Quantitative Amino Acid Analysis Research Articles

High-Throughput Quantitative Analysis of Amino Acids in Freeze-Dried Drops Using Time-of-Flight Secondary Ion Mass Spectrometry.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has become a promising analytical tool for molecular profiling in biological applications. However, its ultrahigh vacuum environment and matrix effects hamper the absolute quantitation of solution samples. Herein, we present a rapid high-throughput platform for quantitative ToF-SIMS analysis of amino acids in matrix deposits formed from freeze-dried solution drops through ice sublimation on a parylene film microarray substrate. Droplets of the amino acid solutions, which were mixed with stable isotope-labeled phenylalanine (F*) of high concentration (10 mM), were loaded on wells of the microarray, then frozen and evaporated slowly below the freezing point, forming continuous solid-phase F* matrix deposits. The amino acids (≤500 μM), adequately well dispersed throughout the F* matrix deposits on each well, were quantitatively analyzed by ToF-SIMS in a rapid and high-throughput fashion. The lower limit of quantitation reached below 10 μM.

Toward multidimensional information: A derivatization-free UHPLC-QqQ MS/MS method for amino acid components of fingerprint.

The analysis of fingerprint chemical composition is a meaningful way to excavate the multidimensional information of fingerprint, including the donor profiling information and the age of a fingerprint, which broadens the evidential values of fingerprint, especially for the partial and distorted fingerprint. But the research remains still in the pilot phases or is ongoing. Amino acids are the dominant organic substances in latent sweat fingerprint and influenced by many donor factors. Hence, their content reflects personal information of donors. Forensic science will be revolutionized if suspects can be individualized by their amino acid content. The diverse nature, distinct physicochemical properties, and ultra-micro levels of amino acids present in fingerprints make it hard to detect. A high sensitivity method for detecting and quantifying multiple amino acid components is required. UHPLC-QqQ MS/MS offers high sensitivity, high separation, simultaneous multicomponents detection, and no derivatization, making it an ideal method for detecting and analyzing amino acids in fingerprints. Therefore, in this study, we propose and validate an efficient UHPLC-QqQ MS/MS method for the extraction and analysis of 13 amino acids from fingerprint. We compared the results of amino acids of 10 different substrates and found that the inherent amino acids in most porous substrates would have been extracted along with the fingerprint amino acids, making them unsuitable for quantitative amino acid analysis. Instead, plastic sheets are ideal substrates for laboratory studies. Then, extensive experiments were conducted among 30 donors for multidimensional information analysis. The type of samples analyzed were eccrine-rich fingerprints. A Binary Logistic Regression (BLR) model was developed, and the female and male donors were successfully differentiated by amino acids in fingerprints. Two other mathematical models were also developed to verify the accuracy, and all three different mathematical models were able to identify donors of different genders with over 90% accuracy. This demonstrates that amino acids have the potential to provide more information for donors as metabolic markers. In the future, we will conduct a series of experiments to analyze more multidimensional information for individual identification by amino acid content in the fingerprint.

Arc-Induced Electrospray Ionization Mass Spectrometry.

Arc-induced electrospray ionization mass spectrometry (AESI-MS) was developed during which alternating current electrospray is simply achieved through the arc plasma. The AESI source exploits the arc's temperature and charge properties to generate aerosols consisting of charged microdroplets. The electrospray region, in which organic molecules are contained within microdroplets, partially overlaps with the arc plasma region. Guided by the electric field, these molecules undergo ionization, yielding ionic target analytes. AESI represents a soft ionization method that combines the mechanisms of atmospheric pressure chemical ionization and electrospray ionization, facilitating the ionization of analytes with wide ranging polarities. The precisely targeted spraying area enhances ion entry into the mass analyzer, thereby enabling excellent ionization efficiency. The AESI source exhibits several notable advantages over the electrospray ionization source, including an elevated but comparable level of active species concentrations and types, simplified mass spectra for direct amino acid analysis, high salt tolerance, versatile analysis of compounds with varying polarities, and reliable quantitative analysis of amino acids in complex matrices. Overall, AESI broadens the methodologies employed to generate microdroplets, providing a technological and scientific framework for creating distinctive electrospray ionization techniques.

