Amino acids tandem mass spectrometry

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... 

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. 

Figure 2.5. Tandem mass spectrometry. A. A peptide mixture is electrosprayed into the mass spectrometer. Individual peptides from the mixture are isolated (circled peptide) and fragmented. B. The fragments from the peptide are mass analyzed to obtain sequence information. The fragments obtained are derived from the N or C terminus of the peptide and are designated "b" or "y" ions, respectively. The spectrum shown indicates peptides that differ in size by the amino acids shown.

Tandem mass spectrometry has become an important tool for determining the sequence of amino acids in protonated peptides98 and the sequence of bases in deprotonated nucleic acids such as DNA.99 Despite the importance and widespread use of CID-MS to sequence peptides and nucleic acids, the mechanistic details of the dissociation processes are poorly understood. A better understanding of the... 

Petritis, K. et al., Simultaneous analysis of underivatized chiral amino acids by liquid chromatography—ionspray tandem mass spectrometry using a teicoplanin chiral stationary phase, J. Chromatogr. A, 913, 331, 2001. 

Soga, T., Kakazu, Y., Robert, M., Tomita, M., andNishioka, T. (2004). Qualitative and quantitative analysis of amino acids by capillary electrophoresis-electrospray ionization-tandem mass spectrometry. Electrophoresis 25, 1964—1972. 

Song, Y, Shenwu, M., Zhao, S., Hou, D., and Liu, Y. M., Enantiomeric separation of amino acids derivatized with 7-fluoro-4-nitrobenzoxadiazole by capillary liquid chromatography/tandem mass spectrometry,/owmaZ o/Chroma/ograp/jy 4 1091(1-2), 102-109,2005. 

Amino Acid Sequencing by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)... 

Table 2.1.2 Gradient elution of the amino acids for tandem mass spectrometry...

Table 2.1.3 Multiple reaction monitoring of amino acids for their tandem mass spectrometry quantitation. In daily practise not all mentioned amino acids are measured in one run, but a set of ten dedicated evaluation programs has been developed, covering groups of amino acids associated with groups of disorders. Amino acids presented in italics indicate stable-isotope-labeled internal standards ... 

Because of these ever-widening interests, the measurement of plasma tHcy is undertaken in many clinical chemistry and routine laboratories. Various methods are employed, including high-performance liquid chromatography (HPLC) assays, conventional amino acid analysis, capillary electrophoresis, gas chromatography with or without mass spectrometry, liquid chromatography with tandem mass spectrometry, and in many routine clinical chemistry laboratories immunoassays. In this chapter, those methods that are often available in laboratories involved in the investigation of inborn errors of metabolism are described, namely HPLC and tandem mass spectrometry. 

McCooeye, M., and Mester, Z. (2006). Comparison of flow injection analysis electrospray mass spectrometry and tandem mass spectrometry and electrospray high-field asymmetric waveform ion mobility mass spectrometry and tandem mass spectrometry for the determination of underivatized amino acids. Rapid Commun. Mass Spectrom. 20 1801-1808. 

Ilg EC, Troxler H, Burgisser DM, Kuster T, Markert M, Guignard F, Hunziker P, Birchler N, Heizmann CW. 1996. Amino acid sequence determination of human S100A12 (P6, calgranulin C, CGRP, CAAF1) by tandem mass spectrometry. Biochem Biophys Res Commun 225(1) 146-150. 

D. Zhang et al., Chiral resolution of D, L-amino acids by tandem mass spectrometry of Ni(II)-bound trimeric complexes. Int. J. Mass Spectrom. 204, 159-169 (2001)... 

D.V. Augusti et al., Rapid quantitative chiral analysis of sugars by electrospray ionization tandem mass spectrometry using modified amino acids as chiral reference compounds. Anal. Chem. 74, 3458-3462 (2002)... 

Although reliable, this technique may lead to false positive results in some cases. To overcome this problem many proteomic companies are now adopting the technique of tandem mass spectrometry to unambiguously identify protein sequences. This technique subjects proteins to successive routines of fragmentation and mass analysis in order to provide the actual amino acid sequence. 

Figure 4.8. MuDPIT platform. The MuDPIT or multidimensional gel protein identification system is an multidimensional liquid chromatography (LC)-based system of separation prior to tandem mass spectrometry (MS/MS). It is not necessary to purify proteins prior MuDPIT, although a reduction in protein complexity by some prior purification is helpful in obtaining interpretable spectra. Protein lysates are digested into peptides that are loaded onto a strong cation exchange (SCX) support. Peptides are sequentially eluted onto a reverse-phase column for a second separation. Eluted peptides from the reverse-phase column are electrosprayed into a tandem mass spectrometer for amino acid sequencing and identification of proteins in the sample.

Hulst, A.G. and Kientz, C.E. (1996) Differentiation between the isomeric amino acids leucine and isoleucine using low-energy collision-induced dissociation tandem mass spectrometry. J. Mass Spectrom., 31 (10), 1188-90. 

Determination of oxidized amino acids in urine is usually performed by isotope dilution gas chromatography-mass spectrometry (L9). DOPA is estimated by HPLC separation of acid protein hydrolysates with fluorescence detection (excitation 280 nm, emission at 320 nm) (A15). Other methods are based on borate-hydrochloric acid difference spectroscopy (this method suffers interference from tyrosine and tryptophan) (W2), derivatization of DOPA with nitrite and subsequent coulometric determination (W3), and fluorometric detection after derivatization with ethylenediamine (A15). 3-Hydroxylysine is quantitated by HPLC with 9-fluorenylmethyl chloroformate precolumn derivatization (M25) of amino acids obtained by gas-phase hydrolysis of proteins (F21). Other general methods to detect amino acid damage are mass spectometry methods applied to protein hydrolysates, such as tandem mass spectrometry (F6). 

Petritis, K. et al. Parameter optimization for the analysis of underivatized protein amino acids by liquid chromatography and ion spray tandem mass spectrometry. /. Chromatogr. A. 2000, 896, 253-263. 

Liu, D.L., Beegle, L.W., and Kanik, I. Analysis of underivatized amino acids in geological samples using ion-pairing liquid chromatography and electrospray tandem mass spectrometry. Asfrofcio/ogy 2008, 8, 229-241. 

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