LC−MS Dotaz Zobrazit nápovědu
The success of bottom-up proteomic analysis frequently depends on the efficient removal of contaminants from protein or peptide samples before LC-MS/MS. For a peptide clean-up workflow, single-pot solid-phase-enhanced peptide sample preparation on carboxylate-modified paramagnetic beads (termed SP2) was evaluated for sodium dodecyl sulfate or polyethylene glycol removal from Arabidopsis thaliana tryptic peptides. The robust and efficient 40-min SP2 protocol, tested for 10-ng, 250-ng, and 10-µg peptide samples, was proposed and benchmarked thoroughly against the ethyl acetate extraction protocol. The SP2 protocol on carboxylated magnetic beads proved to be the most robust approach, even for the simultaneous removal of massive sodium dodecyl sulfate (SDS) and polyethylene glycol (PEG) contaminations from AT peptide samples in respect of the LC-MS/MS data outperforming ethyl acetate extraction.
- Klíčová slova
- Arabidopsis thaliana, LC-MS/MS, SP2, detergent, ethyl acetate extraction, magnetic beads, peptide clean-up, polyethylene glycol, sodium dodecyl sulfate,
- MeSH
- benchmarking MeSH
- chromatografie kapalinová metody MeSH
- dodecylsíran sodný MeSH
- kapalinová chromatografie-hmotnostní spektrometrie * MeSH
- peptidy analýza MeSH
- polyethylenglykoly * MeSH
- proteomika metody MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- dodecylsíran sodný MeSH
- ethyl acetate MeSH Prohlížeč
- peptidy MeSH
- polyethylenglykoly * MeSH
Saxitoxins (STXs) are potent neurotoxins produced by marine dinoflagellates or freshwater cyanobacteria known to cause acute and eventually fatal human intoxications, which are classified as paralytic shellfish poisonings (PSPs). Rapid analysis of STXs in blood plasma can be used for a timely diagnosis and confirmation of PSPs. We developed a fast and simple method of STX extraction based on plasma sample acidification and precipitation by acetonitrile, followed by quantification using liquid chromatography-tandem mass spectrometry (LC-MS-MS). Our approach provides the results ≤30 min, with a limit of detection of 2.8 ng/mL and a lower limit of quantification of 5.0 ng/mL. Within-run and between-run precision experiments showed good reproducibility with ≤15% values. Standard curves for calibration were linear with correlation coefficients ≥0.98 across the assay calibration range (5-200 ng/mL). In an interlaboratory analytical exercise, the method was found to be 100% accurate in determining the presence or absence of STX in human plasma specimens, with recovery values of 86-99%. This simple method for STX determination in animal or human plasma can quickly and reliably diagnose STX exposures and confirm suspected PSP cases to facilitate patient treatment or expedite necessary public health or security actions.
- MeSH
- chromatografie kapalinová MeSH
- kapalinová chromatografie-hmotnostní spektrometrie * MeSH
- krevní plazma MeSH
- lidé MeSH
- reprodukovatelnost výsledků MeSH
- saxitoxin * MeSH
- tandemová hmotnostní spektrometrie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- saxitoxin * MeSH
Microflow liquid chromatography interfaced with mass spectrometry (μLC-MS/MS) is increasingly applied for high-throughput profiling of biological samples and has been proven to have an acceptable trade-off between sensitivity and reproducibility. However, lipidomics applications are scarce. We optimized a μLC-MS/MS system utilizing a 1 mm inner diameter × 100 mm column coupled to a triple quadrupole mass spectrometer to establish a sensitive, high-throughput, and robust single-shot lipidomics workflow. Compared to conventional lipidomics methods, we achieve a ∼4-fold increase in response, facilitating quantification of 351 lipid species from a single iPSC-derived cerebral organoid during a 15 min LC-MS analysis. Consecutively, we injected 303 samples over ∼75 h to prove the robustness and reproducibility of the microflow separation. As a proof of concept, μLC-MS/MS analysis of Alzheimer's disease patient-derived iPSC cerebral organoid reveals differential lipid metabolism depending on APOE phenotype (E3/3 vs E4/4). Microflow separation proves to be an environmentally friendly and cost-effective method as it reduces the consumption of harmful solvents. Also, the data demonstrate robust, in-depth, high-throughput performance to enable routine clinical or biomedical applications.