Dynamic Elevation of Aromatic Amino Acids in Hepatitis C Virus-Induced Cirrhosis After a Standard Meal.

Perturbations in aromatic (AAAs) and branched-chain amino acids (BCAAs) are seen in decompensated liver disease. The aim of this study was to evaluate the dynamic, postprandial relationship between hepatitis C virus-induced liver disease and amino acid concentrations in patients with compensated liver disease. Patients infected with hepatitis C virus underwent a baseline liver biopsy to determine Ishak Fibrosis Score and evaluate the liver transcriptome. Patients ate a standard meal and underwent peripheral vein sampling at defined intervals. Quantitative analysis of amino acids was performed using liquid chromatography-tandem mass spectrometry. At baseline, there was no difference in AAA and BCAA concentrations between patients with cirrhosis and non-cirrhotic patients. After a standard meal, AAAs, but not BCAAs, were elevated in patients with cirrhosis compared with non-cirrhotic patients at every time point. The HepQuant SHUNT fraction was significantly higher in patients with cirrhosis and positively correlated with AAA concentration at all time points, but not BCAA. Analysis of the hepatic transcriptome demonstrated greater downregulation of the AAA degradation pathways than the BCAA degradation pathways. At baseline, cirrhotic patients with compensated liver disease have adequate reserve liver function to metabolize AAAs and BCAAs. When faced with a metabolic stressor, such as a standard meal, patients with cirrhosis are less able to metabolize the increased load of AAAs. This impairment correlates with portosystemic shunting. Further evaluation of AAA levels in compensated liver disease might further the understanding of the liver-muscle axis and the role it may play in the development of sarcopenia in liver disease.

A Robust Capillary Electrophoresis with Laser-Induced Fluorescence Detection (CE-LIF) Method for Quantitative Compositional Analysis of Trace Amino Acids in Hypersaline Samples.

The search for life in our solar system can be enabled by the characterization of extreme environments representing conditions expected on other planets within our solar system. Molecular abundances observed in these environments help establish instrument design requirements, including limits of detection and pH/salt tolerance, and may be used for validation of proposed planetary science instrumentation. Here, we optimize capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) separations for low limit of detection quantitative compositional analysis of amino acids in hypersaline samples using carboxyfluorescein succinimidyl ester (CFSE) as the amine-reactive fluorescent probe. Two methods were optimized for identification and quantification of proteinogenic amino acids, those with and those without acidic side chains, with limits of detection as low as 250 pM, improving on previous CFSE-amino acid CE-LIF methods by an order of magnitude. The resilience of the method to samples with high concentrations of Mg2+ (>4 M diluted to >0.4 M for analysis) is demonstrated on a sample collected from the salt harvesting facility South Bay Salt Works in San Diego, CA, demonstrating the highest Mg2+ tolerance for CE-LIF methods used in amino acid analyses to date. This advancement enables the rapid and robust analysis of trace amino acids and the search for biosignatures in hypersaline systems.

Erratum to “Nutritional profiling and quantitative analysis of amino acids and vitamins using LC–MS/MS in selected raw and germinated ancient grains”

Erratum to “Nutritional profiling and quantitative analysis of amino acids and vitamins using <scp>LC</scp>–<scp>MS</scp>/<scp>MS</scp> in selected raw and germinated ancient grains”

Ionic liquid as regenerable media for simultaneous recovery of melanin and amino acid from waste human hair: An elaborate and efficient biorefining approach

Waste human hair (WHH) is rich in valuable resources like keratin and melanin. However, current resource recovery methods face challenges such as complex procedures, consumption of acidic and alkaline reagents, unsatisfactory yield, and inferior purity of melanin products, primarily due to the difficulty in avoiding the generation of melanin-keratin blends (MKBs). For the first time, this study established an ionic liquid-targeted depolymerization/isolation (IL-TDI) process for the simultaneous recovery of melanin and monomeric amino acids from WHH with both satisfactory yield and superior purity via four steps, including depolymerization, liquid-solid separation, extraction, and recovery of IL. IL-TDI process yielded 6.02% melanin and 34.58% monomeric amino acid, and the environmentally friendly ionic liquid, 1-Butyl-3-methylimidazolium chloride (BMIMCl), was recyclable. SEM and FTIR analyses revealed that the melanin extracted by IL-TDI process retained its native chemical structure and microstructure. In contrast, the traditional base dissolution and acid isolation (BDAI) process achieved only a 2.2% melanin yield and completely destroyed the natural microstructure of melanin. Various characterizations, including UV–visible spectroscopy, quantitative amino acid analysis, and kinetic rate calculations, were conducted to study the separation mechanism of melanin and keratin. In IL-TDI process, disulfide bonds in keratin were initially cleaved by BMIMCl during dissolution. Subsequently, melanin isolation followed two consecutive first-order reactions, and hydrogen ions in BMIMCl preferentially hydrolyzed dissolved keratin, during which preventing the generation of MKB proved essential for achieving high yield of melanin. Economic and E-factor calculations demonstrated this novel biorefining approach's suitability for sustainable WHH recovery.

