Amino Acid Analysis (AAA)

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.

Our amino acid analysis service consists of

• Quantification of amino acid analysis
• Quantification of protein or peptide
• Quantification of protein traces
• Accurate quantification of protein or peptide
• Determine amino acid sequence of protein, peptide and mAb
• Amino acid composition analysis

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

• Sample Format: Solid samples or in solution.
• Sample Amount:
  1) Dry protein/peptide samples: 100 µg of sample or greater at a concentration of approximately 0.5-2 mg/mL.
  2) Liquid material: >100 μL at a minimum concentration of 0.5 mg/mL.
• Please do not hesitate to contact us for any additional sample requirements.

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

1. 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

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.