- MeSH
- apolipoproteiny E MeSH
- chromatografie kapalinová metody MeSH
- fenotyp MeSH
- kapalinová chromatografie-hmotnostní spektrometrie * MeSH
- lidé MeSH
- lipidomika MeSH
- reprodukovatelnost výsledků MeSH
- tandemová hmotnostní spektrometrie * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- apolipoproteiny E MeSH
BACKGROUND: In vivo solid-phase microextraction (SPME) is a minimally invasive, non-exhaustive sample-preparation technique that facilitates the direct isolation of low molecular weight compounds from biological matrices in living systems. This technique is especially useful for the analysis of phytocannabinoids (PCs) in plant material, both for forensic purposes and for monitoring the PC content in growing Cannabis spp. plants. In contrast to traditional extraction techniques, in vivo SPME enables continuous tracking of the changes in the level of PCs during plant growth without the need for plant material collection. In this study, in vivo SPME utilizing biocompatible C18 probes and liquid-chromatography coupled to quadrupole time-of flight mass spectrometry (LC-Q-TOF-MS) is proposed as a novel strategy for the extraction and analysis of the acidic forms of five PCs in growing medicinal cannabis plants. RESULTS: The SPME method was optimized by testing various parameters, including the extraction phase (coating), extraction and desorption times, and the extraction temperature. The proposed method was validated with satisfactory analytical performance regarding linearity (10-3000 ng/mL), limits of quantification, and precision (relative standard deviations below 5.5 %). The proposed method was then successfully applied for the isolation of five acidic forms of PCs, which are main components of growing medicinal cannabis plants. As a proof-of-concept, SPME probes were statically inserted into the inflorescences of two varieties of Cannabis spp. plants (i.e., CBD-dominant and Δ9-THC-dominant) cultivated under controlled conditions for 30 min extraction of tetrahydrocannabinolic acid (Δ9-THCA), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabiviarinic acid (CBVA), and tetrahydrocannabivarinic acid (THCVA). SIGNIFICANCE AND NOVELTY: The results confirmed that the developed SPME-LC-Q-TOF-MS method is a precise and efficient tool that enables direct and rapid isolation and analysis of PCs under in vivo conditions. The proposed methodology is highly appealing option for monitoring the metabolic pathways and compositions of multiple PCs in medicinal cannabis at different stages of plant growth.
- Klíčová slova
- Biocompatible probes, Cannabis spp., In vivo sampling, Phytocannabinoids, Solid-phase microextraction,
- MeSH
- Cannabis * chemie MeSH
- kanabinoidy * analýza MeSH
- kapalinová chromatografie-hmotnostní spektrometrie * metody MeSH
- mikroextrakce na pevné fázi * metody MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- kanabinoidy * MeSH
Organoids are 3D cell cultures with microanatomies mimicking aspects of real organs, useful for e.g. animal-free studies of development, disease, and drug discovery. The cell medium of organoid models of Langerhans islets, regulating blood glucose levels by insulin secretion, can be analyzed by liquid chromatography-mass spectrometry (LC-MS). However, organoid medium complexity is a major challenge, as matrix interferences can reduce sensitivity and selectivity, even with optimized LC-MS conditions. By applying preparative agarose gel electrophoresis-electrodialysis (PGE-ED), we were able to decrease the cell medium background signal, allowing for reduced interferences affecting LC-MS analysis of human insulin.