ADCY5-related dyskinesia: a treatable inherited neurometabolic disorder

Case Report. A 40-year-old Brazilian man presented with long-standing loss of balance, dystonia, and choreoathetosis, with episodic paroxysmal worsening. Neuroimaging and cerebrospinal fluid analysis were both unremarkable. Quantitative urine organic acid analysis, plasma acylcarnitine quantitative profile, and quantitative analysis of plasma amino acids by high performance liquid chromatography were all within normal interval values. Multigene next-generation sequencing panel identified the heterozygous pathogenic variant c.1252C&gt;T (p.Arg418Trp) in the ADCY5 gene, defining a diagnosis of ADCY5-related dyskinesia. Oral caffeine was associated to clonazepam and provided marked improvement of motor symptoms. Conclusion. This report adds evidence to the safety and efficacy of caffeine in the treatment of genetically confirmed cases of ADCY5-related dyskinesia in the context of gain-of-function variants.

Nutritional profiling and quantitative analysis of amino acids and vitamins using LC–MS/MS in selected raw and germinated ancient grains

AbstractBackgroundAncient grains are grains that have remained unchanged over the past decades. Among them, millets and pseudocereals are highly nutritious underutilized grains that are gluten free. The present study compares the nutritional composition of nine important ancient grains in its raw and germinated forms.ResultsAmong the grains studied, amaranth had the highest protein content (14.52%), followed by proso (13.04%) and foxtail millets (12.78%). Amino acid analysis was carried out using LC–MS/MS. Besides amaranth, barnyard, pearl, and proso millets contained considerable amount of essential amino acids. The lysine content of sorghum has increased four folds during germination. Valine, histidine, and glutamic acid contents increased marginally upon sprouting. These grains were also found to contain significant amount of B complex vitamins. The ascorbic acid content of the grains increased considerably upon germination especially in foxtail millet (78.03–30452.90 ng/g).ConclusionThe present study is the first comparative study that has brought out the distinct nutritional superiority of the ancient grains in their raw and germinated forms. These gluten‐free grains can be utilized to improve the nutritional demand and food security of the growing population.

Analysis of Urinary Amino Acids by High-Performance Liquid Chromatography with Fluorescence Detection Using 2,3-Naphthalenedicarboxaldehyde as Fluorescence Derivatization Reagent

Amino acids are involved in various chemical reactions in vivo, and changes in several amino acids in urine are related to certain disease states. Therefore, developing an efficient method to analyze the amino acids in urine is useful in the timely diagnosis of diseases. In this study, we developed a high-performance liquid chromatography (HPLC) fluorescence method for the quantitative analysis of urinary amino acids using the fluorescence derivatization reagent 2,3-naphthalenedicarboxaldehyde (NDA). NDA was selected because it does not require heating for the reaction and can react within a short time, rendering its use in clinical settings feasible. The reaction temperature, reaction time, and other derivatization conditions were optimized, and the reaction was found to be completed in 5 min at 25 °C. The separation of NDA–amino acids was investigated on an octadecylsilyl (ODS) column under gradient conditions. The mobile phase was a mixture of water–acetonitrile–trifluoroacetic acid. Eighteen NDA–amino acids (histidine (His), arginine (Arg), asparagine (Asn), glutamine (Gln), citrulline (Cit), serine (Ser), aspartic acid (Asp), threonine (Thr), glutamic acid (Glu), glycine (Gly), tyrosine (Tyr), alanine (Ala), tryptophan (Trp), valine (Val), phenylalanine (Phe), isoleucine (Ile), leucine (Leu), and 5-aminovaleric acid (internal standard)) were separated within 100 min under optimal conditions. The calibration curves showed good linearity in the range of 0.25–25 pmol per injection with correlation coefficients of &gt;0.998. The limits of quantification for NDA–amino acids were 16.7–74.7 fmol. The developed analytical method was applied to a human urine sample and 16 amino acids (His, Arg, Asn, Gln, Cit, Ser, Thr, Glu, Gly, Tyr, Ala, Trp, Val, Phe, Ile, and Leu) were quantified. The urinary amino acid concentrations were 5–960 μM. Urinary amino acid analysis using this method is expected to be clinically applicable as a novel biomarker for diseases affecting the bladder, urinary tract, and kidneys.

