| 1. |
- Adamczyk, Barbara, 1985, et al.
(författare)
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Pregnancy-Associated Changes of IgG and Serum N-Glycosylation in Camel (Camelus dromedarius).
- 2016
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Ingår i: Journal of proteome research. - : American Chemical Society (ACS). - 1535-3907 .- 1535-3893. ; 15:9, s. 3255-65
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Tidskriftsartikel (refereegranskat)abstract
- The dromedary camel (Camelus dromedarius) is an agriculturally important species of high economic value but of low reproductive efficiency. Serum and IgG N-glycosylation are affected by physiological and pathogenic changes and might therefore be a useful diagnostic tool in camel livestock management. This study presents the first comprehensive annotation of the N-glycome from dromedary camel serum as well as their single-domain and conventional antibodies and its subsequent application for camel pregnancy diagnostics. N-glycans were released by PNGaseF, labeled with 2-aminobenzamide (2-AB), and analyzed by hydrophilic interaction liquid chromatography with fluorescent detection (HILIC-UPLC-FLD), enzymatic sequencing and mass spectrometry (MS). The use of a high-throughput robotic platform for sample preparation allowed the rapid generation of glycomics data from pregnant (n = 8) and nonpregnant (n = 8) camels of the Majaheem and Wadha breed. IgG N-glycans dominate the glycan profile of camel serum and present a mixture of core-fucosylated and noncore-fucosylated N-glycans which can contain N-glycolylneuraminic and N-acetylneuraminic acid. Significant pregnancy-associated but breed-independent increases in galactosylation, core-fucosylation, sialylation, and decreases in serum O-acetylation were observed. The monitoring of IgG and serum N-glycosylation presents an attractive complementary test for camel pregnancy diagnostics and presents an interesting tool for biomarker discovery in camel health and breeding.
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| 2. |
- Aerts, Jordan, et al.
(författare)
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Electrochemically Etched Tapered-Tip Stainless-Steel Electrospray-Ionization Emitters for Capillary Electrophoresis-Mass Spectrometry
- 2023
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 22:4, s. 1377-1380
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Tidskriftsartikel (refereegranskat)abstract
- We have used household consumables to facilitate electrochemical etching of stainless-steel hypodermic tubing to produce tapered-tip emitters suitable for electrospray ionization for use in mass spectrometry. The process involves the use of 1% oxalic acid and a 5 W USB power adapter, commonly known as a phone charger. Further, our method avoids the otherwise commonly used strong acids that entail chemical hazards: concentrated HNO3 for etching stainless steel, or concentrated HF for etching fused silica. Hence, we here provide a convenient and self-inhibiting procedure with minimal chemical hazards to manufacture tapered-tip stainless-steel emitters. We show its performance in metabolomic analysis with CE-MS of a tissue homogenate where the metabolites acetylcarnitine, arginine, carnitine, creatine, homocarnosine, and valerylcarnitine were identified, all with basepeak separated electropherograms, within <6 min of separation. The mass spectrometry data are freely available through the MetaboLight public data repository via access number MTBLS7230.
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| 3. |
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| 4. |
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| 5. |
- Akhtar, Malik N., et al.
(författare)
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Evaluation of Database Search Programs for Accurate Detection of Neuropeptides in Tandem Mass Spectrometry Experiments
- 2012
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 11:12, s. 6044-6055
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Tidskriftsartikel (refereegranskat)abstract
- Neuropeptide identification in mass spectrometry experiments using database search programs developed for proteins is challenging. Unlike proteins, the detection of the complete sequence using a single spectrum is required to identify neuropeptides or prohormone peptides. This study compared the performance of three open-source programs used to identify proteins, OMSSA, X!Tandem and Crux, to identify prohormone peptides. From a target database of 7850 prohormone peptides, 23550 query spectra were simulated across different scenarios. Crux was the only program that correctly matched all peptides regardless of p-value and at p-value < 1 X 10(-2), 33%, 64%, and >75%, of the 5, 6, and >= 7 amino acid-peptides were detected. Crux also had the best performance in the identification of peptides from chimera spectra and in a variety of missing ion scenarios. OMSSA, X!Tandem and Crux correctly detected 98.9% (99.9%), 93.9% (97.4%) and 88.7% (98.3%) of the peptides at E- or p-value < 1 X 10(-6) (< 1 X 10(-2)), respectively. OMSSA and X! Tandem outperformed the other programs in significance level and computational speed, respectively. A consensus approach is not recommended because some prohormone peptides were only identified by one program.
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| 6. |
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| 7. |
- Alm, Henrik, et al.
(författare)
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In Vitro Neurotoxicity of PBDE-99 : Immediate and Concentration-Dependent Effects on Protein Expression in Cerebral Cortex Cells
- 2010
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 9:3, s. 1226-1235
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Tidskriftsartikel (refereegranskat)abstract
- Polybrominated diphenyl ethers (PBDEs) are commonly used flame retardants in various consumer products. Pre- and postnatal exposure to congeners of PBDEs disrupts normal brain development in rodents. Two-dimensional difference gel electrophoresis (2D-DIGE) was used to analyze concentration-dependent differences in protein expression in cultured cortical cells isolated from rat fetuses (GD 21) after 24 h exposure to PBDE-99 (3, 10, or 30 muM). Changes on a post-translational level were studied using a 1 h exposure to 30 muM PBDE-99. The effects of 24 h exposure to 3 and 30 muM PBDE-99 on mRNA levels were measured using oligonucleotide microarrays. A total of 62, 46, and 443 proteins were differentially expressed compared to controls after 24 h of exposure to 3, 10, and 30 muM PDBE-99, respectively. Of these, 48, 43, and 238 proteins were successfully identified, respectively. We propose that the biological effects of low-concentration PBDE-99 exposure are fundamentally different than effects of high-concentration exposure. Low-dose PBDE-99 exposure induced marked effects on cytoskeletal proteins, which was not correlated to cytotoxicity or major morphological effects, suggesting that other more regulatory aspects of cytoskeletal functions may be affected. Interestingly, 0.3 and 3 muM, but not 10 or 30 muM increased the expression of phosphorylated (active) Gap43, perhaps reflecting effects on neurite extension processes.
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| 8. |
- Alm, Rikard, et al.
