Amino acid mass spectrometry

Despite the high efficiency of the present-day chromatographic and electrophoretic techniques and the fast developments in mass spectrometry, amino acid analysis still represents a valuable analytical tool in peptide chemistry for characterization of the final products, but also for monitoring intermediate coupling steps in solution and on solid supports by comparing ratios of diagnostic amino acids. 

Edman degradation was originally developed for determination of the primary structure (i.e., amino acid sequence) of peptides and proteins. Sequence analysis is not regularly performed for quality control in routine peptide synthesis but is more often employed for problem solving. As described earlier in this chapter, efficient characterization of synthetic peptides can be readily obtained by a combination of RP-EDPLC and mass spectrometry. Amino acid analysis is also valuable if MS is not available. If an incorrect mass or a discrepancy in the amino acid composition is found, one obvious alternative is to resynthesize the peptide. But, in order to deduce the cause of a failed synthesis, additional analyses must be performed. Both Edman degradation and tandem MS can be used to obtain sequence information... 

A general outline of the bottom-up mass spectrometry approach for proteome analysis is presented in Figure 3. In general, the mass spectrometry is performed at the peptide level after digesting the protein to obtain the molecular mass and amino acid sequence-specific ions, which are correlated with similar information in the protein or nucleotide database.7 16 Based upon these measurements, the following approaches have evolved. 

Mass spectrometry— Electrospray or MALDl-TOF—could easily distinguish a protein of this approximate mass (101 amino acids x approximately 110 d per amino acid =12 kd) that contained an extra 41 atomic mass units as a result of the substitution of a valine for a glycine residue. 

Liquid Chromatography-Mass Spectrometry Liquid Chromatography-Fourier Transform infrared Liquid Chromatography-Nuciear Magnetic Resonance Spectrometry Amino Acids... 

Mass spectral fragmentation patterns of alkyl and phenyl hydantoins have been investigated by means of labeling techniques (28—30), and similar studies have also been carried out for thiohydantoins (31,32). In all cases, breakdown of the hydantoin ring occurs by a-ftssion at C-4 with concomitant loss of carbon monoxide and an isocyanate molecule. In the case of aryl derivatives, the ease of formation of Ar—NCO is related to the electronic properties of the aryl ring substituents (33). Mass spectrometry has been used for identification of the phenylthiohydantoin derivatives formed from amino acids during peptide sequence determination by the Edman method (34). 

Gas Chromatography (gc). A principal advantage of gas chromatography has been the faciUty with which it can be combined with mass spectrometry for amino acid identification and confirmation of purity. The gc-mass spectrometry combination offers the advantage of obtaining stmctural information rather than the identification by retention time in hplc. 

Spectrometric Analysis. Remarkable developments ia mass spectrometry (ms) and nuclear magnetic resonance methods (nmr), eg, secondary ion mass spectrometry (sims), plasma desorption (pd), thermospray (tsp), two or three dimensional nmr, high resolution nmr of soHds, give useful stmcture analysis information (131). Because nmr analysis of or N-labeled amino acids enables determiaation of amino acids without isolation from organic samples, and without destroyiag the sample, amino acid metaboHsm can be dynamically analy2ed (132). Proteia metaboHsm and biosynthesis of many important metaboUtes have been studied by this method. Preparative methods for labeled compounds have been reviewed (133). 

With the identities and amounts of amino acids known, the peptide is sequenced to find out in what order the amino acids are linked together. Much peptide sequencing is now done by mass spectrometry, using either electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) linked to a time-of-flight (TOF) mass analyzer, as described in Section 12.4. Also in common use is a chemical method of peptide sequencing called the Edman degradation. 

C and 5 N values were determined by mass spectrometry (MM 903, VG Isogas), equipped with a CN elemental analyzer (Roboprep CN, Europa Scientific). As control for sample purity, C N ratios from the elemental analyser were compared with C N ratios as calculated from the amino acid profile of the same sample. 

Petzke, K.J. Lemke, S. (2009). Hair protein and amino acid i C and abundances take more than 4 weeks to clearly prove influences of animal protein intake in young women with a habitual daily protein consumption of more than Ig per kg body weight. Rapid Communications in Mass Spectrometry, Vol. 23, 2411-2420. (http //dx.doi.org/10.1002/ rcm.4025)... 

The Edman reaction enabled amino acid sequence analysis to be automated. Mass spectrometry provides a sensitive and versatile tool for determining primary strucmre and for the identification of post-translational modifications. 

Chance DH, Adam BW, Smith SJ, Alexander JR, Hillman SL, Hannon WH (1999) Validation of accuracy-based amino acid reference materials in dried-blood spots by tandem mass spectrometry for newborn screening assays. Clin Chem 45 1269-1277. 

Gatlin, C.L., Eng, J.K., Cross, S.T., Detter, J.C., and Yates, J.R III, Automated Identification of amino acid sequence variations in proteins by HPLC/mi-crospray tandem mass spectrometry, Anal. Chem., 72, 757, 2000. 

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