scholarly journals High-Throughput Analysis of Amino Acids for Protein Quantification in Plant and Animal-Derived Samples Using High Resolution Mass Spectrometry

Molecules ◽  
2021 ◽  
Vol 26 (24) ◽  
pp. 7578
Author(s):  
Priyanka Reddy ◽  
Aaron Elkins ◽  
Joe Panozzo ◽  
Simone J. Rochfort
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Retention Time ◽  
High Resolution Mass Spectrometry ◽  
Amino Acid Content ◽  
Protein Quantification ◽  
Borate Buffer Solution ◽  
Buffer Solution ◽  
High Throughput Analysis ◽  
Protein Levels

Current methods for measuring the abundance of proteogenic amino acids in plants require derivatisation, extended run times, very sensitive pH adjustments of the protein hydrolysates, and the use of buffers in the chromatographic phases. Here, we describe a fast liquid chromatography–mass spectrometry (LC–MS) method for the determination of amino acids that requires only three steps: hydrolysis, neutralisation, and sample dilution with a borate buffer solution for pH and retention time stability. The method shows excellent repeatability (repeated consecutive injections) and reproducibility (repeated hydrolysis) in the amino acid content, peak area, and retention time for all the standard amino acids. The chromatographic run time is 20 min with a reproducibility and repeatability of <1% for the retention time and <11% for the peak area of the BSA and quality control (QC) lentil samples. The reproducibility of the total protein levels in the hydrolysis batches 1–4 was <12% for the BSA and the lentil samples. The level of detection on column was below 0.1 µM for most amino acids (mean 0.017 µM).

Studies on Chromatographic Fractioning by Cations Exchangers of a Bovine Hemoglobin Hydrolysate

2018 ◽  
Vol 69 (10) ◽  
pp. 2794-2798
Author(s):  
Alina Diana Panainte ◽  
Ionela Daniela Morariu ◽  
Nela Bibire ◽  
Madalina Vieriu ◽  
Gladiola Tantaru ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Retention Time ◽  
Chromatographic Separation ◽  
Bovine Hemoglobin ◽  
Reverse Phase ◽  
Hplc System ◽  
Fast Flow ◽  
Hydrolysis Of ◽  
Phase Column

A peptidic hydrolysate has been obtained through hydrolysis of bovine hemoglobin using pepsin. The fractioning of the hydrolysate was performed on a column packed with CM-Sepharose Fast Flow. The hydrolysate and each fraction was filtered and then injected into a HPLC system equipped with a Vydak C4 reverse phase column (0.46 x 25 cm), suitable for the chromatographic separation of large peptides with 20 to 30 amino acids. The detection was done using mass spectrometry, and the retention time, size and distribution of the peptides were determined.

scholarly journals Magnesium Supplementation Alters Leaf Metabolic Pathways for Higher Flavor Quality of Oolong Tea

Agriculture ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 120 ◽  
Author(s):  
Jiuliang Xu ◽  
Liangquan Wu ◽  
Bingxin Tong ◽  
Jiaxu Yin ◽  
Zican Huang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Free Amino Acids ◽  
Free Amino ◽  
High Resolution Mass Spectrometry ◽  
Southern China ◽  
Substantial Reduction ◽  
Sugar Accumulation ◽  
Limiting Factor ◽  
Oolong Tea

