Amino acid analysis instrumentation

The painstaking procedures and special instrumentation required for manipulating nanoliter range volumes were described for the determination of amino acids in single cells218 and the amino acid analysis of subnanogram amounts of protein219. The amino acid extract... 

The fourth category of protein assay is amino acid analysis. This method is the most accurate and robust method for determination of protein concentration, but is appropriate only for pure proteins. In addition, it is relatively slow and requires specialized instrumentation and knowledge of the target protein s theoretical amino acid composition. 

Some laboratories do not have access to mass spectrometric analysis, but the number is fewer as the cost for this type of instrumentation is decreasing. It is suggested that these laboratories utilize amino acid analysis due to reduced cost and rapid turnaround. Peptide composition and stoichiometry can be determined, the technique is highly reproducible, and can be used to monitor cycle-to-cycle coupling efficiency. However, not all amino acids are recovered quantitatively. Cys and Trp are totally destroyed and must be quantitated using distinctly different hydrolysis procedures. Ser and Thr can be partially destroyed. Some laboratories perform amino acid analysis in addition to mass spectrometric analysis in order to assure peptide composition, stoichiometry, and quantity (see also Sections 7.3, 7.3.1 and 7.3.2). 

Modification of the prcformulation format for biotechnological products from the original guidance must be considered. The sections regarding chemical structure, physicochemical properties, and stability may be revised according to the nature and characteristics of proteins and peptides. Aside from the conventional analytical instruments and techniques used in the study of small molecules, methods such as amino acid analysis, sequence analysis bioassay, immunoassay, and enzymatic assay are commonly used and should be included in the report. 

With the advances achieved in mass spectrometry instrumentation over the past decade, amino acid analysis (AAA) is less frequently utilized for the quality control of synthetic peptides than it was in the past. AAA remains, however, the method of choice for the quantitative analysis of synthetic peptides. Although in most cases AAA is conducted after cleavage of the peptide from the resin, a peptide that is still attached to the solid support can be analyzed as well. Analysis of a peptidyl resin is often facilitated by the fact that many polymeric supports incorporate an internal amino acid reference [30]. 

Precolumn derivatization methods are not free from disadvantages. One is that side-products from the reaction can sometimes be difficult to separate from the peaks of interest. Also with precolumn derivatization, as its name implies, each sample must be derivatized prior to injection. This can add to the total analysis time. However, instrumentation is now available to derivatize several samples simultaneously in a closed system, which not only solves the sample preparation time problem but also results in highly reproducible chemistry. Since the reaction may not go to 100%, an internal standard is frequently necessary. Finally, the addition of a similar tag to all the amino acids may make them more difficult to separate. Table 1 summarizes the properties of several pre- and postcolumn derivatizing reagents employed for amino acid analysis. 

Amino acid analysis is commonly used for the identification of proteins in paint layers, where contamination is low, the conservation state is generally good, and the range of possible proteins is very limited (mainly egg, collagen, or casein). However, the identification of archaeological proteinaceous residues is by no means an easy task and requires peptide mapping using HPLC/ MS or MALDI techniques (see Section 36.5). With the increased availability of advanced instrumentation, pro-teomics may become the favored approach for protein determination also in paint layers [21]. 

High-sensitivity amino acid analysis is usually done by precolumn derivatiza-tion (for a review, see Lottspeich and Henschen, 1982) followed by reverse-phase HPLC in an fully automated and dedicated instrument. The described combined o-phthaldialdehyde (OPA)/9-fluoromethoxycarbonyl (FMOC) derivatization of the amino acid has the advantage of being sensitive and, at the same time, reacting with all amino acids commonly present in proteins (Schuster, 1988). 

Supplies, reagents, and instrumentation for amino-acid analysis (for example Applied Biosystems, Perkin-Elmer, model 420H/130/920 Amino acid analyzer and model 470/120/900 protein sequencer). 

Ammo acid analysis of purified rCRALBP is useful to quantify the protein, and for corroborating the identity of the recombinant protein (Table 1). We typically perform phenylthiocarbamyl (PTC) amino acid analysis on about 1 pg rCRALBP samples (20-30 pmol protein per analysis) using Applied Biosystems instrumentation (26). 

The CML content of the samples was quantified by acid hydrolysis with 6 N of HCl for 24 hr at 1 lO C, followed by amino acid analysis on a Hitachi L-8500A instrument equipped with an ion-exchange HPLC column ( 2622 SC, 4.6 x 80 mm Hitachi). ... 

Chromatographic procedures applied to the identification of proteinaceous paint binders tend to be rather detailed consisting of multiple analytical steps ranging from solvent extractions, chromatography clean up, hydrolysis, derivatisation reactions, and measurement to data analysis. Knowledge of the error introduced at each step is necessary to minimise cumulative uncertainty. Reliable results are consequently obtained when laboratory and field blanks are carefully characterised. Additionally, due to the small amounts of analyte and the high sensitivity of the analysis, the instrument itself must be routinely calibrated with amino acid standards along with measurements of certified reference proteins. All of these factors must be taken into account because many times there is only one chance to take the measurement. 

GC-C-IRMS instrumentation enables the compound-specific isotope analysis of individual organic compounds, for example, n-alkanes, fatty acids, sterols and amino acids, extracted and purified from bulk organic materials. The principle caveat of compound-specific work is the requirement for chemical modification, or derivatisation, of compounds containing polar functional groups primarily to enhance their volatility prior to introduction to the GC-C-IRMS instrument. Figure 14.7 summarises the most commonly employed procedures for derivatisation of polar, nonvolatile compounds for compound-specific stable isotope analysis using GC-C-IRMS. 

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