Amino acids sample contamination

As a result, the baseline enantioseparation was obtained, as shown in Figure 19.1a. Monomeric Pro is one of these rare amino acids, which develop yellow (and not bluish) color when visualized with ninhydrin. l-Pto used as an external standard (Figure 19.1b) confirmed the identity of the lower yellow spot number 2 as enantiomer L and the upper yellow spot number 3 as enantiomer d, as shown in Figure 19.1a. The respective Rp values were 0.57 0.02 and 0.74 0.02. Brownish-purple spot number 1 (Rp = 0.32 0.02 Figure 19.1) apparently originates from the Pro-derived peptides and it is also fully separated from the monomeric l-Pto spot number 2. The presence of peptides in the two freshly prepared Pro samples wimesses to rapid peptidization of this amino acid (although contamination of the commercial monomeric DL-Pro and L-Pro samples with peptides cannot be excluded). 

L-tryptophan is a naturally-produced, natural amino acid used as a dietary supplement. Samples from one manufacturer were found to be contaminated by trace amounts of another naturally occurring compound — l,l -ethylidene bis-[i.-tryptophan]. The latter compound appears to have been responsible for causing a number of deaths and additional cases of chronic eosinophilia-myalgia,111 some of which might have been prevented had there... 

The question was whether impurities were present in the samples analysed (Bada et al., 1983). In a more recent publication, Cronin and Pizzarello (1997) reported amino acid analyses using Murchison material in which an excess of L-enantiomers was present. Contamination with terrestrial biological material can be ruled out, as the amino acids in question are not proteinogenic a-methylamino acids, which occur either extremely seldom or not at all in terrestrial life forms, were detected. GLPC/mass spectrometry (MS) analysis gave the following enantiomeric excess (ee) values ... 

The number of scientific articles published on meteorites has increased dramatically in the last few years few of these, however, concern themselves with small meteorites, the size of which lies between that of the normal meteorites (from centimetres to metres in size) and that of interplanetary dust particles. In the course of an Antarctic expedition, scientists (mainly from French institutions) collected micrometeorites from 100 tons of Antarctic blue ice (Maurette et al 1991). These micrometeorites were only 100 400 pm in size five samples, each consisting of 30-35 particles, were studied to determine the amount of the extraterrestrial amino acids a-aminoisobutyric acid (AIBS) and isovaline—both of which are extremely rare on Earth—which they contained. The analysis was carried out using a well-tested and extremely sensitive HPLC system at the Scripps Institute, La Jolla. Although the micrometeorites came from an extremely clean environment, the samples must have been contaminated, as they all showed traces of L-amino acids. Only one sample showed a significantly higher concentration of AIBS (about 280 ppm). The AIBS/isovaline ratio in the samples also lay considerably above that previously found in CM-chondrites. 

GC requires low molecular weight molecules, and the macromolecular nature of proteinaceous materials (made up of 21 amino acids covalently condensed) means that they are typically too large to be readily identifiable and time consuming pretreatments of the sample are required. In order to free the amino acids, hydrolysis is required. Subsequent steps of purification to eliminate pigment interferences are also often necessary. Consequently sample pretreatments must be carefully carried out to reduce the risk of loss and/or contamination of the sample. 

Figure 9.1a reporting GC-MS results taken on laboratory blank in a GC-MS procedure [87] shows negligible amino acid contamination. Indeed, quantitating a protein as the sum of 14 amino acids, a value of about 200 ng is the minimum required threshold to positively consider the protein identification. Figure 9. lb reports the chromatogram of an amino acid standard solution acquired in the SIM mode and corresponds to the quantitation limit that is a content of about 700 ng in the sample at a confidence level of 95%. This seems amenable when working at the trace level. 

Essentially the same amino acids, and nearly equal quantities of D and L enantiomers, were detected in the Murray meteorite, another type II carbonaceous chondrite [6]. Recent expeditions to Antarctica have returned with a large number of meteorites, many of which are carbonaceous chondrites. These may have been protected from terrestrial contamination by the pristine Antarctic ice. Careful analysis of two of these, the Yamato (74662) and the Allan Hills (77306), both type II carbonaceous chondrites, by ion exchange chromatography, gas chromatography, and GC/MS, have detected a wide variety of both protein and non-protein amino acids in approximately equal D and L abundances [9,10]. Fifteen amino acids were detected in the Yamato meteorite and twenty in the Allan Hills, the most abundant being glycine and alanine. The amino acid content of the Yamato meteorite is comparable with that of the Murchison and Murray, but the Allan Hills contains 1/5 to 1/10 that quantity. Unlike earlier meteorites from other locations, the quantities of amino acids in the exterior and interior portions of the Yamato and Allan Hills meteorites are almost identical [9,10]. Thus, these samples may have been preserved without contamination since their fall in the blue ice of Antarctica, which js 250,000 years old in the region of collection. 

