Simple and Reproducible Sample Preparation for Single-Shot Phosphoproteomics with High Sensitivity

2016 ◽  
pp. 251-260 ◽  
Author(s):  
Rosa R. Jersie-Christensen ◽  
Abida Sultan ◽  
Jesper V. Olsen
Keyword(s):  
Sample Preparation ◽  
High Sensitivity ◽  
Single Shot

scholarly journals A Single LC-MS/MS Analysis to Quantify CoA Biosynthetic Intermediates and Short-Chain Acyl CoAs

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 468
Author(s):  
Anthony E. Jones ◽  
Nataly J. Arias ◽  
Aracely Acevedo ◽  
Srinivasa T. Reddy ◽  
Ajit S. Divakaruni ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Biosynthetic Pathway ◽  
Solid Phase ◽  
High Sensitivity ◽  
Short Chain ◽  
Extraction Column ◽  
Chain Acyl ◽  
Precision And Accuracy ◽  
Mass Spectrometry Mass Spectrometry

Coenzyme A (CoA) is an essential cofactor for dozens of reactions in intermediary metabolism. Dysregulation of CoA synthesis or acyl CoA metabolism can result in metabolic or neurodegenerative disease. Although several methods use liquid chromatography coupled with mass spectrometry/mass spectrometry (LC-MS/MS) to quantify acyl CoA levels in biological samples, few allow for simultaneous measurement of intermediates in the CoA biosynthetic pathway. Here we describe a simple sample preparation and LC-MS/MS method that can measure both short-chain acyl CoAs and biosynthetic precursors of CoA. The method does not require use of a solid phase extraction column during sample preparation and exhibits high sensitivity, precision, and accuracy. It reproduces expected changes from known effectors of cellular CoA homeostasis and helps clarify the mechanism by which excess concentrations of etomoxir reduce intracellular CoA levels.

scholarly journals Rapid and Sensitive Quantification of Intracellular Glycyl-Sarcosine for Semi-High-Throughput Screening for Inhibitors of PEPT-1

Pharmaceutics ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1019
Author(s):  
Teresa von Linde ◽  
Gzona Bajraktari-Sylejmani ◽  
Walter E. Haefeli ◽  
Jürgen Burhenne ◽  
Johanna Weiss ◽  
...  
Keyword(s):  
Sample Preparation ◽  
High Throughput ◽  
High Throughput Screening ◽  
Dynamic Range ◽  
High Sensitivity ◽  
Peptide Bond ◽  
Peptide Transporter ◽  
Systemic Absorption ◽  
Screening Assay ◽  
Limit Of Quantification

The peptide transporter PEPT-1 (SLC15A1) plays a major role in nutritional supply with amino acids by mediating the intestinal influx of dipeptides and tripeptides generated during food digestion. Its role in the uptake of small bioactive peptides and various therapeutics makes it an important target for the investigation of the systemic absorption of small peptide-like active compounds and prodrug strategies of poorly absorbed therapeutics. The dipeptide glycyl-sarcosine (Gly-Sar), which comprises an N-methylated peptide bond that increases stability against enzymatic degradation, is widely utilized for studying PEPT-1-mediated transport. To support experiments on PEPT-1 inhibitor screening to identify potential substrates, we developed a highly sensitive Gly-Sar quantification assay for Caco-2 cell lysates with a dynamic range of 0.1 to 1000 ng/mL (lower limit of quantification 0.68 nM) in 50 µL of cell lysate. The assay was validated following the applicable recommendations for bioanalytic method validation of the FDA and EMA. Sample preparation and quantification were established in 96-well cell culture plates that were also used for the cellular uptake studies, resulting in a rapid and robust screening assay for PEPT-1 inhibitors. This sample preparation principle, combined with the high sensitivity of the UPLC-MS/MS quantification, is suitable for screening assays for PEPT-1 inhibitors and substrates in high-throughput formats and holds the potential for automation. Applicability was demonstrated by IC50 determinations of the known PEPT-1 inhibitor losartan, the known substrates glycyl-proline (Gly-Pro), and valaciclovir, the prodrug of aciclovir, which itself is no substrate of PEPT-1 and consequently showed no inhibition in our assay.

