- Service Details
- Demo
- Case Study
- FAQ
- Publications
What is Amino Acid (AA)?
AAs are the most basic units that make up proteins and polypeptides through peptide bonds. AAs also form the building blocks of products/metabolites involved in cell and energy metabolism, such as fatty acids and ketone bodies. Free AAs and protein hydrolysates are widely distributed in biological fluids and play important roles in various biological processes, particularly in maintaining homeostasis. Changes in AA levels have been found to be closely associated with several diseases, including phenylketonurics, diabetes, kidney disease, liver disease, and cancer (such as adenoma and colorectal cancer). Besides, the scope of AAs of interest is not limited to essential or proteinogenic AAs; there are numerous "unusual amino acids" that constitute microbial peptides or result from cell metabolism or metabolic disorders. AAs are widely used in industries such as bioanalytical and clinical chemistry, medicine, food, and cosmetics. Therefore, in order to study the mechanisms of life or produce polypeptides and proteins, it is essential for us to first understand AAs. The rapid, efficient, and reliable technologies for determining and quantifying the concentrations of AAs and their derivatives in biological samples are crucial in virtually every aspect, including understanding the physiological effects of AAs as well as predicting, diagnosing, and elucidating disease mechanisms.
How to Perform Amino Acid Analysis (AAA)?
Currently, with the advancement of bioanalytical technology, numerous AAA techniques have emerged and found extensive applications in various fields including pharmaceuticals, food industry, animal feed production, agriculture, medical research, environmental analysis, biochemical/biomedical research as well as clinical diagnosis. Typically, AAA for peptides or proteins involves (i) acid hydrolysis of peptides or proteins into their constituent AAs, (ii) the separation of AAs by chromatography, (iii) AAs derivatization (which can be performed before or after chromatography), and (iv) spectrophotometric detection. The development of derivatization-free AAA assays utilizing mass spectrometry (MS) for the detection and utilizing internal standards for the quantification of individual AA residues after chromatographic separation has been aimed at enhancing detection sensitivity and reducing the required amount of protein material. Meanwhile, there are many well-established methods as well for implementing AAA in dry blood spot, serum, plasma, cerebral spinal fluid, urine, and tissues. And the D, L-Amino Acids in samples can also be easily distinguished. These methods accelerated the AAA procedure, significantly improved speed, sensitivity, and accuracy, and reduced the amount of initial sample.
Figure 1. Organigram summarizing the main analytical methods for AAA [1].
Our Amino Acid Analysis service
With over a decade of experience in AAA, Creative Proteomics possesses advanced techniques and expertise in various sample analyses and has aided in many clinical and research situations. With iTRAQ and SRM/MRM technologies, using ion-exchange LC, reverse-phase HPLC, gas chromatography (GC), capillary electrophoresis (CE), strong cation exchange (SCX), and hydrophilic interaction chromatography (HILIC), with ultraviolet (UV), fluorescence (FL) spectrophotometry, ESI-MS/MS, MALDI TOF MS, TOF/TOF MS/MS, Creative Proteomics can obtain accurate data on proteins, peptides, and AAs of a particular sample. In addition to abundant and proficient methodologies, our company is also equipped with automatic amino acid analyzer and high-throughput Liquid Chromatography with tandem mass spectrometry (LC-MS/MS). We can provide comprehensive identification and quantification services for 20 proteinogenic amino acids as well as other amino acids in biological compounds, biological fluids, drugs, tissues, food, microbiology samples, environmental samples, etc.
Figure 2. The methods for hydrolyzed analyzing amino acids in Creative Proteomics.
Amino Acid Analysis (AAA) Services We Can Provide:
Amino Acid Quantification and Identification: Quantitative and qualitative analysis of amino acids in proteins, peptides, and other biological or pharmaceutical samples. This service detects all 20 natural amino acids and various non-standard amino acids.
Protein and Peptide Quantification: Accurate quantification of proteins or peptides, including precise measurement in protein stock solutions, essential for applications in drug development and research.
Amino Acid Composition Analysis: Determines the amino acid composition of proteins and peptides, which supports quantification and structural studies.
Physiological Fluid Analysis: Quantification of free-form amino acids in physiological fluids like plasma, urine, and cerebrospinal fluid, offering insights into metabolic states and health conditions.
Food Content Analysis: Amino acid profile analysis in food samples, including complete and supplemental amino acid profiles, which supports nutritional and dietary research.
Environmental Sample Analysis: Analysis of amino acids in environmental samples, supporting research in fields like microbiology, agriculture, and ecology.