Several factors indicate that the amino acids detected in all of these carbonaceous chondrites are indigenous and that they must have originated abiotically. First, the presence of protein and non-protein amino acids, with approximately equal quantities of D and L enantiomers points to a nonbiological origin and precludes terrestrial contamination. In addition, the non-extractable fraction of the Murchison is significantly heavier in 13C than terrestrial samples. Finally, the relative abundances of some compounds detected resemble those of products formed in prebiotic synthesis experiments. The aliphatic hydrocarbons are randomly distributed in chain length, and the C2, C3, and C4 amino acids have the highest concentrations (i.e., the most easily synthesized amino acids with the least number of possible structures are most abundant) [4]. 

The use of the relative proportions and racemization properties of amino acids as a means of identifying contaminated samples would have several advantages. First of all, modern amino acid analyzers are very sensitive analytical instruments. Systems are... 

Fig. 2. NAPI facilitates H2A, H2B release from nucleosomes that are on positively coiled DNA (A) but not negatively coiled DNA (B). The positively coiled DNA (6.0 kb) with a superhelical density of + 0.05 and negatively coiled DNA (6.0 kb) with a superhelical density of -0.05 were reconstituted with lysine, arginine-labeled histones H3, H4, H2A, H2B by NaCl dialysis from 2.0 M to 1.2 M to 0.6 M to 0.1 M NaCl over a 14 h period. The samples were incubated with NAPI at 35 °C for 5 min and applied to a 5-20% sucrose/100 mM NaCl/40 mM Tris, pH 7.8 gradient. After sedimentation at 200,000 X g for 5 h, fractions were collected and the distribution of DNA (bottom panel) was determined on agarose gel and the distribution of protein (top panel) on SDS-PAGE followed by fluorography. These data are unpublished observations (V. Levchenko and V. Jackson). The deg-H2A is degraded H2A in which a 15 amino acid peptide of the C terminal has been proteolytically removed. When H2A, H2B is no longer present in a nucleosome, the C terminal region is sensitive to proteolysis [126] from a protease which is a minor contaminate in the NAPI preparation.

CSF should be free of any blood contamination because of the marked differences between plasma and CSF amino acid levels. Usually the first milliliter of CSF is used for routine measurements and the second milliliter is used for amino acid analysis. CSF samples are routinely stored at - 80°C. 

Before leaving the aminoacids problem, it is interesting to note that aminoacids have been detected in carbonaceous chondrites found in Antarctica. The risk of contamination is much less important in Antarctica than in Australia and this is one of the reasons why these studies were undertaken. They fully confirm the results obtained on Murchison 54,55), even if in one CM carbonaceous chondrite the amino acid content was only 10% of what was observed in Murchison 56,57). The contamination is in fact lower than in Murchison the aminoacid content was very similar for samples taken near the surface of the Antarctica chondrites or from their bulk. On the other hand, all the significant analyses on Murchison were performed on samples from the interior of the meteoritic fragments due to the high degree of surface contamination. In the case of the Allende meteorite, which has the same terrestrial age as Murchison, contamination was found to extend to a depth of more than 5 mm below the surface 52). 

Next, the removal of any suspected contaminants—such as carbohydrates (e.g., glucose), free amino acids, nucleotides and so on—can be accomplished by the procedure of Wells and Dittmer (1963). The lipid sample, dissolved in a mixture of chloroform-methanol-water (60 30 4.5, v/v), is passed through a previously washed column of Sephadex G-25, the effluent is collected and saved, and the column is then washed with a mixture of chloroform-methanol (2 1, v/v). The second eluent is collected and combined with the first and will contain all the phospholipid, free of contaminants. The two eluates are combined, phased by the addition of water, and then the chloroform-rich layer is removed and evaporated to dryness under nitrogen. The residue is dissolved in chloroform-methanol (2 1, v/v) and made to volume in a glass-stoppered volumetric flask. 

---