Amino Acid Analysis: A Comprehensive Overview

Amino acids, serving as the fundamental building blocks of proteins, are indispensable to biological systems due to their dual roles in structural biology and metabolic regulation. Their comprehensive analysis enables critical insights across scientific domains—from decoding protein architecture in biochemistry to assessing nutritional biomarkers in clinical practice. Methodologically, amino acid characterization encompasses compositional profiling for quantifying proteinogenic ratios, sequential mapping via N-terminal sequencing and mass spectrometry, and quantitation of free amino acid pools in extracellular and intracellular compartments. Advanced strategies include chemical derivatization techniques such as pre-column labeling to enhance chromatographic resolution, alongside specialized analyses like enantiomeric separation of D/L-amino acid isomers and detection of post-translational modifications (e.g., phosphorylated or glycosylated residues). These approaches collectively empower researchers to unravel metabolic network dynamics, diagnose pathological states, and refine therapeutic development through precision analytics.

Amino acid sequences of each motif identified by MEME tools (Zhang L et al., 2020).

Amino Acid Composition Profiling

The quantitative assessment of amino acid composition involves identifying and measuring the relative abundance of individual amino acids within biological samples, providing critical insights into the structural and functional properties of proteins or peptides.

Analytical Methodologies

• Pre-Column Derivatization Coupled with HPLC: Amino acids undergo chemical modification with fluorescent or UV-active tags (e.g., ortho-phthalaldehyde, dansyl chloride) prior to separation via HPLC, enabling precise quantification through optical detection.
• Ion-Exchange Chromatography (IEC): Separation is achieved based on charge differentials using ion-exchange resins, followed by post-separation derivatization (e.g., ninhydrin-based chromogenic reactions) for spectrophotometric detection.
• Mass Spectrometric Approaches: Direct molecular weight determination is performed using mass spectrometry (MS), often integrated with chromatographic techniques (LC-MS or GC-MS) for enhanced sensitivity and high-resolution detection.

Effect of triple-frequency ultrasound assisted fermentation on the amino acid composition profiling of rice lees (Murtaza MS et al., 2025).

Applications

• Proteomic Investigations: Deciphering amino acid profiles to correlate protein structure with biological activity. For example, amino acid analysis revealed the metabolic heterogeneity between clinical isolates of Staphylococcus aureus and standard strain ATCC 29213: in exponential growth period, the glutamic acid level of clinical strains was significantly higher than that of standard strains, histidine was its unique component, and the difference of aspartic acid ratio (44% of clinical strains vs 59% of standard strains) and lysine highlighted the strain-specific metabolic characteristics. These differences reflect the metabolic plasticity of pathogens, which may be related to their virulence regulation (such as glutamic acid promoting biofilm formation), antibiotic resistance (histidine metabolism compensating stress adaptation) and niche adaptation mechanism (lysine pathway optimizing host environment survival), providing molecular basis for analyzing the pathogenic advantages of clinical strains and formulating targeted intervention strategies (Alreshidi M et al., 2023).
• Nutritional Science: Quantifying essential amino acid profiles in food matrices to assess dietary quality. For example, the content and composition of amino acids in Cyclocarya paliurus leaves change dynamically with the development stages (S1-S3), which significantly affects the flavor of tea: the total amount of amino acids in leaves at S1 and S2 stages is high, among which S2 is rich in sweet amino acids (such as alanine) and essential amino acids (methionine, phenylalanine, etc.), so it is recommended to be used for high-quality tea and large-scale production respectively; In S3 stage, due to the decrease of amino acid abundance (related to the decrease of gene expression related to glycolysis and TCA cycle), it is suitable to extract polysaccharides and phenols. Eighteen kinds of amino acids were identified, among which glutamic acid and arginine were the dominant components. The study revealed that four bZIP transcription factors may participate in amino acid biosynthesis by regulating carbohydrate metabolism genes, providing molecular targets for directional improvement of tea flavor (Du Z et al., 2022).
• Plant stress: to explore the changes of amino acid metabolism in plants under stress. For example, the study revealed the mechanism of amino acid metabolism remodeling in Zanthoxylum bungeanum leaves under drought stress: 56 amino acids (including 8 essential amino acids) were identified in the leaves of Fengjiao and Hanjiao respectively, and the contents of most amino acids increased significantly under drought stress, among which there were more differentially accumulated amino acids (DAA) and differentially expressed genes (DEGs) in Fengjiao. Drought induced the accumulation of proline and γ -aminobutyric acid (GABA), which participated in osmotic adjustment and antioxidation. The increase of aromatics (phenylalanine, tyrosine, etc.) and long-chain amino acids (isoleucine, leucine, etc.) is associated with stress resistance and nutritional improvement, suggesting its potential in the development of functional foods (such as sports drinks). This study provides molecular targets for analyzing drought adaptation mechanism and quality improvement of plants (Hu H et al., 2022).