(författare)
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Detection and identification of protein isoforms using cluster analysis of MALDI-MS mass spectra
- 2006
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 5:4, s. 785-792
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Tidskriftsartikel (refereegranskat)abstract
- We describe an approach to screen large sets of MALDI-MS mass spectra for protein isoforms separated on two-dimensional electrophoresis gels. Mass spectra are matched against each other by utilizing extracted peak mass lists and hierarchical clustering. The output is presented as dendrograms in which protein isoforms cluster together. Clustering could be applied to mass spectra from different sample sets, dates, and instruments, revealed similarities between mass spectra, and was a useful tool to highlight peptide peaks of interest for further investigation. Shared peak masses in a cluster could be identified and were used to create novel peak mass lists suitable for protein identification using peptide mass fingerprinting. Complex mass spectra consisting of more than one protein were deconvoluted using information from other mass spectra in the same cluster. The number of peptide peaks shared between mass spectra in a cluster was typically found to be larger than the number of peaks that matched to calculated peak masses in databases, thus modified peaks are probably among the shared peptides. Clustering increased the number of peaks associated with a given protein.
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| 9. |
- Alm, Rikard, et al.
(författare)
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Proteomic variation is as large within as between strawberry varieties
- 2007
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 6:8, s. 3011-3020
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Tidskriftsartikel (refereegranskat)abstract
- in search for a strawberry (Fragaria ananassa) with low allergen content, we determined the proteomic variation within and between different varieties. Proteomics data were generated by DIGE and proteins identified with MALDI-MS/MS. The amount of the strawberry allergen Fra a 1 varied between different strawberry varieties (CV = 39%). The variation was at the same level, or even slightly larger, due to different growth conditions (CV = 43%). For 153 other proteins, the biological variation was more affected by different growth conditions than by different varieties (mean CV = 52% and 43%, respectively) due to variation in a subset of proteins. Thus, the allergen variation due to growth conditions must be taken into consideration in attempts to obtain a low-allergen strawberry. However, the allergen content was always lower in colorless (white) strawberry varieties than in the red ones. Moreover, of the spots whose expression correlated with the allergen and the color (32 and 68, respectively), only 3 were the same. This implies that these two phenotypic traits are not inseparable, and it may be possible to breed a red strawberry with low amount of allergen.
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| 10. |
- Alm, Tove, et al.
(författare)
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A Chromosome-Centric Analysis of Antibodies Directed toward the Human Proteome Using Antibodypedia
- 2014
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 13:3, s. 1669-1676
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies are crucial for the study of human proteins and have been defined as one of the three pillars in the human chromosome-centric Human Proteome Project (CHPP). In this article the chromosome-centric structure has been used to analyze the availability of antibodies as judged by the presence within the portal Antibodypedia, a database designed to allow comparisons and scoring of publicly available antibodies toward human protein targets. This public database displays antibody data from more than one million antibodies toward human protein targets. A summary of the content in this knowledge resource reveals that there exist more than 10 antibodies to over 70% of all the putative human genes, evenly distributed over the 24 human chromosomes. The analysis also shows that at present, less than 10% of the putative human protein-coding genes (n = 1882) predicted from the genome sequence lack antibodies, suggesting that focused efforts from the antibody-based and mass spectrometry-based proteomic communities should be encouraged to pursue the analysis of these missing proteins. We show that Antibodypedia may be used to track the development of available and validated antibodies to the individual chromosomes, and thus the database is an attractive tool to identify proteins with no or few antibodies yet generated.
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| 11. |
- Almanza-Aguilera, Enrique, et al.
(författare)
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Impact in Plasma Metabolome as Effect of Lifestyle Intervention for Weight-Loss Reveals Metabolic Benefits in Metabolically Healthy Obese Women
- 2018
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3907 .- 1535-3893. ; 17:8, s. 2600-2610
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Tidskriftsartikel (refereegranskat)abstract
- Little is known regarding metabolic benefits of weight loss (WL) on the metabolically healthy obese (MHO) patients. We aimed to examine the impact of a lifestyle weight loss (LWL) treatment on the plasma metabolomic profile in MHO individuals. Plasma samples from 57 MHO women allocated to an intensive LWL treatment group (TG, hypocaloric Mediterranean diet and regular physical activity, n = 30) or to a control group (CG, general recommendations of a healthy diet and physical activity, n = 27) were analyzed using an untargeted1H NMR metabolomics approach at baseline, after 3 months (intervention), and 12 months (follow-up). The impact of the LWL intervention on plasma metabolome was statistically significant at 3 months but not at follow-up and included higher levels of formate and phosphocreatine and lower levels of LDL/VLDL (signals) and trimethylamine in the TG. These metabolites were also correlated with WL. Higher myo-inositol, methylguanidine, and 3-hydroxybutyrate, and lower proline, were also found in the TG; higher levels of hippurate and asparagine, and lower levels of 2-hydroxybutyrate and creatine, were associated with WL. The current findings suggest that an intensive LWL treatment, and the consequent WL, leads to an improved plasma metabolic profile in MHO women through its impact on energy, amino acid, lipoprotein, and microbial metabolism.
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| 12. |
- Alocci, D., et al.
(författare)
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GlyConnect: Glycoproteomics Goes Visual, Interactive, and Analytical
- 2019
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 18:2, s. 664-677
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Tidskriftsartikel (refereegranskat)abstract
- Knowledge of glycoproteins, their site-specific glycosylation patterns, and the glycan structures that they present to their recognition partners in health and disease is gradually being built on using a range of experimental approaches. The data from these analyses are increasingly being standardized and presented in various sources, from supplemental tables in publications to localized servers in investigator laboratories. Bioinformatics tools are now needed to collect these data and enable the user to search, display, and connect glycomics and glycoproteomics to other sources of related proteomics, genomics, and interactomics information. We here introduce GlyConnect (https://glyconnect.expasy.org/), the central platform of the Glycomics@ExPASy portal for glycoinformatics. GlyConnect has been developed to gather, monitor, integrate, and visualize data in a user-friendly way to facilitate the interpretation of collected glycoscience data. GlyConnect is designed to accommodate and integrate multiple data types as they are increasingly produced.
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| 13. |
- Amelina, Hanna, et al.
(författare)
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Proteomics-based method for the assessment of marine pollution using liquid chromatography coupled with two-dimensional electrophoresis
- 2007
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 6:6, s. 2094-2104
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Tidskriftsartikel (refereegranskat)abstract
- Using a proteomic approach, we have developed a new method for the assessment of marine pollution that generates highly reproducible protein expression patterns and it is simple and scalable. The protocol is based on applying liquid chromatography (LC) coupled with two-dimensional electrophoresis (2-DE) to analyze changes in the protein expression pattern after exposure to marine pollution. The digestive gland of the sentinel “blue mussel” (Mytilus edulis) was batch-processed through a simple cell fractionation followed by ion-exchange chromatography and 2-DE. The selection of ligands, elution method, and small volume design was carefully considered to define a protocol that could be mainly robotized. A pilot study with samples collected from different Gothenburg harbor areas indicated that the clean area could be distinguished from the polluted ones based on a protein expression pattern (PES) composed of 13 proteins. Principal component analysis (PCA) and hierarchical clustering confirmed that the PES was sufficient to discriminate polluted and unpolluted areas and to provide a spatial gradient from the polluted source. Several proteins from the PES were identified by electrospray ionization tandem mass spectrometry (ESI−MS/MS), and they are involved in β-oxidation, amino acid metabolism, detoxification, protein degradation, organelle biogenesis, and protein folding. In the near future, this methodology could show potential advantages to assess marine pollution and could become a stable platform to elucidate ecotoxicological questions.
