Protein and peptide samples are aliquoted and mixed with Internal Standards (see Quantitation), dried in glass tubes (6 x 50 mm, Fisher PN 14-957AA) in a vacuum concentrator and subjected to vapor phase hydrolysis by 6N HCl (Thermo Sci # 24308) at 150ºC for 1.5 hours under argon atmosphere in the presence of phenol (2%, Sigma #P5566) which limits the halogenation of Tyrosine residues. The samples are subsequently reconstituted in 0.4 N Borate Buffer (Agilent # 5061-3339) to bring the eventual pH to 10 for optimum derivatization and transferred to the Agilent G1367E autosampler for automated derivatization and loading. In cases where Cysteine data is requested, Dithiodiproprionic Acid (Acros Organics # 14800500) is added at the beginning of the assay to convert Cysteine to S-2-carboxyethylthio-L-cysteine (Cys-MPA) which is preserved during hydrolysis.
Insoluble samples (feeds, waste, cloth etc.) are weighed, Internal Standards added, and liquid-phase hydrolysis is used. Enough 6N HCl is added to cover the sample and samples are exposed to 100ºC for 22 hours. Since the alkylation reagent for Cysteine is not soluble in acid, cysteine conversion and quantitation is not available for this assay. Liquid HCl samples are filtered after hydrolysis and a portion of the filtrate is dried down, the HCl is pulled off, and reconstituted in 0.4 M Borate buffer (pH 10) prior to derivatization.
Note: In cases where a proteinaceous sample undergoes hydrolysis, any free amino acids that are present in the sample matrix and survive the hydrolysis will be included in the quantitation. While this is not a consideration for purified proteins and peptides, it is a consideration in complex matrices like serum or culture media.
Note: In all cases where hydrolysis is used, Asparagine and Glutamine are deamidated to their respective acids. Results for these residues are reported as ASX and GLX to denote that these data contain the combined amounts from both the amide and the acid. Acid hydrolysis also destroys Tryptophan. If it is necessary to quantitate this residue, a separate assay is performed using 4 N Methansulfonic Acid, Sigma # M4141(with 0.2%w/v tryptamine) for hydrolysis. The Tryptophan analysis has a very high %RSD (~25%) and requires ~50-100 micrograms of sample. It requires two assays and there is an additional charge.
Physiological samples or samples where only the free amino acids are of concern might be filtered to remove proteins or dried down and then reconstituted in 0.4 N Borate Buffer to bring the eventual pH to 10 for optimum derivatization. No hydrolysis is involved for free amino acid determination and Tryptophan can be measured. Cystine can be measured using a different detector but Cysteine cannot be measured.
The Agilent 1260 HPLC analyzes all samples by pre-column derivatization of the amino acids with o-phthalaldehyde (OPA), Agilent #5061-3335 and 9-fluoromethyl-chloroformate (FMOC) Agilent #5061-3337. OPA reacts with primary amino acids and FMOC with secondary amino acids (Proline). Both reagents react rapidly and quantitatively and give highly fluorescent and UV-absorbing isoindole derivatives. The derivatized amino acids are separated by reverse phase HPLC and detected by UV absorbance(primaries at 338/390 nm and secondaries at 266/324 nm) with a variable wavelength detector (G1365D) or by fluorescence (primaries at excitation/emission 340/450 and secondaries at 266/305) using an in-line fluorescence detector (G1321B).