Protein Sequence Analysis

Protein sequence analysis stands as a foundational technology in life sciences, enabling the elucidation of amino acid arrangements while uncovering structural, functional, and mechanistic insights into biological macromolecules. Advancements in analytical methodologies have transformed this field into an indispensable tool for biomedicine, therapeutic development, and disease mechanism studies.

Approaches

Edman Degradation

A classical N-terminal sequencing technique that iteratively cleaves amino acids from peptide chains for sequential identification. While effective for short peptides and N-terminal profiling, its labor-intensive workflow and single-residue resolution limit utility to small-scale analyses, primarily in early-stage protein characterization.

Mass Spectrometry-Based Sequencing

Modern workflows employ tandem mass spectrometry (MS/MS), where proteins are enzymatically digested (e.g., trypsinization) into peptides, ionized, and fragmented for spectral analysis. Coupled with database matching, MS/MS achieves high sensitivity and specificity, enabling:

• Large-scale proteomic profiling in complex biological matrices
• Detection of post-translational modifications (e.g., phosphorylation, glycosylation)
• Quantitative analyses in systems biology studies

Genomic Sequencing Integration

Leveraging high-throughput DNA sequencing platforms, inferred protein sequences are derived from coding gene regions. This approach benefits from:

• Cost-effective scalability via next-generation sequencing (NGS)
• Identification of pathogenic mutations linked to aberrant protein function
• Integration with transcriptomic data for expression-level insights

Applications

• Proteomics: Mapping sequence-structure relationships to decipher protein roles in cellular pathways. For example, CheW and CheY, as the core proteins of bacterial chemotactic signal transduction, have significant differences in their functional evolution: CheW interacts with chemotactic receptor and CheA kinase through highly conserved residues such as Gly57 and Arg62 to maintain cross-species signal transmission function; CheY is involved in the interaction of CheA, FliM and CheZ, but its active site is less conserved between Escherichia coli and Aspergillus brasiliensis, suggesting functional differentiation. Evolutionary analysis showed that CheY had more amino acid substitutions and functional remodeling than CheW, and assumed a more complex role in signal regulation (Alexandre G et al., 2003).
• Drug Discovery: Target validation through epitope mapping and binding site characterization. For example, protein sequence analysis revealed the core role of Mur family enzymes in cell wall synthesis and their potential as antibacterial targets: Mur family was divided into transferase (MurG et al.), ligase (MurC-F) and oxidoreductase (MurB) by sequence characteristics, and it was inferred that MurG/MraY had the best activity in alkaline environment based on pI value. The distribution of polar/nonpolar amino acids endows these proteins with hydrophobicity, suggesting that they participate in peptidoglycan synthesis through membrane binding mechanism. Sequence conservation not only analyzes the stability of functional structure, but also provides a key basis for the design of broad-spectrum inhibitors targeting multi-drug resistant bacteria. Conservative functional domains can be used as drug action sites, while hydrophobic characteristics guide the molecular optimization of penetrating cell membranes, highlighting the dual value of protein sequence analysis in analyzing evolutionary mechanism and developing new antibacterial strategies (Amera GM et al., 2020).
• Analysis of sequence characteristics: machine learning-driven design and engineering application of protein abundance regulation. For example, protein sequence analysis reveals that amino acid composition is the decisive factor of protein abundance variation across species (prokaryotic to mammalian), and evolutionary pressure optimizes expression level by balancing structural stability and metabolic efficiency. The machine learning framework based on Transformer accurately identifies highly expressed related sequence motifs, carries out virtual mutagenesis by combining with Mutation-Guided Abundance Engineering (MGEM), and analyzes the regulation mechanism of physical and chemical properties such as hydrophobicity and polarity on abundance (Buric F et al., 2025).