Comparative Study and the Optimum Conditions On the Use of Certain Bacterial Strains For the Production of Some Amino Acids

This study aims to use some strains (Corynebacterium glutamicum, Lactobacillus bulgaricus, and Brevibacterium linens for the production of some amino acids (lysine, methionine, threonine, arginine, tyrosine, aspartic acid, glutamic acid, alanine, and proline) on synthetic fermentation media under different conditions (incubation periods; incubation temperatures; pH values; media volume and different sources of carbon and nitrogen) on the course of fermentation reactions of amino acids, and then quantitative analysis of amino acids by HPLC for choose the best conditions for increasing production of amino acids. The results showed that in the best conditions for three strains, the highest production of amino acids was recorded for Brevibacterium linens the pH (7.5), incubation temperature (30°C), medium volume (50 ml), and incubation period (48 hr), while for Corynebacterium glutamicum observed on pH (7), incubation temperature (30°C), medium volume (50 ml) and incubation period (48 hr), however, for Lactobacillus bulgaricus observed on pH 6.5, incubation temperature 37°C, medium volume (50 ml) and incubation period (48 hr). The best carbon and nitrogen sources were recorded for glucose and ammonium sulfate for three tested strains.

On-chip microfluidic dual detection of amino acid metabolism disorders using cell-free protein synthesis

Various metabolic diseases are associated with the accumulation of specific amino acids due to abnormal metabolic pathways, and thus can be diagnosed by measuring the level of amino acids in body fluids. However, present methods for amino acid analysis are not readily accessible because they require a complex experimental setup, expensive equipment, and a long processing time. Here, we present a dual sensing microfluidic device that enables fast, portable, and quantitative analysis of target amino acids, harnessing the biological mechanism of protein synthesis. In this device, the working principle of a finger-actuated pumping unit is applied, and the microchannels are designed to perform cell-free synthesis of a reporter protein in response to the target amino acids in the assay samples. Multiple steps required for the translational assay are controlled by the simple operation of two pushbuttons on the device. It is demonstrated that the developed microfluidic device provides precise quantification of two amino acids (methionine and phenylalanine) within 30 min at room temperature. We expect that the application of the presented device can be readily extended to the point-of-care testing of other metabolic compounds.

Stroke Literature Synopsis (Preclinical).

Stroke Literature Synopsis (Preclinical).

A Racemic Naphthyl-Coumarin-Based Probe for Quantitative Enantiomeric Excess Analysis of Amino Acids and Enantioselective Sensing of Amines and Amino Alcohols.

A new racemic naphthyl‐coumarin‐based probe was found to bind covalently with amino acids in MeOH‐KOH system and thereby generates distinct CD responses. The induced strong CD signals allowed quantitative enantiomeric excess analysis of amino acids and enantioselective sensing of amines and amino alcohols. The mechanism for the reaction of the coumarin‐aldehyde probe with an amino acid was investigated by CD, UV‐Vis, NMR, ESI‐MS analyses and ECD calculation.

Application of Untargeted Metabolomics to Reveal the Taste-Related Metabolite Profiles during Mandarin Fish (Siniperca chuatsi) Fermentation.

