Kairos Amino Acid Sample PrepMetabolomics, Biopharma, Biomedical research, Proteomics
Analysis of Amino Acids is Essential to the Research of Various Disease States
The amino acid sequence is the primary structure of a protein and is unique to particular peptide and protein structures. Determining the structure of protein/peptides is important in biomedical research. Amino acids play an important role in physiological analysis. For example, the analysis of amino acids in subjects in the control group can provide baseline data to compare disease states against.
LC-MS, Automation, and Amino Acid Analysis (AAA)
Application benefits of the Kairos Amino Acid workflow include the following:
- Comparable to manual preparations, with the added benefit of automation using the Andrew+ Pipetting Robot freeing up analyst time to perform other tasks,
- OneLab is a user-friendly, intuitive cloud-native software,
- Fast analytical run times (< 10 mins),
- Confidence in peak detection,
- Greater flexibility with laboratory resources. Kairos Amino Acids workflow removes the need for a dedicated amino acid system,
- Analytical selectivity of the chromatographic method provides separation of isobaric species.
Figure 1: Kairos Amino Acid sample preparation workflow. Step 1 corresponds to the "Sample Transfer & Protein Precipitation" protocol. Step 2 corresponds to the "Derivatization" protocol.
This workflow describes the sample transfer, protein precipitation, and derivatization of amino acid samples for quantitation on an LC-MS platform, for instance, the ACQUITY UPLC I-Class Xevo TQ-S micro.
➤ STEP 1 | Sample Transfer & Protein Precipitation (T&P)
Calibrators, QCs, and Internal Standard are reconstituted manually, and along with the samples, are aliquoted into 1.5 mL microtubes. Subsequently, the Andrew+ Pipetting Robot transfers the samples and performs the protein precipitation for up to 96 samples at a time. For reconstitution details, please follow the link in the resources section below.
Kairos T&P Workflow
- Add 50 μL of calibrator or QC or sample
- Add 50 μL of Internal Standard (mix)
- Add 50 μL of water (mix)
- Normalize samples if required (recommended for samples at a concentration of ≥ 500 μM)
- Centrifuge for 15 minutes at 3000 x g
Figure 2: Andrew+ deck configuration for the automated "Kairos T&P, 96 Samples - Andrew+" protocol. Domino/Device positions on the Andrew+ working deck: [1&2] Tip Insertion System Domino, [3-6] Microtube Domino,  Deepwell Microplate Domino,  Microplate Shaker+.
➤ STEP 2 | Amino Acid Derivatization
Precipitated samples are derivatized using the well-known AccQ•Tag Ultra "3X" Derivitization Kit (Waters, p/n 186004535). Kairos Derivatization is compatible with both "Kairos T&P, Samples - Andrew+" and "Kairos T&P, Samples, Normalization - Andrew+" protocols.
Kairos Derivatization Workflow
- Add 70 μL of Borate Buffer
- Add 10 μL of the supernatant obtained in STEP 1 - Kairos T&P (mix)
- Add 20 μL of AccQ•Tag reagent (mix)
- Heat for 10 minutes at 55°C
- Samples are sealed using cap mats and then transferred to LC-MS for analysis
Figure 3: Andrew+ deck configuration for the automated "Kairos Derivatization, 96 Samples - Andrew+" protocol. Domino/Device positions on the Andrew+ working deck: [1-4] Tip Insertion System Domino,  Storage Plate Domino,  Microtube Domino,  Deepwell Microplate Domino,  Microplate Shaker+,  96-PCR Plate Peltier+,  8-Channel Pipette Reservoir Domino.
- "Kairos T&P, Samples, Normalization - Andrew+" protocols (x48/96) are used to normalize samples across the entire plate, thereby automating the whole process. Once samples are prepared, a 950 μL volume of 0.1 M HCl is dispensed across all samples (low and high).
- Anserine, Hydroxyproline and, Argininosuccinic Acid are affected at the lower end of the calibration line by the normalization across all samples. When measuring samples at this level, it is recommended to use "Kairo T&P, Samples - Andrew+" protocols (x48/96). These protocols have been optimized to provide the best results for these amino acids at lower levels (it is recommended to normalize standards/QCs/samples at a concentration of ≥ 500 μM).
- In the "Kairos T&P, Samples - Andrew+" protocols (x48/96), samples are processed without normalization. A user's intervention offers the possibility to normalize samples at a concentration of ≥ 500 μM prior to centrifugation.
- One vial of "3X" AccQ•Tag reagent, one vial of Reagent Diluent, and two vials of Borate Buffer are required for the "Kairos Derivatization, 48 Samples - Andrew+" protocol.
- Two vials of "3X" AccQ•Tag reagent, Reagent Diluent, and Borate Buffer are required for the "Kairos Derivatization, 96 Samples - Andrew+" protocol. This is due to the 5 mL dead volume of the Agilent 6-column reagent reservoir.
- The Borate Buffer should be pH-adjusted with 0.5 M NaOH prior to use at the ratio 430 μL of 0.5 M NaOH to 1 vial of Borate buffer.
- The "3X" AccQ•Tag reagent should be heated for 10 minutes shortly before commencing the following steps to ensure adequate dissolution of the material.
Application Kits and Standards
- Kairos Amino Acid Calibrator Set (500+) by Waters | p/n 186009048
- Kairos Amino Acid QC Set (500+) by Waters | p/n 186009049
- Kairos Amino Acid Internal Standard Set (500+) by Waters | p/n 186009050
- CORTECS C18 Column by Waters | p/n 186007096
- AccQ•Tag Ultra "3X" Derivitization Kit by Waters | p/n 186004535
- Kairos Amino Acid Reconstitution Reagents by Waters | p/n 186009103
- AccQ-Tag Ultra Borate Buffer by Waters | p/n 186009283
- Waters 2 mL 96-square well collection plate, cut corner H1 by Waters | p/n 186002482
- Fisherbrand™ Premium 1.5 mL microtube by Fisher Scientific | p/n 11926955
- Agilent 6-column reagent reservoir by Agilent Technologies | p/n 201284-100
- Eppendorf twin.tec® 96-well skirted LoBind® PCR plate by Eppendorf | p/n 0030129555
- INTEGRA 10 mL multichannel reservoir by INTEGRA Biosciences | p/n 4332
- 6 mm pre-slit Silicone/PFTE cap mats by Analytical Sales and Services Inc. | p/n 96727
Recommended Reagents and Chemicals
- 0.1 M Hydrochloric acid (HCl) solution by Honeywell | p/n 35335 or equivalent
- 0.5 M Sodium hydroxide (NaOH) solution Titripur® by Supelco/Merck | p/n 109138 or equivalent
- CHROMASOLV™ LC-MS grade water by Honeywell | P/N: 39253 or equivalent