Analytical Profiling of Free Amino Acids

The quantification of free amino acids serves as a critical tool across diverse disciplines, including nutritional science, metabolic investigations, and food quality assessment. Technological advancements have expanded its scope beyond basic quantification to inform health diagnostics, mechanistic studies of disease pathways, and precision food engineering.

Analytical Methodologies

HPLC

HPLC remains a cornerstone technique for separating and quantifying free amino acids in complex matrices. Utilizing pre- or post-column derivatization (e.g., with fluorescent tags like OPA or dansyl chloride), this method enhances detectability by conferring chromophoric properties to amino acids. Key features include:

• Exceptional sensitivity: Capable of detecting trace-level analytes in biological fluids (e.g., serum, urine) and food extracts.
• Multiplex capacity: Simultaneous analysis of 20+ amino acids in a single run.
• Adaptability: Compatible with diverse sample types, from fermentation broths to clinical specimens.
• Limitations: Requires specialized expertise for derivatization protocols, and reagent-dependent workflows may introduce operational complexity.

GC-MS

GC-MS excels in analyzing volatile amino acid derivatives, offering superior resolving power for structural identification. Non-volatile analytes necessitate derivatization (e.g., silylation) prior to separation. Advantages include:

• Analytical precision: High mass accuracy enables unambiguous compound identification.
• Broad applicability: Effective for profiling amino acids in environmental samples and metabolomic studies.
• Limitations: Demands rigorous sample preparation and advanced instrumentation maintenance.

Enzymatic Assays

Enzyme-based methods employ substrate-specific catalysts (e.g., L-amino acid oxidase) to generate quantifiable signals (e.g., NADH fluorescence). Notable strengths:

• Target specificity: Ideal for analyzing individual amino acids like glutamine or phenylalanine.
• Cost efficiency: Minimal equipment requirements compared to chromatographic systems.
• Limitations: Susceptibility to matrix interference and restricted multiplexing capability.

If you want to know more about amino acid analysis methods, please refer to "Amino Acid Analysis Methods".