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| 14. |
- Andersson, Tomas, et al.
(författare)
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Automating MALDI Sample Plate Loading
- 2007
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 6:2, s. 894-896
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Tidskriftsartikel (refereegranskat)abstract
- We describe the design and implementation of a generic robotic solution to automate the loading of MALDI sample plates into a mass spectrometer. The soft- and hardware aspects are described together with the various safety issues that need to be addressed. The automation increases thoughput by a factor of between 5- and 80-fold.
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| 15. |
- Antberg, Linn, et al.
(författare)
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Critical Comparison of Multidimensional Separation Methods for Increasing Protein Expression Coverage
- 2012
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 11:5, s. 2644-2652
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Tidskriftsartikel (refereegranskat)abstract
- We present a comparison of two-dimensional separation methods and how they affect the degree of coverage of protein expression in complex mixtures. We investigated the relative merits of various protein and peptide separations prior to acidic reversed-phase chromatography directly coupled to an ion trap mass spectrometer. The first dimensions investigated were density gradient organelle fractionation of cell extracts, 1D SDS-PAGE protein separation followed by digestion by trypsin or GluC proteases, strong cation exchange chromatography, and off-gel isoelectric focusing of tryptic peptides. The number of fractions from each first dimension and the total data accumulation RP-HPLC-MS/MS time was kept constant and the experiments were run in triplicate. We find that the most critical parameters are the data accumulation time, which defines the level of under-sampling and the avoidance of peptides from high expression level proteins eluting over the entire gradient.
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| 16. |
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| 17. |
- Arrigoni, Giorgio, et al.
(författare)
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Mass Spectrometry Analysis of a Protein Kinase CK2 beta Subunit Interactome Isolated from Mouse Brain by Affinity Chromatography.
- 2008
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 7:3, s. 990-1000
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Tidskriftsartikel (refereegranskat)abstract
- CK2, an acronym derived from the misnomer "casein kinase 2", denotes a ubiquitous and extremely pleiotropic Ser/Thr protein kinase, the holoenzyme of which is composed of two catalytic (alpha and/or alpha') and two noncatalytic beta subunits acting as a docking platform and the multifarious functions of which are still incompletely understood. By combining affinity chromatography and mass spectrometry, we have identified 144 mouse brain proteins that associate with immobilized CK2beta. A large proportion (60%) of the identified proteins had been previously reported to be functionally related to CK2, and a similar proportion have been classified as phosphoproteins with approximately half of these having the features of CK2 targets. A large number of the identified proteins ( approximately 40%) either are nuclear or shuttle between the nucleus and cytoplasm, and the biggest functional classes of CK2beta interactors are committed to protein synthesis and degradation (32 proteins) and RNA/DNA interaction (20 proteins). Also well represented are the categories of cytoskeletal/structural proteins (19), trafficking proteins (17), and signaling proteins (14). The identified proteins are examined in relation to their functions and potential as targets and/or regulators of CK2, disclosing in some cases unanticipated links between this kinase and a variety of biochemical events.
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| 18. |
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| 19. |
- Azimzadeh, Omid, et al.
(författare)
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PPAR Alpha: A Novel Radiation Target in Locally Exposed Mus musculus Heart Revealed by Quantitative Proteomics
- 2013
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 12:6, s. 2700-2714
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Tidskriftsartikel (refereegranskat)abstract
- Radiation exposure of the thorax is associated with a markedly increased risk of cardiac morbidity and mortality with a latency period of decades. Although many studies have confirmed the damaging effect of ionizing radiation on the myocardium and cardiac endothelial structure and function, the molecular mechanism behind this damage is not yet elucidated. Peroxisome proliferator-activated receptor alpha (PPAR alpha), a transcriptional regulator of lipid metabolism in heart tissue, has recently received great attention in the development of cardiovascular disease. The goal of this study was to investigate radiation-induced cardiac damage in general and the role of PPAR alpha in this process in particular. C57BL/6 mice received local heart irradiation with X-ray doses of 8 and 16 gray (Gy) at the age of 8 weeks. The mice were sacrificed 16 weeks later. Radiation-induced changes in the cardiac proteome were quantified using the Isotope Coded Protein Label (ICPL) method followed by mass spectrometry and software analysis. Significant alterations were observed in proteins involved in lipid metabolism and oxidative phosphorylation. Ionizing radiation markedly changed the phosphorylation and ubiquitination status of PPAR alpha. This was reflected as decreased expression of its target genes involved in energy metabolism and mitochondrial respiratory chain confirming the proteomics data. This study suggests that persistent alteration of cardiac metabolism due to impaired PPAR alpha activity contributes to the heart pathology after radiation.
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| 20. |
- Bakshi, Mayur V, et al.
(författare)
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Total body exposure to low-dose ionizing radiation induces long term alterations to the liver proteome of neonatally exposed mice
- 2015
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 14:1, s. 366-373
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Tidskriftsartikel (refereegranskat)abstract
- Tens of thousands of people are being exposed daily toenvironmental low-dose gamma radiation. Epidemiological data indicate thatsuch low radiation doses may negatively affect liver function and result in thedevelopment of liver disease. However, the biological mechanisms behindthese adverse effects are unknown. The aim of this study was to investigateradiation-induced damage in the liver after low radiation doses. Neonatal maleNMRI mice were exposed to total body irradiation on postnatal day 10 usingacute single doses ranging from 0.02 to 1.0 Gy. Early (1 day) and late (7months) changes in the liver proteome were tracked using isotope-codedprotein label technology and quantitative mass spectrometry. Our dataindicate that low and moderate radiation doses induce an immediateinhibition of the glycolysis pathway and pyruvate dehydrogenase availability inthe liver. Furthermore, they lead to significant long-term alterations in lipidmetabolism and increased liver inflammation accompanying inactivation of thetranscription factor peroxisome proliferator-activated receptor alpha. This study contributes to the understanding of the potentialrisk of liver damage in populations environmentally exposed to ionizing radiation.