Technique development of high-throughput and high-sensitivity sample preparation and separation for proteomics

Bioanalysis ◽  
2021 ◽  
Author(s):  
Zhenbin Zhang ◽  
Minyang Zheng ◽  
Yufen Zhao ◽  
Perry G Wang
Keyword(s):  
Sample Preparation ◽  
High Throughput ◽  
High Sensitivity ◽  
Proteomics Analysis ◽  
Comprehensive Review ◽  
Proteomics Research ◽  
Quantification Accuracy ◽  
Technique Development

Sample preparation and separation methods determine the sensitivity and the quantification accuracy of the proteomics analysis. This article covers a comprehensive review of the recent technique development of high-throughput and high-sensitivity sample preparation and separation methods in proteomics research.

Imatinib quantification in human serum with LC-MS3 as an effective way of protein kinase inhibitor analysis in biological matrices

2017 ◽  
Vol 32 (3) ◽  
Author(s):  
Marek Dziadosz ◽  
Michael Klintschar ◽  
Jörg Teske
Keyword(s):  
Protein Kinase ◽  
Sample Preparation ◽  
Human Serum ◽  
Kinase Inhibitor ◽  
Limit Of Detection ◽  
High Sensitivity ◽  
Protein Kinase Inhibitor ◽  
Preparation Technique ◽  
Analytical Strategy ◽  
Inhibitor Analysis

AbstractBackground:As imatinib gained a lot of attention in the field of medicine, appropriate methods are needed for drug analysis. LC-MS/MS combined with complex sample preparation and column enrichment is usually the method of choice when high sensitivity is necessary. The application of LC-MSMethods:An LC-MSResults:The conditions applied resulted in a limit of detection/quantification value of 0.14/0.45 ng/mL reached without a sophisticated sample preparation technique or enrichment column application. It could be demonstrated that MSConclusions:The presented analytical strategy is an effective way of protein kinase inhibitor analysis in human serum.

scholarly journals Myocardial injury after pulmonary vein isolation: fire versus ice

2021 ◽  
Vol 42 (Supplement_1) ◽  
Author(s):  
A R Morgado Gomes ◽  
N S C Antonio ◽  
S Silva ◽  
M Madeira ◽  
P Sousa ◽  
...  
Keyword(s):  
Pulmonary Vein ◽  
Pulmonary Vein Isolation ◽  
Troponin I ◽  
Surrogate Marker ◽  
High Sensitivity ◽  
Cryoballoon Ablation ◽  
Single Shot ◽  
High Sensitivity Troponin ◽  
Ablation Index ◽  
Significant Difference

Abstract Introduction The cornerstone of atrial fibrillation (AF) catheter ablation is pulmonary vein isolation (PVI), either using point-by-point radiofrequency ablation (RF) or single-shot ablation devices, such as cryoballoon ablation (CB). However, achieving permanent transmural lesions is difficult and pulmonary vein (PV) reconnection is common. Elevation of high-sensitivity Troponin I (hsTnI) may be used as a surrogate marker for transmural lesions. Still, data regarding the comparison of hsTnI increase after PVI with RF or cryo-energy is controversial. Purpose The aim of this study is to compare the magnitude of hsTnI elevation after PVI with CB versus RF using ablation index guidance. Methods Prospective study of 60 patients admitted for first ablation procedure of paroxysmal or persistent AF in a single tertiary Cardiology Department. Thirty patients were submitted to PVI using CB and 30 patients were submitted to RF, using CARTO® mapping system and ablation index guidance. Patients with atrial flutter were excluded. Baseline characteristics were compared between groups, as well as hsTnI before and after the procedure. Results Mean age was 57.9±12.3 years old, 62% of patients were male and 77% had paroxysmal AF. There were no significant differences between groups regarding gender, age, prevalence of hypertension, dyslipidaemia, diabetes, obesity or AF type. There was also no significant difference in electrical cardioversion need during the procedure. HsTnI median value before ablation was 1.90±1.98 ng/dL. Postprocedural hsTnI was significantly higher in CB-group (6562.7±4756.2 ng/dL versus 1564.3±830.7 ng/dL in RF-group; P=0.001). Regarding periprocedural complications, there was only one case of mild pericardial effusion in RF-group associated with postablation hsTnI of 1180.0 ng/dL. Conclusions High-sensitivity Troponin I was significantly elevated after PVI, irrespective of the ablation technique. In CB-group, hsTnI elevation was significantly higher than in RF-group. This disparity may reflect more extensive lesions with cryoablation, without compromising safety. Longterm studies are needed to understand whether this hsTnI elevation is predictive of a lower AF recurrence rate. FUNDunding Acknowledgement Type of funding sources: None.

scholarly journals High-speed Analysis of Large Sample Sets – How Can This Key Aspect of the Omics Be Achieved?