High-Throughput and High-Sensitivity Detection: Utilizes advanced techniques, including isotope internal standards and multiple chromatography methods (ion-exchange LC, reverse-phase HPLC, GC, CE, LC-MS/MS), to achieve high sensitivity and reproducibility.
Amino Acids and Derivatives Analysis: Uses pre-column derivatization, such as AccQTag, combined with reverse-phase chromatography for high-resolution separation and quantification of amino acids and derivatives.
Protein and Peptide Sequence Analysis: Determines the amino acid sequence of proteins, peptides, and monoclonal antibodies (mAbs), supporting structural analysis and research.
Customized Hydrolysis and Analysis: Offers specialized hydrolysis for complex proteins or modified amino acids, facilitating the recovery of amino acids like tryptophan and cysteine that may be lost in standard processes.
Brochures
Proteomics Services
Welcome to the uncharted territory of proteomics, a revolutionary journey led by Creative Proteomics. Our latest brochure is your gateway to understanding the significance and potential of proteomics and how Creative Proteomics is poised to provide you with exceptional proteomics solutions.
Technological superiority
- Professional detection and analysis capability: Experienced technical team, strict quality control system, together with ultra-high resolution detection system and professional data pre-processing and analysis capability, ensure reliable and accurate data.
- Reproducible: By adding isotope internal standard for correction, various samples can be accurately quantified.
- High-throughput: Deeper coverage for protein or peptide sequence.
- High resolution and sensitivity: Over 100 types of amino acids and derivatives can be quantitatively analyzed at the same time.
Sample Requirements for Amino Acid Analysis
| Sample Type | Sample Format | Sample Amount | Preparation Requirements | Notes |
|---|---|---|---|---|
| Dry Protein/Peptide Samples | Solid | ≥100 µg at 0.5-2 mg/mL | Ensure high purity; lyophilized and free of contaminants | Store dry; avoid moisture exposure |
| Liquid Protein/Peptide Samples | Solution | >100 µL at minimum 0.5 mg/mL | Deionized water or suitable buffer; avoid interfering agents | Fresh or properly preserved |
| Biological Fluids (e.g., plasma, urine) | Solution | 50-200 µL | Centrifuged to remove particulate matter | Use fresh or flash-frozen samples |
| Tissue Samples | Solid or Solution | 10-50 mg | Homogenized, lyophilized, or flash-frozen | Avoid repeated freeze-thaw cycles |
| Food Samples | Solid | 100-200 mg | Dried and ground into fine powder | Avoid additives or preservatives |
| Environmental Samples (e.g., soil, water) | Varies | Varies based on sample type | Pre-treated to remove any interfering substances | Filtered and clarified if in solution |
How to place an order
The provision of comprehensive support tailored to your specific requirements for Amino Acid Analysis is our area of expertise. Please feel free to contact us via email whenever you need to discuss your specific requirements. Our customer service representatives are available 24 hours a day, from Monday to Sunday.
Reference
- Ferré S, González-Ruiz V, Guillarme D, et al. Analytical strategies for the determination of amino acids: Past, present and future trends. Journal of Chromatography B. 2019 Nov 15, 1132: 121819
PCA chart
KEGG pathway enrichment analysis
Sample clustering heatmap
Amino acid analysis for peptide quantitation using reversed-phase liquid chromatography combined with multiple reaction monitoring mass spectrometry
Journal: Analytical and Bioanalytical Chemistry
Published: 2023
Main Technology: Reversed-phase liquid chromatography, MRM-MS.
Abstract
Amino acid analysis (AAA) can be used for absolute quantitation of standard peptides after acid hydrolysis using 6 M HCl. Obtained individual amino acids can then be quantified by liquid chromatography-mass spectrometry (LC–MS). Achieving baseline separation of non-derivatized amino acids is challenging when reversed-phase (RP) chromatography is used. Several derivatization methods are commonly utilized to address this issue; however, derivatization has several drawbacks, such as derivative instability and lack of reproducibility. Currently, separation of non-derivatized amino acids is typically done using HILIC, but HILIC has problems of poor reproducibility and long column equilibration times. We developed a method to quantify non-derivatized amino acids, including methionine and cysteine, from peptide hydrolysates by RP-LC-MS without special pre-treatment of the samples. Samples were spiked with certified isotopically labeled (13C- and/or 15N-) amino acids as internal standards. The amino acids released from acid hydrolysis were then analyzed by RP-UPLC-MRM-MS and quantified using the analyte/internal standard chromatographic peak area ratios. Peptide quantitation was based on the sum of the individual amino acid concentrations from the known peptide sequences. The resulting method did not require derivatization, used standard C18-based reversed-phase liquid chromatography, did not require external calibration, was robust, and was able to quantify all 17 amino acids for which we had internal standards, including the sulfur-containing amino acids, cysteine and methionine, in their respective oxidized forms. This simple and robust method enabled the absolute quantitation of standard peptides using only acid hydrolysis and a standard RP-UPLC-MRM-MS setup.