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| 21. |
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| 22. |
- Bendz, Maria, et al.
(författare)
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Quantification of Membrane Proteins Using Nonspecific Protease Digestions
- 2009
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 8:12, s. 5666-5673
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Tidskriftsartikel (refereegranskat)abstract
- We present a mass spectrometry-based method for the identification and quantification of membrane proteins using the low-specificity protease Proteinase K, at very high pH, to digest proteins isolated by a modified SDS-PAGE protocol. The resulting peptides are modified with a fragmentation-directing isotope labeled tag. We apply the method to quantify differences in membrane protein expression of Bacillus subtilis grown in the presence or absence of glucose.
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| 23. |
- Bengtsson, Sofia, et al.
(författare)
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Large-scale proteomics analysis of human ovarian cancer for biomarkers
- 2007
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 6:4, s. 1440-1450
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Tidskriftsartikel (refereegranskat)abstract
- Ovarian cancer is usually found at a late stage when the prognosis is often bad. Relative survival rates decrease with tumor stage or grade, and the 5-year survival rate for women with carcinoma is only 38%. Thus, there is a great need to find biomarkers that can be used to carry out routine screening, especially in high-risk patient groups. Here, we present a large-scale study of 64 tissue samples taken from patients at all stages and show that we can identify statistically valid markers using nonsupervised methods that distinguish between normal, benign, borderline, and malignant tissue. We have identified 217 of the significantly changing protein spots. We are expressing and raising antibodies to 35 of these. Currently, we have validated 5 of these antibodies for use in immunohistochemical analysis using tissue microarrays of healthy and diseased ovarian, as well as other, human tissues.
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| 24. |
- Bentz, Maria, et al.
(författare)
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Membrane protein identification: N-terminal labeling of nontryptic membrane protein peptides facilitates database searching
- 2008
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 7:2, s. 659-665
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Tidskriftsartikel (refereegranskat)abstract
- Membrane proteins are fairly refractory to digestion especially by trypsin, and less specific proteases, such as elastase and pepsin, are much more effective. However, database searching using nontryptic peptides is much less effective because of the lack of charge localization at the N and C termini and the absence of sequence specificity. We describe a method for N-terminal-specific labeling of peptides from nontryptic digestions of membrane proteins, which facilitates Mascot database searching and can be used for relative quantitation. The conditions for digestion have been optimized to obtain peptides of a suitable length for mass spectrometry (MS) fragmentation. We show the effectiveness of the method using a plasma membrane preparation from a leukemia cell line and demonstrate a large increase in the number of membrane proteins, with small extra-membranar domains being identified in comparison to previous published methods.
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| 25. |
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| 26. |
- Berggård, Tord, et al.
(författare)
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140 mouse brain proteins identified by Ca2+-calmodulin affinity chromatography and tandem mass spectrometry.
- 2006
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 5:3, s. 669-687
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Tidskriftsartikel (refereegranskat)abstract
- Calmodulin is an essential Ca2+-binding protein that binds to a variety of targets that carry out critical signaling functions. We describe the proteomic characterization of mouse brain Ca2+-calmodulin-binding proteins that were purified using calmodulin affinity chromatography. Proteins in the eluates from four different affinity chromatography experiments were identified by 1-DE and in-gel digestion followed by LC-MS/MS. Parallel experiments were performed using two related control-proteins belonging to the EF-hand family. After comparing the results from the different experiments, we were able to exclude a significant number of proteins suspected to bind in a nonspecific manner. A total of 140 putative Ca2+-calmodulin-binding proteins were identified of which 87 proteins contained calmodulin-binding motifs. Among the 87 proteins that contained calmodulin-binding motifs, 48 proteins have not previously been shown to interact with calmodulin and 39 proteins were known calmodulin-binding proteins. Many proteins with ill-defined functions were identified as well as a number of proteins that at the time of the analysis were described only as ORFs. This study provides a functional framework for studies on these previously uncharacterized proteins.
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| 27. |
- Bergström Lind, Sara, et al.
(författare)
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Immunoaffinity Enrichments Followed by Mass Spectrometric Detection for Studying Global Protein Tyrosine Phosphorylation
- 2008
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 7:7, s. 2897-2910
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Tidskriftsartikel (refereegranskat)abstract
- Phosphorylation of protein tyrosine residues regulates important cell functions and is, when dysregulated, often crucially involved in oncogenesis. It is therefore important to develop and evaluate methods for identifying and studying tyrosine phosphorylated (P-Tyr) proteins. P-Tyr proteins are present at very low concentrations within cells, requiring highly selective enrichment methods to be detected. In this study, we applied immunoaffinity as enrichment step for P-Tyr proteins. Five selected anti-phosphotyrosine antibodies (monoclonal antibodies 4G10, PY100, PYKD1, 13F9 and one polyclonal antiserum) were evaluated with respect to their capability to enrich P-Tyr proteins from cell extracts of the K562 leukemia cell line. The enrichment resulted in the detection of a group of proteins that potentially were tyrosine-phosphorylated (putative P-Tyr proteins). High accuracy identification of actual P-Tyr sites were performed using a highly selective and sensitive liquid chromatography Fourier transform mass spectrometer (LC-FTMS) setup with complementary collision activated dissociation (CAD) and electron capture dissociation (ECD) fragmentations. 4G10 and PY100 antibodies recognized the greatest number of putative P-Tyr proteins in initial screening experiments and were therefore further evaluated and compared in immunoaffinity enrichment of both P-Tyr proteins and peptides. Using the 4G10 antibody for enrichment of proteins, we identified 459 putative P-Tyr proteins by MS. Out of these proteins, 12 were directly verified as P-Tyr proteins by MS analysis of the actual site. Using the PY100 antibody for enrichment of peptides, we detected 67 P-Tyr peptides (sites) and 89 putative P-Tyr proteins. Generally, enrichment at the peptide level made it difficult to reliably determine the identity of the proteins. In contrast, protein identification following immunoaffinity enrichment at the protein level gave greater sequence coverage and thus a higher confidence in the protein identification. By combining all available information, 40 proteins were identified as true P-Tyr proteins from the K562 cell line. In conclusion, this study showed that a combination of immunoaffinity enrichment using multiple antibodies of both intact and digested proteins in parallel experiments is required for best possible coverage of all possible P-Tyr proteins in a sample.
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| 28. |
- Betancourt, Lazaro, et al.
(författare)
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Quantitative Assessment of Urea In-Solution Lys-C/Trypsin Digestions Reveals Superior Performance at Room Temperature over Traditional Proteolysis at 37 °C.