- Klíčová slova
- Insulin, Liquid chromatography-mass spectrometry, Preparative gel electrophoresis, Stem cell-derived islet organoids,
- MeSH
- chromatografie kapalinová MeSH
- elektroforéza v agarovém gelu MeSH
- inzulin * MeSH
- kapalinová chromatografie-hmotnostní spektrometrie * MeSH
- lidé MeSH
- organoidy MeSH
- tandemová hmotnostní spektrometrie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- inzulin * MeSH
We have developed and validated a novel LC-MS/MS method for simultaneously analyzing amino acids, biogenic amines, and their acetylated and methylated derivatives in plants. This method involves a one-step extraction of 2-5 mg of lyophilized plant material followed by fractionation of different biogenic amine forms and exploits an efficient combination of hydrophilic interaction chromatography (HILIC), reversed phase (RP) chromatography with pre-column derivatization, and tandem mass spectrometry. This approach enables high-throughput processing of plant samples, significantly reducing the time needed for analysis and its cost. We also present a new synthetic route for deuterium-labelled polyamines. The LC-MS/MS method was rigorously validated by quantifying levels of nitrogen-related metabolites in seedlings of seven plant species including Arabidopsis, maize, and barley, all of which are commonly used model organisms in plant science research. Our results revealed substantial variations in the abundance of these metabolites between species, developmental stages, and growth conditions, particularly for the acetylated and methylated derivatives and the various polyamine fractions. However, the biological relevance of these plant metabolites is currently unclear. Overall, this work contributes significantly to the field of plant science by providing a powerful analytical tool and setting the stage for future investigations into the functions of these nitrogen-related metabolites in plants.
- Klíčová slova
- LC-MS/MS, acetylated amino acids, acetylated biogenic amines, amino acids, biogenic amines, methylated amino acids, plant metabolism,
- Publikační typ
- časopisecké články MeSH
We have recently demonstrated the ability of a C18 stationary phase with a positively charged surface (PCS-C18) to provide superior chromatographic separation of peptides using mobile phase acidified with a mere 0.01 % formic acid, significantly improving MS sensitivity. Here, we examined three columns packed with different PCS-C18 phases using the MS-favorable mobile phase acidified with low formic acid concentrations to establish the impact of separation performance and better MS sensitivity on peptide identifications. The surface charge interaction was evaluated using the retention of nitrate. The highest interaction was observed for the AdvanceBio Peptide Plus column. A surface charge-dependent shift in the retention time of peptides was confirmed with a change in formic acid concentration in the mobile phase. The separation performance of the columns with MS-favorable mobile phase acidified with low concentrations of formic acid was evaluated using well-characterized peptides. The loading capacity was assessed using a basic peptide with three lysine residues. Good chromatographic peak shapes and high loading capacity were observed for the Acquity Premier CSH C18 column, even when using a mobile phase acidified with 0.01 % formic acid. The extent of improvement in peptide identification when using reduced formic acid concentration was evaluated by analyzing the tryptic digest of trastuzumab and tryptic digest of whole bacteria cell lysate. Each column provided improved MS signal intensity and peptide identification when using the mobile phase with 0.01 % formic acid. The ability of the Acquity Premier CSH C18 column to provide better separation of peptides, even with a reduced formic acid concentration in the mobile phase, boosted MS signal intensity by 65 % and increased the number of identified peptides from the bacterial sample by 19 %. Our study confirms that significant improvement in the proteomic outputs can be achieved without additional costs only by tailoring the chemistry of the stationary phase to the composition of the mobile phase. Our results can help researchers understand the retention mechanism of peptides on the PCS-C18 stationary phases using low-ionic strength mobile phases and, more importantly, select the best-suited stationary phases for their LC-MS proteomic applications.