The goal of our current study was to develop a RP-UPLC-MRM-MS method for the quantitation of underivatized amino acids from peptide hydrolysates, using simple sample preparation and robust chromatography, and to use this method in a high-throughput format for routine analysis.
Figure 1. The Graphical Abstract of Amino Acid Analysis.
What sample types are suitable for amino acid analysis?
Amino acid analysis can be performed on various sample types, including purified proteins, peptides, biological fluids (like plasma and urine), food products, and environmental samples. It's important to note that samples should be free from contaminants, and specific preparation steps, such as homogenization for tissues or drying for food samples, are crucial to ensure accurate results.
How does acid hydrolysis affect amino acid analysis results?
Acid hydrolysis is often used to break down proteins and peptides into their constituent amino acids. While this method is effective for most amino acids, some, like tryptophan and cysteine, are sensitive to acidic conditions and may degrade during the hydrolysis process. To mitigate this, alternative hydrolysis methods or derivatization techniques may be employed to preserve these amino acids for accurate quantification.
How do sample preparation techniques affect amino acid analysis results?
Sample preparation is critical in amino acid analysis, as it influences the accuracy and reliability of the results. For protein samples, acid hydrolysis is commonly employed to break down proteins into their constituent amino acids. However, this process can lead to the degradation of sensitive amino acids like tryptophan and cysteine. Therefore, optimizing hydrolysis conditions, such as temperature, time, and the type of acid used, is essential. Additionally, contaminants in samples, such as lipids or carbohydrates, can interfere with chromatographic analysis. Proper purification techniques, including centrifugation and filtration, are necessary to eliminate these potential interferences before analysis.
Can amino acid analysis detect non-proteinogenic amino acids? If so, how?
Yes, amino acid analysis can detect non-proteinogenic amino acids, which are often present in various biological samples due to metabolic processes or as byproducts of microbial metabolism. Advanced techniques like pre-column derivatization and mass spectrometry enhance the detection of these non-standard amino acids. Derivatization improves the chromatographic separation and detection sensitivity. The choice of derivatizing agents can be crucial; for example, using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) allows for the effective detection of a broader range of amino acids, including those that may not be readily identifiable through standard methods.
What techniques are used to differentiate between D- and L-amino acids in samples?
Differentiating between D- and L-amino acids typically involves using chiral chromatography or mass spectrometry techniques. Chiral chromatography utilizes stationary phases that can separate D- and L-forms based on their stereochemistry. Mass spectrometry can also help identify and quantify the different isomers by their unique mass-to-charge ratios. This is particularly important in studying metabolic pathways and understanding specific biochemical processes.
How do you ensure the accuracy and reproducibility of amino acid quantification?
Accuracy and reproducibility in amino acid quantification are achieved through stringent quality control measures, including the use of internal standards and calibration curves. By incorporating isotopically labeled amino acids as internal standards, any variability in sample preparation or analysis can be accounted for, ensuring consistent results. Additionally, replicating measurements across multiple runs helps to confirm reproducibility. Our experienced technical team continually reviews protocols and results to maintain high standards in data quality.
Learn about other Q&A about proteomics technology.
High-throughput method for Peptide mapping and Amino acid sequencing for Calcitonin Salmon in Calcitonin Salmon injection using Ultra High Performance Liquid Chromatography–High Resolution Mass Spectrometry (UHPLC-HRMS) with the application of Bioinformatic tools.
KURIL, Akhilesh Kumar, and K. SARAVANAN.
Journal: Journal of Pharmaceutical and Biomedical Analysis
Year: 2024
Trypanosoma cruzi DNA Polymerase β Is Phosphorylated In Vivo and In Vitro by Protein Kinase C (PKC) and Casein Kinase 2 (CK2).
MALDONADO, Edio, et al.
Journal: Cells
Year: 2022
Reducing branched-chain amino acids improves cardiac stress response in mice by decreasing histone H3K23 propionylation.
YANG, Zhi, et al.
Journal: The Journal of Clinical Investigation
Year: 2023
Confirmation of translatability and functionality certifies the dual endothelin1/VEGFsp receptor (DEspR) protein.
HERRERA, Victoria LM, et al.
Journal: BMC Molecular Biology
Year: 2016
See more articles published by our clients.