- 2018
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 17:7, s. 2556-2561
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Tidskriftsartikel (refereegranskat)abstract
- Urea-containing buffer solutions are generally used in proteomic studies to aid protein denaturation and solubilization during cell and tissue lysis. It is well-known, however, that urea can lead to carbamylation of peptides and proteins and, subsequently, incomplete digestion of proteins. By the use of cells and tissues that had been lysed with urea, different solution digestion strategies were quantitatively assessed. In comparison with traditional proteolysis at 37 °C, urea in-solution digestion performed at room temperature improved peptide and protein identification and quantitation and had a minimum impact on miscleavage rates. Furthermore, the signal intensities and the number of carbamylated and pyroglutamic acid-modified peptides decreased. Overall, this led to a reduction in the negative effects often observed for such modifications. Data are available via ProteomeXchange with identifier PXD009426.
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| 29. |
- Bhattacharjee, Payel, 1984, et al.
(författare)
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Mass Spectrometric Analysis of Lewy Body-Enriched alpha-Synuclein in Parkinson's Disease
- 2019
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 18:5, s. 2109-2120
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Tidskriftsartikel (refereegranskat)abstract
- Parkinson's disease (PD) is characterized by intraneuronal inclusions of aggregated alpha-synuclein protein (so-called Lewy bodies) in distinct brain regions. Multiple posttranslational modifications may affect the structure and function of alpha-synuclein. Mass spectrometry-based analysis may be useful for the characterization and quantitation of alpha-synuclein forms, but has proven challenging, mainly due to the insolubility of Lewy bodies in aqueous buffer. In the present study, we developed a novel method by combining differential solubilization with immunoprecipitation and targeted proteomics using liquid chromatography and tandem mass spectrometry. Brain tissue homogenization and sample preparation were modified to facilitate analysis of soluble, detergent-soluble, and detergent-insoluble protein fractions (Lewy body-enriched). The method was used to compare alpha-synuclein forms from cingulate cortex (affected) and occipital cortex (unaffected) in two study sets of PD patients and controls. We identified similar to 20 modified alpha-synuclein variants, including species with N-terminal acetylation and C-terminal truncations at amino acids 103 and 119. The levels of alpha-synuclein forms Ac-alpha-syn(1-6), alpha-syn(13-21), alpha-syn(35-43), alpha-syn(46-58), alpha-syn(61-80), and alpha-syn(81-96) except alpha-syn(103-119) were significantly increased in PD cingulate region compared to controls in the Lewy body-enriched alpha-synuclein fraction. In the soluble fraction, only Ac-alpha-syn(1-6) was significantly increased in PD compared to controls. None of the detected alpha-synuclein variants were Lewy body-specific, but acetylated forms should be examined further as potential biomarkers for abnormal alpha-synuclein accumulation.
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| 30. |
- Bichmann, Leon, et al.
(författare)
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DIAproteomics : A Multifunctional Data Analysis Pipeline for Data-Independent Acquisition Proteomics and Peptidomics
- 2021
-
Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 20:7, s. 3758-3766
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Tidskriftsartikel (refereegranskat)abstract
- Data-independent acquisition (DIA) is becoming a leading analysis method in biomedical mass spectrometry. The main advantages include greater reproducibility and sensitivity and a greater dynamic range compared with data-dependent acquisition (DDA). However, the data analysis is complex and often requires expert knowledge when dealing with large-scale data sets. Here we present DIAproteomics, a multifunctional, automated, high-throughput pipeline implemented in the Nextflow workflow management system that allows one to easily process proteomics and peptidomics DIA data sets on diverse compute infrastructures. The central components are well-established tools such as the OpenSwathWorkflow for the DIA spectral library search and PyProphet for the false discovery rate assessment. In addition, it provides options to generate spectral libraries from existing DDA data and to carry out the retention time and chromatogram alignment. The output includes annotated tables and diagnostic visualizations from the statistical postprocessing and computation of fold-changes across pairwise conditions, predefined in an experimental design. DIAproteomics is well documented open-source software and is available under a permissive license to the scientific community at https://www. openms.de/diaprotcoinics/.
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| 31. |
- Bock, Thomas, et al.
(författare)
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Proteomic Analysis Reveals Drug Accessible Cell Surface N-Glycoproteins of Primary and Established Glioblastoma Cell Lines
- 2012
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 11:10, s. 4885-4893
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Tidskriftsartikel (refereegranskat)abstract
- Glioblastoma is the most common primary Glioblastoma Cell Surface Capturing brain tumor in adults with low average survival time after diagnosis. In order to improve glioblastoma treatment, new drug-accessible targets need to be identified. Cell surface glycoproteins are prime drug targets due to their accessibility at the surface of cancer cells. To overcome the limited availability of suitable antibodies for cell surface protein detection, we performed a comprehensive mass spectrometric investigation of the glioblastoma surfaceome. Our combined cell surface capturing analysis of primary ex vivo glioblastoma cell lines in combination with established glioblastoma cell lines revealed 633 N-glycoproteins, which vastly extends the known data of surfaceome drug targets at subcellular resolution. We provide direct evidence of common glioblastoma cell surface glycoproteins and an approximate estimate of their abundances, information that could not be derived from genomic and/or transcriptomic glioblastoma studies. Apart from our pharmaceutically valuable repertoire of already and potentially drug-accessible cell surface glycoproteins, we built a mass-spectrometry-based toolbox enabling directed, sensitive, and repetitive glycoprotein measurements for clinical follow-up studies. The included Skyline Glioblastoma SRM assay library provides an elevated starting point for parallel testing of the abundance level of the detected glioblastoma surfaceome members in future drug perturbation experiments.
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| 32. |
- Boersema, P. J., et al.
(författare)
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Biology/Disease-Driven Initiative on Protein-Aggregation Diseases of the Human Proteome Project: Goals and Progress to Date
- 2018
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 17:12, s. 4072-4084
-
Tidskriftsartikel (refereegranskat)abstract
- The Biology/Disease-driven (B/D) working groups of the Human Proteome Project are alliances of research groups aimed at developing or improving proteomic tools to support specific biological or disease-related research areas. Here, we describe the activities and progress to date of the B/D working group focused on protein aggregation diseases (PADs). PADs are characterized by the intra- or extracellular accumulation of aggregated proteins and include devastating diseases such as Parkinson's and Alzheimer's disease and systemic amyloidosis. The PAD B/D working group aims for the development of proteomic assays for the quantification of aggregation-prone proteins involved in PADs to support basic and clinical research on PADs. Because the proteins in PADs undergo aberrant conformational changes, a goal is to quantitatively resolve altered protein structures and aggregation states in complex biological specimens. We have developed protein-extraction protocols and a set of mass spectrometric (MS) methods that enable the detection and quantification of proteins involved in the systemic and localized amyloidosis and the probing of aberrant protein conformational transitions in cell and tissue extracts. In several studies, we have demonstrated the potential of MS-based proteomics approaches for specific and sensitive clinical diagnoses and for the subtyping of PADs. The developed methods have been detailed in both protocol papers and manuscripts describing applications to facilitate implementation by nonspecialized laboratories, and assay coordinates are shared through public repositories and databases. Clinicians actively involved in the PAD working group support the transfer to clinical practice of the developed methods, such as assays to quantify specific disease related proteins and their fragments in biofluids and multiplexed MS-based methods for the diagnosis and typing of systemic amyloidosis. We believe that the increasing availability of tools to precisely measure proteins involved in PADs will positively impact research on the molecular bases of these diseases and support early disease diagnosis and a more-confident subtyping.
