Attomole

Analytical Applications. Chemiluminescence and bioluminescence are useful in analysis for several reasons. (/) Modem low noise phototubes when properly instmmented can detect light fluxes as weak as 100 photons/s (1.7 x 10 eins/s). Thus luminescent reactions in which intensity depends on the concentration of a reactant of analytical interest can be used to determine attomole—2eptomole amounts (10 to 10 mol). This is especially useful for biochemical, trace metal, and pollution control analyses (93,260—266) (see Trace and residue analysis). (2) Light measurement is easily automated for routine measurements as, for example, in clinical analysis. 

The most widely appHed colorimetric assay for amino acids rehes upon ninhydrin-mediated color formation (129). Fluorescamine [38183-12-9] and (9-phthalaldehyde [643-79-8] are popular as fluorescence reagents. The latter reagent, ia conjunction with 2-mercaptoethanol, is most often used ia post-column detection of amino acids separated by conventional automated amino acid analysis. More recently, determiaation by capillary 2one electrophoresis has been developed and it is possible to determine attomole quantities of amino acids (130). 

Simple etching of the capillary end served to decouple the electrophoretic current from that of amperometric detection, permitting quantitation of attomole levels of catecholamines from brain microdialyzates.24 A postcolumn reactor using bromine generated electrochemically in situ has been used in the detection of peptide thiols, such as glutathione and cysteine, separated by capillary electrophoresis.25... 

The sensitivity of electrophoretic methods extends to the attomole range, which is particularly useful for scarce samples (seldom a problem in additive analysis) and degradation products. 

Three different lots of a standard 3.2-kb HCV RNA were serially diluted and quantified over a thousandfold range. The average signal per attomole of target varied by less than 20% among the three lots. 

Ivanov, A.R., Zang, L., Karger, B. L. (2003). Low-attomole electrospray ionization MS and MS/ MS analysis of protein tryptic digests using 20 pm-i.d. polystyrene-divinylbenzene monolithic capillary columns. Anal. Chem. 75, 5306-5316. 

Sensitivity. MS is an extremely sensitive method. Nowadays, substances over a wide range of molecular weight can be measured [at the attomol (10 18 mol) or subattomol level]. 

Primary amines are derivatized readily and quantitatively as illustrated in reaction 15. CE and detection by LIF had LOD in the low attomol (1 x 10-18) range for amino acids and amino sugars320 321. 

For SDS-protein complexes, the proteins can be derivatized with fluorescent dyes prior to the analysis. Derivatized proteins can be detected in the attomole (amol) level, and because the complex is resolved by sieving, multiple reaction products are detected as peak broadening instead of multiple peaks.19... 

Note Modem FT-ICR mass spectrometers offer ultrahigh resolving power (R= 10 -10 ) [193,194] and highest mass accuracy (Am = 10" -10 u, cf. examples in Chaps. 3.3.2 and 3.4.1), attomol detection limits (with nanoESI or MALDI sources), high mass range and MS capabilities. [195]... 

Chatman K, Hollenbeck T, Hagey L, Vallee M, Purdy RH, et al. 1999. Nanoelectrospray mass spectrometry and precursor ion monitoring for quantitative steroid analysis and attomole sensitivity. Anal Chem 71 2358-2363. 

Valaskovic M, KeUeher NK, McLafferty FW. 1996. Attomole protein characterization by capillary electrophoresis—mass spectrometry. Science 273 1199-1202. 

An interesting study by Tetter et al. indicates that reducing the dimensions of the capillary results in better operation and sensitivity (17). Another paper describes protein analysis at the attomole level using 5-p.m-ID capillaries... 

JH Wahl, DR Goodlett, HR Udseth, RD Smith. Attomole-level capillary elec-trophoresis-mass spectrometric protein analysis using 5-/rm-i.d. capillaries. Anal Chem 64 3194-3196, 1992. 

H3. Hashida, S., Tanaka, K., Yamamoto, N., Uno, T, Yamaguchi, K., and Ishikawa, E., Detection of one attomole of [Arg ]-vasopressin by novel noncompetitive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay). J. Biochem. (Tokyo) 110, 486-492 (1991). 

Many of the advantages that MALDI offers for peptide analysis are equally applicable to proteins. Protein analysis is similar to peptide analysis, in which ionization usually occurs through the addition of one, two, or three protons. However, since proteins are significantly bigger than peptides, ion detection is typically less efficient. Therefore, while peptides are measured at the femtomole or even attomole level with MALDI, proteins are usually measured at the high femtomole to low picomole level. 

In 1974, Comarisov and Marshall60 developed Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). This technique allows mass spectrometric measurements at ultrahigh mass resolution (R = 100000-1000000), which is higher than that of any other type of mass spectrometer and has the highest mass accuracy at attomole detection limits. FTICR-MS is applied today together with soft ionization techniques, such as nano ESI (electrospray ionization) or MALDI (matrix assisted laser/desorption ionization) sources. 

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