Oolong tea, one of the most famous tea beverages in China, contains specialized metabolites contributing to rich flavors and human health. Accumulation patterns of such metabolites and underlying regulatory mechanisms significantly vary under different growth conditions. To optimize quality and yield while minimizing environmental effects, three treatments were designed in this study: Conventional fertilization, optimized fertilization, and optimized fertilization supplemented with magnesium (Mg). We investigated the yield, taste quality, primary and secondary metabolites of oolong tea, and found that a substantial reduction in chemical fertilizers (nutrient optimization by reducing 43% N, 58% P2O5 and 55% K2O) did not affect the tea yield in this study. Interestingly, Mg fertilization is an important factor influencing amino acid and sugar accumulation in oolong tea, resulting in higher concentrations of total free amino acids and a lower ratio of tea polyphenols (TP) to free amino acids (FAA). Gas chromatography-time-of-flight mass spectrometry (GC-TOF-MS) and liquid chromatography-high resolution mass spectrometry (LC-HRMS) combined multivariate analyses revealed distinct features of metabolite accumulation in leaves of three different treatments, as indicated by 34 differentially accumulated characteristic compounds. The levels of serine, aspartic acid, isoleucine, phenylalanine, theanine, and proline were reduced by fertilizer optimization and increased by Mg supplementation. Mg particularly promoted theanine accumulation favoring a stronger umami taste of oolong tea, while decreasing astringency and bitter metabolites. Thus, Mg application paves a new path for tea quality improvement in Southern China where Mg deficiency in the soil is a frequent limiting factor for crop production.

Interpretable machine learning model to detect chemically adulterated urine samples analyzed by high resolution mass spectrometry

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Gabriel L. Streun ◽  
Andrea E. Steuer ◽  
Lars C. Ebert ◽  
Akos Dobay ◽  
Thomas Kraemer
Keyword(s):  
Machine Learning ◽  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
High Resolution ◽  
Retention Time ◽  
Drug Testing ◽  
High Resolution Mass Spectrometry ◽  
Urine Samples ◽  
Machine Learning Model ◽  
Resolution Mass

Abstract Objectives Urine sample manipulation including substitution, dilution, and chemical adulteration is a continuing challenge for workplace drug testing, abstinence control, and doping control laboratories. The simultaneous detection of sample manipulation and prohibited drugs within one single analytical measurement would be highly advantageous. Machine learning algorithms are able to learn from existing datasets and predict outcomes of new data, which are unknown to the model. Methods Authentic human urine samples were treated with pyridinium chlorochromate, potassium nitrite, hydrogen peroxide, iodine, sodium hypochlorite, and water as control. In total, 702 samples, measured with liquid chromatography coupled to quadrupole time-of-flight mass spectrometry, were used. After retention time alignment within Progenesis QI, an artificial neural network was trained with 500 samples, each featuring 33,448 values. The feature importance was analyzed with the local interpretable model-agnostic explanations approach. Results Following 10-fold cross-validation, the mean sensitivity, specificity, positive predictive value, and negative predictive value was 88.9, 92.0, 91.9, and 89.2%, respectively. A diverse test set (n=202) containing treated and untreated urine samples could be correctly classified with an accuracy of 95.4%. In addition, 14 important features and four potential biomarkers were extracted. Conclusions With interpretable retention time aligned liquid chromatography high-resolution mass spectrometry data, a reliable machine learning model could be established that rapidly uncovers chemical urine manipulation. The incorporation of our model into routine clinical or forensic analysis allows simultaneous LC-MS analysis and sample integrity testing in one run, thus revolutionizing this field of drug testing.

scholarly journals Thermal stability of amino acids in meals and feeds used in shrimp farming

Gaia Scientia ◽  
2016 ◽  
Vol 10 (4) ◽  
pp. 372-389
Author(s):  
João Paulo de Sousa Prado ◽  
José Marcelino Oliveira Cavalheiro ◽  
Thiago Brandão Cavalheiro ◽  
Fernanda Vanessa Gomes da Silva
Keyword(s):  
Amino Acids ◽  
Elevated Temperatures ◽  
Amino Acid Content ◽  
Storage Conditions ◽  
Shrimp Farming ◽  
Protein Levels ◽  
Commercial Feed ◽  
The Stability ◽  
The Right ◽  
And Control

