Amino acids selective reaction monitoring

It is important to first note that many classical clinical assays have traditionally measured one metabolite to detect one disease. Consequently, many of the rules of method validation were designed around this premise. MS/MS, as originally designed, detected two classes of compounds, amino acids and acylcamitines, in four to five different MS methods (known as scan modes such as neutral loss, precursor ion, or selected reaction monitoring), for approximately 500 distinct masses, more than 70 known compounds, and 20-30 stable isotope internal standards. How then did one approach such a complicated validation to gain acceptance as a reliable, useful method The answer is quite simple - start simply and compare to what was already established. 

For application with triple-quadrapole and especially (J-LIT instruments, a selected-reaction monitoring (SRM) procedure was developed for the sensitive and selective detection of phosphopeptides in proteomes with known amino-acid sequences [26]. A list of SRM transitions of potential phosphopeptides is generated for all expected tryptic peptides in the mixtme with Ser, Thr, or Tyr and for double-and triple-charge ions in the mass range of m/z 400-1600. The number of transitions included is limited by the maximum cycle time of 10 s, which assures that peptides in a 30 s wide peak are at least analysed twice. The procedure was applied to the cell cycle regulatory protein Cyclin B from Schizosaccharomyces pombe. 

This same full-scan and SRM approach used for ACs is also done for amino acids. Basic amino acids such as citrulline and arginine are characterized by loss of 102 Da and ammonia or other basic amino side chain. Selective reaction monitoring (SRM) is used rather than full scan and is based on NL of 119 for citrulline (102 -I-17, where 17 is ammonia) and NL of 161 for arginine (102 -i- 59, where 59 is the amino side chain). These SRMs are often grouped together in the visual spectrum as shown in Figure 13.12. Note that citrulline can also be acquired in an NL 102 scan since source-induced dissociate may cause the ammonia to be lost and hence detection of m/z 215 (MH+ minus 17) versus m/z 232. Details of this fragmentation are described elsewhere. 

There are no special requirements in the selection of an N-terminal amino acid residue in a segment with which the carboxy component of a segment is to be coupled, unless a highly hindered amino acid or a secondary amino acid is selected. If a Pro or Hyp residue is located at the N-terminus of the segment, monitoring of the coupling reaction with ninhydrin or fluorescamine is extremely difficult. 

Following extractive deproteinization of the plasma, the amino acids (and their stable-isotope-labeled internal standards) are separated by HPLC and introduced into the mass spectrometer. Electrospray ionization results in the formation of electrically charged molecules, which are separated on the basis of their mass/charge (m/z) ratio in the first quadrupole. Following fragmentation in the collision cell, the characteristic fragment for each amino acid is selected in the second quadrupole. This process is named multiple reaction monitoring. 

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