Applications

• Clinical Nutrition: Monitoring amino acid imbalances to guide dietary interventions in malnutrition or metabolic disorders. The analysis of free amino acids revealed the protein nutritional characteristics of Lentinus edodes mycelia (strains 18 and 18N44): the contents of total amino acids (TAA:21.62%) and essential amino acids (EAA:7.13%) of strain 18 were significantly higher than those of conventional Lentinus edodes, and its E/T and E/N values were close to the ideal standards of FAO/WHO, and SRC(68.07) and EAAI(54.86%). The analysis showed that mycelium was rich in lysine, which could effectively supplement the lysine deficiency of plant food, but the restrictive amino acids were Met+Cys (strain 18) and Leu(18N44). The difference in the distribution of flavor amino acids (strain 18 is mainly bitter, and 18N44 is bitter/sweet) provides sensory optimization basis for the development of functional foods (Yu CX et al., 2023).
• Disease Pathogenesis: Identifying dysregulated pathways in conditions like phenylketonuria or cancer cachexia. For example, dynamic monitoring of free amino acids By quantifying the changes of plasma amino acid concentration in children with PKU before and after meals, the absorption characteristics of different protein substitutes (CGMP-AA1/AA2) were analyzed: CGMP-AA2 significantly increased the postprandial levels of tyrosine (neurotransmitter synthesis associated with 120-minute peak concentration), histidine and leucine, but there was no difference in total amino acids and total essential amino acids between groups. This analysis reveals that the types and proportions of specific amino acids (such as tyrosine) in protein substitutes directly affect the absorption rate and physiological effects, which provides key data support for optimizing nitrogen utilization rate and neurological function of children with PKU and lays a scientific foundation for personalized formula design (Daly A et al., 2020).
• Food Industry: Optimizing protein quality in fermented products and assessing adulteration in dietary supplements. For example, free amino acid analysis provides key strategies for potato quality improvement and safe production by analyzing crop metabolic characteristics in multiple dimensions: locating key metabolic nodes formed by processing carcinogens through quantitative analysis of acrylamide precursors such as asparagine, and guiding genetic breeding to reduce risk components; At the same time, the distribution characteristics of essential amino acids and flavor components were evaluated, and the nutritional quality evaluation system was constructed. QTL loci and genome prediction model (GEBV) based on genome-wide association positioning can realize the precision of parent selection and optimize the yield, disease resistance and nutritional quality simultaneously. This strategy is from the analysis of metabolic regulation network to molecular marker-assisted breeding, which forms a closed-loop research paradigm and promotes the development of functional potato varieties and precision agriculture (Pandey J et al., 2023).

Enhancing Amino Acid Analysis Through Amino acid derivatization treatment

Chemical modification of amino acids serves as a pivotal strategy to optimize their detection and separation in analytical workflows, particularly when employing chromatographic techniques such as high-performance liquid chromatography (HPLC) and gas chromatography (GC). By introducing functional groups that enhance spectral properties, volatility, or ionization efficiency, this process significantly improves both sensitivity and resolution, enabling precise quantification even in complex matrices.

Optimization of Detection Sensitivity

Amino acids inherently exhibit limited detectability due to weak inherent spectral signals. Chemical modification addresses this limitation by incorporating fluorophores or chromophores. For instance, o-phthalaldehyde (OPA) reacts with primary amines under controlled pH to generate highly fluorescent derivatives, amplifying signals in HPLC systems. Similarly, dansyl chloride forms stable fluorescent adducts with amino groups, facilitating sensitive detection in both HPLC and mass spectrometry. These modifications elevate detection thresholds, enabling accurate measurement of trace analytes in biological and environmental samples.

Chromatographic Resolution Enhancement

Beyond sensitivity gains, chemical modification alters amino acid physicochemical properties—such as hydrophobicity or volatility—to enhance chromatographic performance. In reversed-phase HPLC, dansyl chloride increases analyte hydrophobicity, improving retention and separation. For GC applications, trifluoroacetic anhydride (TFAA) converts amino acids into volatile esters, enabling efficient gas-phase separation. Such tailored modifications ensure baseline resolution of structurally similar analytes, critical for high-fidelity analyses.

Reagent-Specific Applications

o-Phthalaldehyde (OPA)

• Mechanism: Forms fluorescent isoindole derivatives with primary amines.
• Application: Ideal for HPLC-based detection of low-concentration amino acids in biological fluids.
• Optimization: Requires precise pH control (typically alkaline conditions) for efficient conjugation.

Dansyl Chloride

• Advantage: Broad pH compatibility and solvent stability.
• Utility: Effective in complex matrices like food extracts or tissue homogenates.

Trifluoroacetic Anhydride (TFAA)

• Role: Enhances volatility for GC-MS applications.
• Outcome: Facilitates analysis of non-volatile amino acids through esterification.

Technical Challenges and Mitigation Strategies

While chemical modification enhances analytical performance, challenges include:

• Reaction Condition Sensitivity: Temperature, pH, and solvent composition must be rigorously optimized to ensure reproducibility.
• Matrix Interference: Competing reactions with non-target compounds may elevate background noise. Mitigation involves selective reagent choice and sample pre-purification.