Spontaneous fermentation is a critical processing step that determines the taste quality of fermented mandarin fish (Siniperca chuatsi). Here, untargeted metabolomics using ultra-high-performance liquid chromatography coupled with Q Exactive tandem mass spectrometry was employed to characterize the taste-related metabolite profiles during the fermentation of mandarin fish. The results demonstrated that the taste profiles of mandarin fish at different stages of fermentation could be distinguished using an electronic tongue technique. Sixty-two metabolites, including amino acids, small peptides, fatty acids, alkaloids, and organic acids, were identified in fermented mandarin fish samples. Additional quantitative analysis of amino acids revealed glutamic acid and aspartic acid as significant contributors to the fresh flavor. Furthermore, the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that amino acid metabolism was the dominant pathway throughout the fermentation process. This study provides a scientific and theoretical reference for the targeted regulation of the quality of fermented mandarin fish.

A UHPLC-PDA method for the quantitative analysis of total amino acids in infant formula with microwave-assisted acid hydrolysis

A UHPLC-PDA method for the quantitative analysis of total amino acids in infant formula with microwave-assisted acid hydrolysis

The use of HPLC-MS/MS for the quantitative analysis of neuroactive amino acids in rat brain homogenates after derivatization with 9-fluorenylmethyl chloroformate

The use of HPLC-MS/MS for the quantitative analysis of neuroactive amino acids in rat brain homogenates after derivatization with 9-fluorenylmethyl chloroformate

Preservation of collagen in the soft tissues of frozen mammoths

We investigated the characteristics of extracellular matrix (ECM) in the soft tissue of two frozen baby woolly mammoths (Mammuthus primigenius) that died and were buried in Siberian permafrost approximately 40,000 years ago. Morphological and biochemical analyses of mammoth lung and liver demonstrated that those soft tissues were preserved at the gross anatomical and histological levels. The ultrastructure of ECM components, namely a fibrillar structure with a collagen-characteristic pattern of cross-striation, was clearly visible with transmission and scanning electron microscopy. Type I and type IV collagens were detected by immunohistochemical observation. Quantitative amino acid analysis of liver and lung tissues of the baby mammoths indicated that collagenous protein is selectively preserved in these tissues as a main protein. Type I and type III collagens were detected as major components by means of liquid chromatography-mass spectrometry analysis after digestion with trypsin. These results indicate that the triple helical collagen molecule, which is resistant to proteinase digestion, has been preserved in the soft tissues of these frozen mammoths for 40,000 years.

Modic changes are associated with activation of intense inflammatory and host defense response pathways -- molecular insights from proteomic analysis of human intervertebral discs

<h3>BACKGROUND CONTEXT</h3> Patients with Modic changes form a distinct clinical subset with reports of higher intensity of pain, poor clinical and surgical outcomes and higher incidence of recurrence. MC also is an independent risk factor for increased postoperative surgical site infection. <h3>PURPOSE</h3> This study aimed to investigate the biological changes at molecular level, in discs with Modic changes. <h3>STUDY DESIGN</h3> Experimental analysis. <h3>MATERIALS AND METHODS</h3> Nucleus pulposus (NP) from 24 patients undergoing microdiscectomy for disc herniation [14 discs with Modic changes and 10 without Modic changes] were procured. The overall expression of proteins, biological processes, protein-protein and metabolite interactions were analyzed and compared. Host defense response proteins (HDRPs) and immunological pathways activated in patients with Modic changes were documented and analyzed. <h3>RESULTS</h3> Label-free proteomic approach with stringent filters revealed a total of 208 proteins in Modic changes and 193 in non-Modic change groups. Forty-five proteins were specific to Modic changes; 30 to non-Modic changes and 163 common to both. Downregulated proteins in Modic changes belonged to components of extracellular matrix such as collagens (COL- 6A1, 6A2, 6A3, 11A1, 12A1, and 20A1), and proteoglycans (versican [VCAN], and biglycan [BGN]). Inflammatory molecules [plasminogen (PLG), angiogenin (ANG), fibroblast growth factor-binding protein 2 (FGFBP2), tetranectin (CLEC3B), cartilage acidic protein 1(CRTAC1), kininogen (KNG1), chitinase-3-like protein 2 (CHI3L2), and ferritin (FTL) were expressed only in the Modic changes group. The significantly altered pathways in Modic changes included Fc Fragment of IgG Receptor IIIa (FCGR3A)-mediated phagocytosis, regulation of Toll-like receptors (TLR) by endogenous ligand, neutrophil and platelet degranulation. 50 HDRPs were identified in the study, 14 of which were specific to Modic changes and included acute phase reactants, antimicrobial peptides, complement cascade proteins, inflammatory molecule and stress response proteins. Metabolite-protein interaction analysis revealed a significant interaction between 19 proteins, specifically involving ubiquitin mediating proteasome degradative pathway and an association with the metabolite-glutamic acid in the Modic changes group. Accumulation of glutamic acid in Modic changes discs was confirmed by quantitative amino acid analysis using High-performance liquid chromatography. <h3>CONLCUSION</h3> Our study confirms that Modic changes represent an intense inflammatory status due to activation of host defense response and immunological pathways. Downstream effects leading to ubiquitin mediated proteasomal degradation of ECM proteins and the resulting metabolites such as glutamic acid could cause excessive pain and needs further investigation. <i>Clinical Significance:</i> We have documented the expression of inflammatory molecules, immune mechanisms and host defense response proteins which throw molecular insights into the pathological mechanisms of Modic changes. Further, ubiquitin mediated proteasomal degradation and accumulation of glutamate in discs with Modic changes might serve as targets for molecular therapy. <h3>FDA DEVICE/DRUG STATUS</h3> This abstract does not discuss or include any applicable devices or drugs.

