Proteomics > Which service should I request? > Amino Acid Analysis > Amino acid - Composition / Quantification
The workflow consists of four major steps:
Amino acid - Composition / Quantification
Table of contents
General description
There are many different methodologies to hydrolyze protein/peptide into amino acids. The main protocol used at FGCZ is using acid hydrolysis. This procedure allows to obtain higher efficiency of acid hydrolysis but the side chains of Gln and Asn will convert to Glu and Asp. In addition, tryptophan would be destroyed under this condition. Therefore, only 17 amino acids will be reported in this analysis.Workflow
The workflow consists of four major steps:
- Acid hydrolysis: proteins/peptides are hydrolyzed together with isotope labelled amino acids under 6M HCl with 0.1% w/v phenol stream at 110°C for 24 hours
- Labeling: reaction with Accq Tag Ultra (aminoquinolyl-N-hydroxysuccinimidyl carbamate). The reaction is shown below:
- LC-QDA analysis: Cortex UPLC reverse-phase separation of the amino acid derivatives
- Data analysis: Data are processed by TargetLynx™ of MassLynx™ Software (Waters)(MassLynx)
- Calibration curve establishment: 2.5 uM to 250 uM per amino acid (7 points) by Standard H and isotope-labeled amino acid mixture
- amino acid quantitifcaiton
Requirements and considerations
- volume: > 5 uL
- minimal amount: 1-5 ug; optimal amount: 10 ug
- dissolve samples in volatile solvents or free of non-volatile buffers, salts, detergents, and primary amine