Multidisciplinary Applications

• Food Science: Quantifying essential amino acids in fortified products to assess nutritional quality. For example, the research system optimized the reaction conditions of amino acid derivatization: the molar ratio of EASC reagent to amino acid was 1:5, the reaction time was 10 minutes at 65℃, and the fluorescence signal was the strongest at pH9.0. Akasil-C18 column achieved the baseline separation of 19 amino acids within 20 minutes. The structure of phenylalanine derivatives was confirmed by electrospray ionization mass spectrometry (m/z 222.0 and 303.9 characteristic peaks). Tissue analysis of Nitraria tangutorum showed that the amino acid content in pericarp and pulp was the highest, mainly proline (Pro) and alanine (Ala), and its accumulation was related to drought resistance and salt tolerance, which met the FAO/WHO ideal protein standard and had high nutritional development value. Although the proportion of essential amino acids in seeds and leaves is lower than the reference value, the total amount is rich, which is suitable for medicine or agricultural raw materials (Zhou W et al., 2019).
• Environmental Monitoring: Identifying amino acid-based biomarkers of pollution in aquatic ecosystems. For example, this study reveals the mechanism of β-Ala and γ-ABA generated by abiotic decomposition of Asp and Glu in low-temperature geochemical environment, and provides a reasonable explanation for the enrichment of n-ω amino acids relative to precursor amino acids in carbonaceous meteorites (such as CI chondrite and Longgong asteroid samples). The concentration of amino acids and the proportion of their derivatives in different meteorites (Murchison, Orgueil, etc.) are significantly different, which is related to the water/rock (W/R) ratio of the parent body: the enrichment of mineral iron ions in low W/R environment inhibits the preservation of amino acids, while high W/R promotes the homogenization of fluid mixing. For example, the amino acid distribution of CI-type meteorites is related to the water-rich mantle, while the CM-type meteorites may originate from the core. The experiment confirmed that the electrochemical decomposition of Asp and Glu did not cause chiral asymmetry, suggesting that amino acids may not have undergone significant chiral selection in the pre-life chemical environment (Li Y et al., 2023).
• Analytical Innovation: High-resolution amino acid profiling in aquatic products via advanced derivatization and UHPLC-HRMS/MS technology. For example, in this study, a new derivatization reagent combined with UHPLC-HRMS/MS technology was developed to realize the efficient differential detection of taurine and other amino acids. Eighteen amino acids were separated by octadecyl silica gel column, and the analysis was completed within 10 minutes. The characteristic fragment ion (C6H4N5O1S) in negative ion mode significantly improved the detection selectivity and sensitivity. The parallel reaction monitoring (PRM) showed that the calibration curves of amino acids such as valine and glutamic acid were linear (R²>0.99), and the coefficient of variation was less than 10%. The method was accurate and reliable. The application showed that taurine content in Ruditapes philippinarum was prominent, and the amino acid composition of edible parts with different specifications was significantly different, which provided an efficient analysis scheme for nutritional value evaluation and quality control of aquatic products (Uekusa S et al., 2021).

Chiral Amino Acid Profiling

Stereospecific Amino Acid Characterization

Chiral amino acid analysis is critical in biochemistry and pharmacology, as the biological activity of enantiomers (L- and D-forms) depends on their stereochemical configuration. Precise discrimination of these mirror-image isomers enables insights into their distinct roles in metabolic pathways and therapeutic interventions.

Analytical Approaches

Enantioselective Chromatography

• Principle: Specialized stationary phases or chiral derivatizing agents differentiate enantiomers via HPLC or GC. These phases exploit stereochemical interactions to achieve baseline separation of D- and L-amino acids under identical chromatographic conditions.
• Utility: Essential in pharmaceutical quality control, where enantiomeric purity directly impacts drug efficacy and safety.

Enzyme-Mediated Discrimination

• Mechanism: Enzymes like D-amino acid oxidase (DAAO) selectively catalyze specific enantiomers, enabling precise identification through substrate-specific reactions.
• Application: High-throughput screening of biological fluids for enantiomeric imbalances in clinical diagnostics.

