for environmental analysis, were investigated and compared for their selectivity and stability, respectively. Comparisons were made with respect to selectivity and efficiency for the SPE columns separately and in combination with analytical columns. Recovery data and matrix dependencies were studied at concentration levels ranging between 0.1-10 μg/l. The RA pre-columns showed superior stability in complex environmental matrices, such as reference humic water and river water, due to the restriction of macromolecules. Retention properties with the RA alkyl-diol-silica were not sufficient for the most polar metabolites. However, efficient sorption/desorption kinetics were found with medium-polar and non-polar compounds. LiChrolut EN was found to strongly retain these polar metabolites as well as medium and non-polar solutes, and was selected for a deeper investigation (e.g. salinity dependence and pH). For a 10 x 2 mm I.D. pre- column, the breakthrough volume for the polar metabolite DIA was at least 150 ml, while for the RA pro-columns it was less than 10 ml. Unfortunately, this polymeric support was strongly effected, in terms of stability, in complex matrices which lead to repeated and expensive exchanging of pre-columns. However, in order to detect even the polar metabolites these pre-columns were finally used on-line together with liquid chromatography PDAD and thermospray mass spectrometry for the determination of triazines in water samples from the Ebro river during different seasons of the year.
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