2020 ◽  
Vol 19 (11) ◽  
pp. 1760-1766
Author(s):  
Rainer Cramer
Keyword(s):  
Sample Preparation ◽  
High Speed ◽  
High Sensitivity ◽  
Great Depth ◽  
Sample Introduction ◽  
New Developments ◽  
High Performing ◽  
High Speed Analysis ◽  
Ionization Methods ◽  
Line Sample

High-speed analysis of large (prote)omics sample sets at the rate of thousands or millions of samples per day on a single platform has been a challenge since the beginning of proteomics. For many years, ESI-based MS methods have dominated proteomics because of their high sensitivity and great depth in analyzing complex proteomes. However, despite improvements in speed, ESI-based MS methods are fundamentally limited by their sample introduction, which excludes off-line sample preparation/fractionation because of the time required to switch between individual samples/sample fractions, and therefore being dependent on the speed of on-line sample preparation methods such as liquid chromatography. Laser-based ionization methods have the advantage of moving from one sample to the next without these limitations, being mainly restricted by the speed of modern sample stages, i.e. 10 ms or less between samples. This speed matches the data acquisition speed of modern high-performing mass spectrometers whereas the pulse repetition rate of the lasers (>1 kHz) provides a sufficient number of desorption/ionization events for successful ion signal detection from each sample at the above speed of the sample stages. Other advantages of laser-based ionization methods include the generally higher tolerance to sample additives and contamination compared with ESI MS, and the contact-less and pulsed nature of the laser used for desorption, reducing the risk of cross-contamination. Furthermore, new developments in MALDI have expanded its analytical capabilities, now being able to fully exploit high-performing hybrid mass analyzers and their strengths in sensitivity and MS/MS analysis by generating an ESI-like stable yield of multiply charged analyte ions. Thus, these new developments and the intrinsically high speed of laser-based methods now provide a good basis for tackling extreme sample analysis speed in the omics.

Optimization of Surface Properties for Nanoindentation Studies on Nuclear Structural Materials

2017 ◽  
Vol 891 ◽  
pp. 67-72
Author(s):  
Petra Bublíková ◽  
Hygreeva Kiran Namburi ◽  
Daniela Marušáková
Keyword(s):  
Mechanical Properties ◽  
Sample Preparation ◽  
Radiation Damage ◽  
High Sensitivity ◽  
Structural Materials ◽  
Thin Layers ◽  
Standard Methods ◽  
Electrolytic Polishing ◽  
Preparation Step ◽  
Extent Of Damage

Nanoindentation using static loading of indenter tip is one of versatile methods used for evaluation of materials in smaller volume. It includes investigation of structural phases and thin layers on substrates etc. In the field of nuclear core and structural materials, nanoindentation has become an important tool to assess mechanical properties and correlate to the level of radiation damage at elevated and room temperatures. Nanoindentation, ideally in combination with Transmission Electron Microscopy, can describe the extent of damage and behaviour from the nanoand micro scale. Due to the high sensitivity of nanoindentation system, that typically uses loads in the range of tens of nN up to several tens of mN, the precise sample preparation is challenging and to be performed especially to understand behaviour of bulk materials. In the current study, samples from Austenitic Stainless Steel (ASS) 321, which is the representative structural material used for reactor internals, were prepared by standard methods - fine polishing, chemical etching, electrolytic etching, electrolytic polishing, electrolytic polishing & etching and ion polishing. Firstly, non-irradiated samples were used for optimization of the sample preparation methodology and then it will be applied on irradiated samples to obtain local mechanical properties. After each preparation step, nanoindentation was performed and load was optimized leading to the minimum standard deviation, also taking into account an indent size and pile-up mechanism. Scanning Probe Microscopy (SPM imaging) and nanoindentation results showed the multi-grained austenitic structure with minimal roughness. Local mechanical properties can be measured according to the knowledge of radiation damage type and location, with focus on grain boundaries to be evaluated. This study shows that advanced methods such as ion polishing are not suitable for ASS preparation, but standard methods based on chemical reaction with structure, especially electrolytic polishing and etching, are highly recommended.

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