The Brazilian shrimp farming uses mainly commercial feed for shrimp nutrition. This choice occurs because of the advantages related to convenience and good adaptation of Litopenaeus vannanmei to feed intake. Thus, the quality of feed is a determining factor for maximum performance of the shrimp farms, making the right selection of suppliers and control of the storage conditions as ways to prevent contamination and spoilage of feed. The objective of this study was to evaluate the stability of amino acids in meals and commercial feed with different protein levels, subjected to high-temperature storage. The samples were exposed to temperature of 50 oC and evaluated every 5 days for 30 days.The analyses of the degradation of amino acids were performed using an elution gradient in HPLC system.In evaluated meals it was observed that valine and arginine were the amino acids that suffered greater loss during the experiment and histidine and alanine suffered less degradation.Significant difference was observed in the content of all amino acids analyzed after exposure of the feed to the temperature of 50 oC; with reduce in values of its amino acid content. The results obtained in this study indicate that meals and feed exposed to elevated temperatures significantly reduced the content of its amino acids.

scholarly journals Identification of Auxin Metabolites in Brassicaceae by Ultra-Performance Liquid Chromatography Coupled with High-Resolution Mass Spectrometry

Molecules ◽  
2019 ◽  
Vol 24 (14) ◽  
pp. 2615 ◽  
Author(s):  
Panagiota-Kyriaki Revelou ◽  
Maroula G. Kokotou ◽  
Violetta Constantinou-Kokotou
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Liquid Chromatography ◽  
High Resolution ◽  
Plant Extracts ◽  
High Resolution Mass Spectrometry ◽  
Ultra Performance Liquid Chromatography ◽  
Charge Ratio ◽  
High Resolution Mass ◽  
Resolution Mass

Auxins are signaling molecules involved in multiple stages of plant growth and development. The levels of the most important auxin, indole-3-acetic acid (IAA), are regulated by the formation of amide and ester conjugates with amino acids and sugars. In this work, IAA and IAA amide conjugates with amino acids bearing a free carboxylic group or a methyl ester group, along with some selected IAA metabolites, were studied in positive and negative electrospray ionization (ESI) modes, utilizing high-resolution mass spectrometry (HRMS) as a tool for their structural analysis. HRMS/MS spectra revealed the fragmentation patterns that enable us to identify IAA metabolites in plant extracts from eight vegetables of the Brassicaceae family using a fast and reliable ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QToF-MS) method. The accurate m/z (mass to charge) ratio and abundance of the molecular and fragment ions of the studied compounds in plant extracts matched those obtained from commercially available or synthesized compounds and confirmed the presence of IAA metabolites.

scholarly journals Relative Quantification of Several Plasma Proteins during Liver Transplantation Surgery

2011 ◽  
Vol 2011 ◽  
pp. 1-12 ◽  
Author(s):  
Ville Parviainen ◽  
Sakari Joenväärä ◽  
Eija Tukiainen ◽  
Minna Ilmakunnas ◽  
Helena Isoniemi ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liver Transplantation ◽  
Plasma Proteins ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Systemic Circulation ◽  
Protein Quantification ◽  
Immunological Methods ◽  
High Throughput Analysis ◽  
Transplantation Surgery

Plasma proteome is widely used in studying changes occurring in human body during disease or other disturbances. Immunological methods are commonly used in such studies. In recent years, mass spectrometry has gained popularity in high-throughput analysis of plasma proteins. In this study, we tested whether mass spectrometry and iTRAQ-based protein quantification might be used in proteomic analysis of human plasma during liver transplantation surgery to characterize changes in protein abundances occurring during early graft reperfusion. We sampled blood from systemic circulation as well as blood entering and exiting the liver. After immunodepletion of six high-abundant plasma proteins, trypsin digestion, iTRAQ labeling, and cation-exchange fractionation, the peptides were analyzed by reverse phase nano-LC-MS/MS. In total, 72 proteins were identified of which 31 could be quantified in all patient specimens collected. Of these 31 proteins, ten, mostly medium-to-high abundance plasma proteins with a concentration range of 50–2000 mg/L, displayed relative abundance change of more than 10%. The changes in protein abundance observed in this study allow further research on the role of several proteins in ischemia-reperfusion injury during liver transplantation and possibly in other surgery.

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