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| 33. |
- Boja, Emily S., et al.
(författare)
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Analytical Validation Considerations of Multiplex Mass-Spectrometry-Based Proteomic Platforms for Measuring Protein Biomarkers
- 2014
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 13:12, s. 5325-5332
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Protein biomarker discovery and validation in current omics era are vital for healthcare professionals to improve diagnosis, detect cancers at an early stage, identify the likelihood of cancer recurrence, stratify stages with differential survival outcomes, and monitor therapeutic responses. The success of such biomarkers would have a huge impact on how we improve the diagnosis and treatment of patients and alleviate the financial burden of healthcare systems. In the past, the genomics community (mostly through large-scale, deep genomic sequencing technologies) has been steadily improving our understanding of the molecular basis of disease, with a number of biomarker panels already authorized by the U.S. Food and Drug Administration (FDA) for clinical use (e.g., MammaPrint, two recently cleared devices using next-generation sequencing platforms to detect DNA changes in the cystic fibrosis transmembrane conductance regulator (CFTR) gene). Clinical proteomics, on the other hand, albeit its ability to delineate the functional units of a cell, more likely driving the phenotypic differences of a disease (i.e., proteins and protein-protein interaction networks and signaling pathways underlying the disease), staggers to make a significant impact with only an average similar to 1.5 protein biomarkers per year approved by the FDA over the past 15-20 years. This statistic itself raises the concern that major roadblocks have been impeding an efficient transition of protein marker candidates in biomarker development despite major technological advances in proteomics in recent years.
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| 34. |
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| 35. |
- Bostanci, Nagihan, et al.
(författare)
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Label-free quantitative proteomics reveals differentially regulated proteins in experimental gingivitis
- 2013
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 2:12, s. 657-678
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Tidskriftsartikel (refereegranskat)abstract
- We investigated the sequential protein expression in gingival crevicular fluid samples during the induction (I) and resolution (R) of experimental gingivitis. Periodontally and systemically healthy volunteers (n = 20) participated in a three-week experimental gingivitis protocol, followed by debridement and two weeks of regular plaque control. Gingival crevicular fluid (GCF) samples were collected at baseline, Day 7, 14, and 21 (induction; I-phase), and at Day 21, 25, 30, and 35 (resolution; R-phase). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for label-free quantitative proteomics was applied. A total of 287 proteins were identified including 254 human, 14 bacterial, 12 fungal, and 7 yeast proteins. Ontology analysis revealed proteins primarily involved in cytoskeletal rearrangements, immune response, antimicrobial function, protein degradation, and DNA binding. There was considerable variation in the number of proteins identified, both among subjects and within subjects across time points. After pooling of samples between subjects at each time point, the levels of 59 proteins in the I-phase and 73 proteins in the R-phase were quantified longitudinally. Our data demonstrate that LC-MS/MS label-free quantitative proteomics is valuable in the assessment of the protein content of the GCF and can facilitate a better understanding of the molecular mechanisms involved in the induction and resolution of plaque-induced gingival inflammation in humans.
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| 36. |
- Boström, Tove, et al.
(författare)
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Investigating the Applicability of Antibodies Generated within the Human Protein Atlas as Capture Agents in Immunoenrichment Coupled to Mass Spectrometry
- 2014
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 13:10, s. 4424-4435
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Tidskriftsartikel (refereegranskat)abstract
- For identification and characterization of proteins in complex samples, immunoenrichment coupled to mass spectrometry is a good alternative due to the sensitivity of the affinity enrichment and the specificity of mass spectrometry analysis. Antibodies are commonly used affinity agents; however, for high-throughput analysis, antibody availability is usually a bottleneck. Here we present a protocol for immunoenrichment coupled to mass spectrometry in a high-throughput setup, where all steps from bead coupling to mass spectrometry sample preparation are performed in parallel in a 96-well format. Antibodies generated within the Human Protein Atlas project were tested for applicability as capture agents. The antibodies were covalently attached to protein A beads, making it possible to reuse the coupled beads at least three times without destroying the antibody binding efficiency. Target proteins were captured from a U251 MG cell lysate, eluted, digested, and analyzed using mass spectrometry. Of 30 investigated antibodies, around 50% could successfully capture the corresponding native target protein, making the available library of more than 21 000 antibodies a valuable resource for immunoenrichment assays. Due to the diversity of different antibodies regarding affinity and specificity, analyzing antibodies in a high-throughput format is challenging. Even though protocol optimization for individual antibodies can be advantageous for future studies, our method enables a fast screening strategy to determine the usefulness of antibodies in immunoenrichment setups. In addition, we show that the specificity of the antibodies can be investigated by using label-free quantification.
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| 37. |
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| 38. |
- Brockmöller, Scarlet F., et al.
(författare)
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Integration of metabolomics and expression of glycerol-3-phosphate acyltransferase (GPAM) in breast cancer-link to patient survival, hormone receptor status, and metabolic profiling
- 2012
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 11:2, s. 850-60
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Tidskriftsartikel (refereegranskat)abstract
- Changes in lipid metabolism are an important but not well-characterized hallmark of cancer. On the basis of our recent findings of lipidomic changes in breast cancer, we investigated glycerol-3-phosphate acyltransferase (GPAM), a key enzyme in the lipid biosynthesis of triacylglycerols and phospholipids. GPAM protein expression was evaluated and linked to metabolomic and lipidomic profiles in a cohort of human breast carcinomas. In addition, GPAM mRNA expression was analyzed using the GeneSapiens in silico transcriptiomics database. High cytoplasmic GPAM expression was associated with hormone receptor negative status (p = 0.013). On the protein (p = 0.048) and mRNA (p = 0.001) levels, increased GPAM expression was associated with a better overall survival. Metabolomic analysis by GC-MS showed that sn-glycerol-3-phosphate, the substrate of GPAM, was elevated in breast cancer compared to normal breast tissue. LC-MS based lipidomic analysis identified significantly higher levels of phospholipids, especially phosphatidylcholines in GPAM protein positive tumors. In conclusion, our results suggest that GPAM is expressed in human breast cancer with associated changes in the cellular metabolism, in particular an increased synthesis of phospholipids, the major structural component of cellular membranes.
