Amino Acid Analytes

6 AMINO ACID, PEPTIDE, AND PROTEIN ANALYTES 4.6.1 Amino Acid Analytes 

Amino acid analysis is important not only from a clinical aspect— amino acid metabolism disorders can be fatal if not diagnosed early—but also as the basis for protein and peptide sequencing. Due to the combination of the small amounts of material used in an actual analysis and the lack of chromophores on most amino acid residues, derivatization is common practice. Derivatization may be done prior to either separation or detection. 

Heam and co-workers [460,461] have published a series of results relating amino acid hydrophobicity to reversed-phase retention of amino acids and peptides. The authors used wide-pore C4 or C]g columns and a gradient of IPA/acetonitrile/water (0.1% TEA) in generating results. A total of 1738 peptides covering 12 previously generated amino acid hydrophobicity scales were part of the study. Predicted and actual retention times of overlapping heptamers in myohemerythrin were presented. This study and previous studies cited therein offer excellent theoretieal and experimental bases for the predictive chromatography of amino acids and peptides. 

The isoaspartic acid released during the deamidation of asparginine from peptides and proteins was monitored on a C g column (A = 254nm) using an 85/15- 50/50 (at 10 min hold 5 min)- 0/100 (at 18 min hold 3 min) (90/10 water [25 mM KH2PO4 with octanesulfonic acid to pH 3.2]/methanol)/methanol 

Plasma amino acids were separated as their o-phtfaalaldehyde derivatives in 20 min on a C g column (A = 230 nm, ex 389 nm, em) using a 98/2 - 0/100 (50/50 water/water [9mM KH2PO4 with 0.5 M TEA to pH 6.9])/(35/15/50 methanol/acetonitrile [9mM KH2PO4 with 0.5 M TEA to pH 6.9]) gradient [464], Linear plots of fluorescence emission versus concentration from 5 to 800pmol/L were obtained. It should be noted that separation was complete with baseline resolution in 12 min but significant column reequilibration time between injections was necessary. 

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CSP, beads chiral monomers (from amino acids) analytical to preparative fair, moderate to low... 

For general biochemical implications of amino acid transport, see ref. Ic. For problems relevant to amino acid analytical chemistry, see Amino Acid Analysis, Rattenbury, J. M., Ed, Ellis Horwood, Chichester, 1981. 

Reference values of this approach are not different from those for other amino acid analyses. An example of a mass chromatogram, representing the plasma of a PKU patient, is shown in Fig. 2.1.1. When evaluating the results of MS/MS amino acid analyses, one has to reahze that the hquid chromatographic separation is by far less efficient that the AAA separation. For this reason, any amino acid may (partly) coelute with other amino acid(s), which potentially interferes with its mass spectromet-ric behavior. This effect is known as quenching. In order to overcome this as much as possible, stable-isotope-labeled internal standards (as many as possible) should be used. However, this matrix effect of ion suppression is the major pitfall in the MS/MS analysis of amino acids. Consequently, the MS/MS analysis of amino acids cannot be regarded as a reference method, similar to all other amino acid analytical methods. 

Mayne PD, Roche G, Deverell D (2001) Amino acids analytical aspects, Workshop report. J Inherit Metab Dis 24 305-308... 

Figure 2 (Opposite) RP HPLC chromatograms of the Alzheimer s Ap 1-42 peptide synthesized by different protocols for activation of the Fmoc-amino acids. Analytical RP HPLC employed a Waters system with a Vydac C4 (214TP54) column at a flow rate of 1.0 ml/min. The peptides were eluted by gradient (5-95 % B, 60 min) with 0.1 % TFA (buffer A) and 0.1 % TFA / acetonitrile (buffer B). a. Crude product obtained using BOP/HOBt/NMM 40 °C acylations + 40 °C deprotections, b. Cmde product obtained using BOP/HOBt/NMM 40 °C acylations 55 °Cdeprotections. c. Crude product obtained using Preformed Fmoc-aminoacyl fluorides 40 °C acylations 55 C deprotections, d. RP HPLC purified peptide.

In order to calculate amino acid residence times from Equation 21, the ratio of the d to l enantiomers of the various amino acids are required as a function of depth in the oceanic water column. Unfortunately, there have been no investigations of the amino acid enantiomers dissolved in any natural waters. The analyses are difficult because most d- and l-amino acids are not separable by the usual amino acid analytical techniques. One exception is isoleucine, which forms alloisoleucine when it racemizes (Equation 13). Isoleucine and alloisoleucine are separable on the buffered columns of the automatic amino acid analyzer (88). However, as can be seen from Table V, only very small amounts of alloisoleucine would be produced from the racemization of isoleucine, unless... 

D.H. Spackmann, W.H. Stein, S. Moore, Automatic recording apparatus for the use in the chromatography of amino acids, Analyt. Chem. 30 1190-1206 (1958)... 

Grigorean, G. Ramirez, J. Ahn, S. H. Lebrilla, C. B. A mass spectrometry method for the determination of enantiomeric excess in mixtures of D,L-amino acids. Analytical Chemistry 2000, 72, 4275 281. 

Since the focus has been on the ten essential amino acids, most nutritional laboratories have optimized their methods around these plus some of the other protein amino acids. Recently, nutritional supplements have begun to include some of the nonprotein amino acids such as ornithine in their formulations. These nonprotein amino acids may cause interference when analysis is performed by methods optimized for standard nutritional work. There are amino acid analytical methods developed for physiological matrices, which include many of the nonprotein amino acids. These methods may be adapted for nutritional matrices. There is also some... 

Reference has already been made to recent work in which the tyrosine and tryptophan content of proteins was estimated by microbiological methods after hydrolysis by alkali. The assumption is made that, with the conditions selected, the two amino acids are completely racemized before hydrolysis is complete. This may not be quite correct in all cases, and, since the concentration of alkali and the period of heating chosen may not be sufficient to racemize the free amino acids, analytical errors may arise. The work of Dakin and Dudley has clearly shown that even closely related proteins behave differently in this respect and caution must therefore be exercised in accepting generalizations as to the behavior of different amino acid residues in the various proteins to alkali. The racemization of proteins by acid will be discussed in the next section, page 373-375. 

Chemical methods for determining the sequence of amino acids in a protein couples identification of the end N-terminal amino acid by an automated amino acid sequencer to an automated amino acid analyTer. Modern instruments can determine sequences for protein or peptide samples that contain 10-100 picomoles, pm (1 pm = lO moles). The overall process requires several automated steps. 

Imaging/Labeling Applications Amino acids " analytes azobenzene photoswitching in vivo, leucine lysines " small molecules metal ions transition metal complexes " nucleic acids peptide analog proteins " " silane coupling agent " sUica particles eads " uronium salts ... 

Imaging/Labeling Applications Amino acids analytes " lysines peptides " silane coupling agent silica particles/beads ... 

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