Modic changes are associated with activation of intense inflammatory and host defense response pathways – molecular insights from proteomic analysis of human intervertebral discs

Patients with modic changes (MC) form a distinct clinical subset with reports of higher intensity of pain, poor clinical and surgical outcomes and higher incidence of recurrence. MC also is an independent risk factor for increased post-operative surgical site infection. This study aimed to investigate the biological changes at molecular level, in discs with MCs. We also aim to identify biological biomarkers and potential targets for molecular therapy. Experimental analysis MATERIALS AND METHODS: Nucleus pulposus (NP) from 24 patients undergoing microdiscectomy for disc herniation [14 discs with MC and 10 without modic changes (NMC)] were procured. The overall expression of proteins, biological processes, protein-protein and metabolite interactions were analysed and compared. Host defense response proteins (HDRPs) and immunological pathways activated in patients with MC were documented and analysed. Label-free proteomic approach with stringent filters revealed a total of 208 proteins in MC and 193 in NMC groups. 45 proteins were specific to MC; 30 to NMC and 163 common to both. Downregulated proteins in MC belonged to components of extracellular matrix such as collagens (COL- 6A1, 6A2, 6A3, 11A1, 12A1, and 20A1), and proteoglycans (versican (VCAN), and biglycan (BGN)). Inflammatory molecules [plasminogen (PLG), angiogenin (ANG), fibroblast growth factor-binding protein 2 (FGFBP2), tetranectin (CLEC3B), cartilage acidic protein 1(CRTAC1), kininogen (KNG-1), chitinase-3-like protein 2 (CHI3L2), and ferritin (FTL) were expressed only in the MC group. The significantly altered pathways in MC included Fc Fragment of IgG Receptor IIIa (FCGR3A)-mediated phagocytosis, regulation of Toll-like receptors (TLR) by endogenous ligand, neutrophil and platelet degranulation. 50 HDRPs were identified in the study, 14 of which were specific to MC and included acute phase reactants, antimicrobial peptides, complement cascade proteins, inflammatory molecule and stress response proteins. Metabolite-protein interaction analysis revealed a significant interaction between 19 proteins, specifically involving ubiquitin mediating proteasome degradative pathway and an association with the metabolite-glutamic acid in the MC group. Accumulation of glutamic acid in MC discs was confirmed by quantitative amino acid analysis using High-performance liquid chromatography. Our study confirms that MC represents an intense inflammatory status and activation of host defense response and immunological pathways. Downstream effects leading to ubiquitin mediated proteasomal degradation of ECM proteins and the resulting metabolites such as glutamic acid could cause excessive pain and needs further investigation. We have documented the expression of inflammatory molecules, immune mechanisms and host defense response proteins which throw molecular insights into the pathological mechanisms of MC. Further, ubiquitin mediated proteasomal degradation and accumulation of glutamate in discs with MC might serve as targets for molecular therapy.