Relevance Across Disciplines

• Drug Development: Enantiopure drugs (e.g., L-DOPA) require stringent chiral analysis to optimize pharmacokinetics and minimize off-target effects. For example, chiral analysis ensures the safety and efficacy of drugs by separating and identifying enantiomers. Different enantiomers may have opposite pharmacological effects (such as teratogenic S- body and sedative R- body of thalidomide) or different activities (such as ibuprofen is only effective in S- body). Accurate separation can optimize the efficacy and reduce the toxicity. Global regulatory requirements (such as FDA) force chiral drugs to specify the enantiomer ratio, and strictly monitor the impurity content and batch stability by means of high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). In drug development, chiral analysis helps targeted design (such as conformational optimization of anticancer drug Venetoclax) and personalized therapy (adjusting warfarin dosage according to patients' metabolic genes), while microfluidic system and AI are combined to accelerate Qualcomm screening and condition optimization (Al-Sulaimi S et al., 2023).
• Neurobiology: D-amino acids regulate neurotransmission and synaptic plasticity, necessitating precise detection to study neurological disorders. For example, schizophrenia, as a neurodevelopmental disorder, its core pathology involves glutamate dysfunction driven by NMDA receptor dysfunction, but the current dopamine D2 receptor antagonists have no significant effect on it. D- amino acid (D- serine/aspartic acid/alanine), as a co-agonist of NMDA receptor, has demonstrated its ability to regulate synaptic plasticity and repair the structural abnormality of dendritic spines in preclinical studies, providing a new therapeutic direction for 30-40% of refractory patients (TRS) (de Bartolomeis A et al., 2022).

PTM Detection

PTMs—such as phosphorylation, glycosylation, and acetylation—modulate protein function, stability, and cellular localization. Their detection is pivotal for understanding proteomic regulation in health and disease.

Methodologies

MS

• Workflow: MS identifies PTMs by detecting mass shifts corresponding to chemical modifications. Tandem MS (MS/MS) localizes modification sites via peptide fragmentation.
• Advantage: Enables system-wide PTM profiling in proteomic studies, revealing dynamic changes during cellular signaling or disease progression.

Immunoaffinity Techniques

• Principle: Modification-specific antibodies (e.g., anti-phosphotyrosine) bind target epitopes for detection via Western blot, ELISA, or immunoprecipitation.
• Utility: Validates PTM involvement in pathological processes, such as oncogenic kinase activation in cancer.

Biomedical Applications

• Disease Mechanisms: Aberrant PTMs (e.g., hyperphosphorylation in Alzheimer's tau proteins) serve as biomarkers and therapeutic targets. For example, by integrating 320,000 human protein post-translational modifications (PTM) data with 4 million non-synonymous DNA variants, the study reveals the association between PTM removal and genetic diseases: 215 pairs of PTM types and diseases were identified in 59 types of PTM (42% were new findings), among which the loss of lysine deacetylation was the most significant, and PTM (such as S- glutathionylation) which was less concerned was also related to diseases. Pathogenicity prediction tools suggest that protecting specific PTM loci may prevent diseases (Vellosillo P et al., 2021).

If you want to know more about the application of amino acid analysis, please refer to "Application of Amino Acid Analysis in Protein: a Comprehensive Overview".

Methodological Innovations

• Chiral Analysis: Coupling capillary electrophoresis with laser-induced fluorescence enhances enantiomer detection limits in complex matrices.
• PTM Quantification: Isobaric tagging (e.g., TMT) enables multiplexed, quantitative PTM analysis across experimental conditions.

Challenges and Solutions

• Enantiomer Cross-Reactivity: Hybrid methods integrating enzymatic pretreatment with MS improve specificity.
• PTM Transience: Chemical stabilization techniques (e.g., phosphatase inhibitors) preserve labile modifications during sample processing.

References

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