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| 39. |
- Burg, Dominic, et al.
(författare)
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Large-Scale Label-Free Quantitative Mapping of the Sputum Proteome
- 2018
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 17:6, s. 2072-2091
-
Tidskriftsartikel (refereegranskat)abstract
- Analysis of induced sputum supematant is a minimally invasive approach to study the epithelial lining fluid and, thereby, provide insight into normal lung biology and the pathobiology of lung diseases. We present here a novel proteomics approach to sputum analysis developed within the U-BIOPRED (unbiased biomarkers predictive of respiratory disease outcomes) international project. We present practical and analytical techniques to optimize the detection of robust biomarkers in proteomic studies. The normal sputum proteome was derived using data-independent HDMSE applied to 40 healthy nonsmoking participants, which provides an essential baseline from which to compare modulation of protein expression in respiratory diseases. The "core" sputum proteome (proteins detected in >= 40% of participants) was composed of 284 proteins, and the extended proteome (proteins detected in >= 3 participants) contained 1666 proteins. Quality control procedures were developed to optimize the accuracy and consistency of measurement of sputum proteins and analyze the distribution of sputum proteins in the healthy population. The analysis showed that quantitation of proteins by HDMSE is influenced by several factors, with some proteins being measured in all participants' samples and with low measurement variance between samples from the same patient. The measurement of some proteins is highly variable between repeat analyses, susceptible to sample processing effects, or difficult to accurately quantify by mass spectrometry. Other proteins show high interindividual variance. We also highlight that the sputum proteome of healthy individuals is related to sputum neutrophil levels, but not gender or allergic sensitization. We illustrate the importance of design and interpretation of disease biomarker studies considering such protein population and technical measurement variance.
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| 40. |
- Bylesjö, Max, et al.
(författare)
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Integrated analysis of transcript, protein and metabolite data to study lignin biosynthesis in hybrid aspen
- 2009
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 8:1, s. 199-210
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Tidskriftsartikel (refereegranskat)abstract
- Tree biotechnology will soon reach a mature state where it will influence the overall supply of fiber, energy and wood products. We are now ready to make the transition from identifying candidate genes, controlling important biological processes, to discovering the detailed molecular function of these genes on a broader, more holistic, systems biology level. In this paper, a strategy is outlined for informative data generation and integrated modeling of systematic changes in transcript, protein and metabolite profiles measured from hybrid aspen samples. The aim is to study characteristics of common changes in relation to genotype-specific perturbations affecting the lignin biosynthesis and growth. We show that a considerable part of the systematic effects in the system can be tracked across all platforms and that the approach has a high potential value in functional characterization of candidate genes.
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| 41. |
- Byström, Sanna, et al.
(författare)
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Affinity Proteomic Profiling of Plasma, Cerebrospinal Fluid, and Brain Tissue within Multiple Sclerosis
- 2014
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 13:11, s. 4607-4619
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Tidskriftsartikel (refereegranskat)abstract
- The brain is a vital organ and because it is well shielded from the outside environment, possibilities for noninvasive analysis are often limited. Instead, fluids taken from the spinal cord or circulatory system are preferred sources for the discovery of candidate markers within neurological diseases. In the context of multiple sclerosis (MS), we applied an affinity proteomic strategy and screened 22 plasma samples with 4595 antibodies (3450 genes) on bead arrays, then defined 375 antibodies (334 genes) for targeted analysis in a set of 172 samples and finally used 101 antibodies (43 genes) on 443 plasma as well as 573 cerebrospinal spinal fluid (CSF) samples. This revealed alteration of protein profiles in relation to MS subtypes for IRF8, IL7, METTL14, SLC30A7, and GAP43. Respective antibodies were subsequently used for immunofluorescence on human post-mortem brain tissue with MS pathology for expression and association analysis. There, antibodies for IRF8, IL7, and METTL14 stained neurons in proximity of lesions, which highlighted these candidate protein targets for further studies within MS and brain tissue. The affinity proteomic translation of profiles discovered by profiling human body fluids and tissue provides a powerful strategy to suggest additional candidates to studies of neurological disorders.
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| 42. |
- Bäck, Peter, et al.
(författare)
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Automated PreScan function for scanning fluorescently stained 2D-PAGE gels
- 2005
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 4:5, s. 1511-1515
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Tidskriftsartikel (refereegranskat)abstract
- To automate the acquisition of images from fluorescently stained gels, the power of the excitation laser(s) must be optimized for each sample to prevent spot saturation (or to allow unimportant spots to saturate) yet still retaining sensitivity. In this work, we describe the implementation and effectiveness of a pre-scan function in a robotic solution for the automation of 2D gel scanning.
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| 43. |
- Bäck, Peter, et al.
(författare)
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Automating gel image acquisition
- 2003
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 2:6, s. 662-664
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Tidskriftsartikel (refereegranskat)abstract
- We describe the design and implementation of a robotic solution to automate the acquisition of gel images. The soft- and hardware aspects are outlined together with the various safety aspects that need to be addressed.
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| 44. |
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| 45. |
- Cabras, T., et al.
(författare)
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Marked Differences in the Submandibular Salivary Proteome between Sardinian Alcohol-Preferring and Sardinian Alcohol-Non Preferring Rats Revealed by an Integrated Top-Down-Bottom-Up Proteomic Platform
- 2018
-
Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 17:1, s. 455-469
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Tidskriftsartikel (refereegranskat)abstract
- Sardinian alcohol-preferring (sP) and Sardinian alcohol-non preferring (sNP) rats have been selectively bred for opposite alcohol preference and consumption. Aiming to verify possible differences at the proteomics level between sP and sNP rats, we investigated the salivary proteome by a a liquid chromatography-mass spectrometry top-down-bottom-up integrated approach. For this purpose, submandibular saliva was collected from alcohol-naive sP and sNP rats under isoprenaline stimulation. A total of 200 peptides and proteins were detected and quantified in the two rat lines, 149 of which were characterized in their naturally occurring structure. The data are available via ProteomeXchange with identifier PXD006997. Surprisingly, sP rats exhibited marked quantitative and qualitative differences with respect to sNP rats, namely higher levels of proteoforms originating from submandibular gland protein C, and from submandibular rat protein 2, as well as those of several unidentified peptides and proteins. sP rats expressed some proteins not detectable in sNP rats such as the glutamine and glutamic acid-rich protein (GRP)-CB. The isoform GRP-B, detectable in both rat lines, was more abundant in sNP rats. The submandibular saliva of sNP rats was also characterized by very high levels of GRP-B proteolytic peptides and rat salivary protein 1. Whether these differences could contribute to the opposite alcohol preference and consumption of sP and sNP rats is currently unknown and requires further investigation. © 2017 American Chemical Society.