- Klíčová slova
- Formic acid, Peptide separation, Proteomics, Reversed-phase liquid chromatography, Stationary phases,
- MeSH
- chromatografie kapalinová metody MeSH
- formiáty * chemie MeSH
- kapalinová chromatografie-hmotnostní spektrometrie MeSH
- peptidy chemie analýza izolace a purifikace MeSH
- proteomika * metody MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- trastuzumab chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- formiáty * MeSH
- formic acid MeSH Prohlížeč
- peptidy MeSH
- trastuzumab MeSH
INTRODUCTION: Helichrysum leucocephalum Boiss. (Asteraceae) is an endemic plant to Iran. No reports have studied the cytotoxicity of the plant. The current study aimed to evaluate the cytotoxicity of H. leucocephalum collected from Fars province (Iran) against MCF-7 and HDF cell lines using HPLC-based activity profiling and to annotate the active constituents by LC-ESIQTOF-MS/MS. METHODS: H. leucocephalum was collected from three locations in Fars province. The dried flowers and leaves were separately extracted by percolation using methanol. The crude extracts were fractionated by liquid-liquid partitioning with dichloromethane (DCM) and aqueous methanol. The cytotoxicity of the fractions was evaluated against MCF-7 and HDF cells by Alamarblue assay. HPLC-based activity profiling was used to track the active constituents. LC-MS dereplication strategy was used for the annotation of the compounds in the active time window. LC-MS data were preprocessed by MZmine 3.3.0 and submitted to multivariate analysis to compare the differences and similarities in the metabolites of the samples. RESULTS: The DCM fractions showed a dose-dependent cytotoxicity against the cancerous cells (IC50s, 9.8-105.1 μg/ml). In general, the metabolites of the flowers and their cytotoxicity were higher than the leaves. LCESIMS/MS analyses revealed that prenylated and geranylated α,β-unsaturated spiroketal phloroglucinols were among the active constituents. CONCLUSION: It can be concluded that H. leucocephalum is a rich source of phloroglucinol derivatives with cytotoxic activities. Further phytochemical analysis is needed to characterize the bioactive components.
- Klíčová slova
- Asteraceae, Cytotoxicity, HPLC, Helichrysum leucocephalum, LC-ESIQTOF-MS/MS,
- Publikační typ
- časopisecké články MeSH
OBJECTIVE: The laboratory diagnosis of inherited metabolic disorders (IMD) has undergone significant development in recent decades, mainly due to the use of mass spectrometry, which allows rapid multicomponent analysis of a wide range of metabolites. Combined with advanced software tools, the diagnosis becomes more efficient as a benefit for both physicians and patients. METHODS: A hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry assay for determination of urinary purines, pyrimidines, N-acylglycines, N-acetylated amino acids, sugars, sugar alcohols and other diagnostically important biomarkers was developed and validated. Evaluation of the results consisting of utilisation of robust scaling and advanced visualization tools is simple and even suitable for urgent requirements. RESULTS: The developed method, covering 65 biomarkers, provides a comprehensive diagnostic platform for 51 IMD. For most analytes, linearity with R2 > 0.99, intra and inter-day accuracy between 80 and 120 % and precision lower than 20 % were achieved. Diagnostic workflow was evaluated on 47 patients and External Quality Assurance samples involving a total of 24 different IMD. Over seven years, more than 2300 urine samples from patients suspected for IMD have been routinely analysed. CONCLUSIONS: This method offers the advantage of a broad coverage of intermediate metabolites of interest and therefore may be a potential alternative and simplification for clinical laboratories that use multiple methods for screening these markers.
- Klíčová slova
- Diagnosis, Hydrophilic interaction chromatography, Inherited metabolic disorders, Liquid chromatography, Mass spectrometry,
- MeSH
- biologické markery moč MeSH
- chromatografie kapalinová metody MeSH
- kapalinová chromatografie-hmotnostní spektrometrie MeSH
- lidé MeSH
- metabolické nemoci * MeSH
- tandemová hmotnostní spektrometrie * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- biologické markery MeSH
The main analytical benefit of this study is the development of methods enabling a rapid determination of total lipids of algae by lipidomic analysis and detailed identification and quantification of a complex mixture of natural TAGs by silver-LC/APCI-MS and NARP-LC/APCI-MS. Both types of chromatography can readily identify, both qualitatively and semiquantitatively, triacylglycerols containing 16:3 and 16:4 acids in the molecule. We conclude that the genus Chloromonas is a major producer of C16 PUFAs mostly contained in TAGs. Since more detailed studies in this field have been stymied by the shortage of 16:3 and 16:4 FAs, we decided to study the alga Chloromonas as a potential biotechnological source of C16 PUFAs.
- Klíčová slova
- Chloromonas pichinchae, ESI, Lipidomics, NARP-LC/APCI-MS, Silver-LC/APCI-MS, Snow alga, Triacylglycerols,
- MeSH
- chromatografie kapalinová metody MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody MeSH
- lipidy analýza chemie MeSH
- stříbro chemie MeSH
- triglyceridy analýza chemie MeSH
- Volvocida chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- lipidy MeSH
- stříbro MeSH
- triglyceridy MeSH