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| 46. |
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| 47. |
- Carapito, Christine, et al.
(författare)
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Validating Missing Proteins in Human Sperm Cells by Targeted Mass-Spectrometry- and Antibody-based Methods
- 2017
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 16:12, s. 4340-4351
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Tidskriftsartikel (refereegranskat)abstract
- The present study is a contribution to the "neXt50 challenge", a coordinated effort across C-HPP teams to identify the SO most tractable missing proteins (MPs) on each chromosome. We report the targeted search of 38 theoretically detectable MPs from chromosomes 2 and 14 in Triton X-100 soluble and insoluble sperm fractions from a total of 15 healthy donors. A targeted mass spectrometry-based strategy consisting of the development of LC-PRM assays (with heavy labeled synthetic peptides) targeting 92 proteotypic peptides of the 38 selected MPs was used. Out of the 38 selected MPs, 12 were identified with two or more peptides and 3 with one peptide after extensive SDS-PAGE fractionation of the two samples and with overall low-intensity signals. The PRM data are available via ProteomeXchange in PASSEL (PASS01013). Further validation by immunohistochemistry on human testes sections and cytochemistry on sperm smears was performed for eight MPs with antibodies available from the Human Protein Atlas. Deep analysis of human sperm still allows the validation of MPs and therefore contributes to the C-HPP worldwide effort. We anticipate that our results will be of interest to the reproductive biology community because an in-depth analysis of these MPs may identify potential new candidates in the context of human idiopathic infertilities.
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| 48. |
- Carayol, Marion, et al.
(författare)
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Blood Metabolic Signatures of Body Mass Index : A Targeted Metabolomics Study in the EPIC Cohort
- 2017
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 16:9, s. 3137-3146
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Tidskriftsartikel (refereegranskat)abstract
- Metabolomics is now widely used to characterize metabolic phenotypes associated with lifestyle risk factors such as obesity. The objective of the present study was to explore the associations of body mass index (BMI) with 145 metabolites measured in blood samples in the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Metabolites were measured in blood from 392 men from the Oxford (UK) cohort (EPIC-Oxford) and in 327 control subjects who were part of a nested case-control study on hepatobiliary carcinomas (EPIC-Hepatobiliary). Measured metabolites included amino acids, acylcarnitines, hexoses, biogenic amines, phosphatidylcholines, and sphingomyelins. Linear regression models controlled for potential confounders and multiple testing were run to evaluate the associations of metabolite concentrations with BMI. 40 and 45 individual metabolites showed significant differences according to BMI variations, in the EPIC-Oxford and EPIC-Hepatobiliary subcohorts, respectively. Twenty two individual metabolites (kynurenine, one sphingomyelin, glutamate and 19 phosphatidylcholines) were associated with BMI in both subcohorts. The present findings provide additional knowledge on blood metabolic signatures of BMI in European adults, which may help identify mechanisms mediating the relationship of BMI with obesity-related diseases.
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| 49. |
- Carlsohn, Elisabet, et al.
(författare)
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Characterization of the outer membrane protein profile from disease-related Helicobacter pylori isolates by subcellular fractionation and nano-LC FT-ICR MS analysis
- 2006
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Ingår i: J Proteome Res. - : American Chemical Society (ACS). - 1535-3893. ; 5:11, s. 3197-204
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Because of the important role of membrane proteins in adhesion, invasion, and intracellular survival of pathogens in the host, membrane proteins are of potential interest in the search for drug targets or biomarkers. We have established a mass spectrometry-based method that allows characterization of the outer membrane protein (OMP) profile of clinical isolates from of the human gastric pathogen Helicobacter pylori. Subcellular fractionation and one-dimensional gel electrophoresis (1D-GE) analysis was combined with nano-liquid chromatography Fourier transform-ion cyclotron resonance mass spectrometry (nano-LC FT-ICR MS) and tandem mass spectrometry (MS/MS) analysis of fifteen H. pylori strains associated either with duodenal ulcers, gastric cancer, or isolated from asymptomatic H. pylori infected carriers. Over 60 unique membrane or membrane-associated proteins, including 30 of the 33 theoretically predicted OMPs, were identified from the strains. Several membrane proteins, including Omp11 and BabA, were found to be expressed by all strains. In the search for clinical markers we found that Omp26 was expressed by all disease-related strains but was only present in one out of five strains from asymptomatic carriers, which makes Omp26 a potential target for further investigation in the search for proteins unique to disease-related H. pylori strains. In addition, presence of Omp30 and absence of Omp6 seemed to be associated with H. pylori strains causing duodenal ulcer.
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| 50. |
- Cesnik, Anthony J., et al.
(författare)
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Spritz : A Proteogenomic Database Engine
- 2021
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 20:4, s. 1826-1834
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Tidskriftsartikel (refereegranskat)abstract
- Proteoforms are the workhorses of the cell, and subtle differences between their amino acid sequences or post-translational modifications (PTMs) can change their biological function. To most effectively identify and quantify proteoforms in genetically diverse samples by mass spectrometry (MS), it is advantageous to search the MS data against a sample-specific protein database that is tailored to the sample being analyzed, in that it contains the correct amino acid sequences and relevant PTMs for that sample. To this end, we have developed Spritz (https://smith-chem-wisc.github.io/Spritz/), an open-source software tool for generating protein databases annotated with sequence variations and PTMs. We provide a simple graphical user interface for Windows and scripts that can be run on any operating system. Spritz automatically sets up and executes approximately 20 tools, which enable the construction of a proteogenomic database from only raw RNA sequencing data. Sequence variations that are discovered in RNA sequencing data upon comparison to the Ensembl reference genome are annotated on proteins in these databases, and PTM annotations are transferred from UniProt. Modifications can also be discovered and added to the database using bottom-up mass spectrometry data and global PTM discovery in MetaMorpheus. We demonstrate that such sample-specific databases allow the identification of variant peptides, modified variant peptides, and variant proteoforms by searching bottom-up and top-down proteomic data from the Jurkat human T lymphocyte cell line and demonstrate the identification of phosphorylated variant sites with phosphoproteomic data from the U2OS human osteosarcoma cell line.
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