Protein peptide lability

The ECD process, by its nature, is a very rapid process and bond dissociation occurs faster than the redistribution of intramolecular vibrational energy that occurs with CID. This explains the dissociation of the strong N-C, amine bonds in the presence of the weaker C—N amide bonds in peptides and proteins.93,94 Consequently, any labile PTMs (e.g., phosphorylation, sulfation, 7-carboxylation, N- and O-glycosylation) are preserved and may be unequivocally located in the peptide/protein sequence. See also discussion in Section 9.10.3.2.6 on the use of ECD/ETD and CID/IRMPD for protein/peptide sequencing, and Table 4. 

It is unusual for either CID or ETD to provide complete sequence information from any one peptide but the use of both techniques provides complementary information, which can gready extend the sequence coverage (Table 4). In addition, because the energy from the ETD process is directed into cleaving the Ca—N bond, the labile PTMs are preserved and their location in the peptide sequence can then be determined90 (see also discussion in Section 9.10.3.2.6 on the use of CID/IRMPD and ECD/ETD for protein/peptide sequencing, and Table 4). 

Evaporation from a spray of charged droplets produced from a stream of liquid yields ions that can be analyzed in a mass spectrometer. Thermally labile and normally nonvolatile substances such as sugars, peptides, and proteins can be examined successfully. 

The FAB source operates near room temperature, and ions of the substance of interest are lifted out from the matrix by a momentum-transfer process that deposits little excess of vibrational and rotational energy in the resulting quasi-molecular ion. Thus, a further advantage of FAB/LSIMS over many other methods of ionization lies in its gentle or mild treatment of thermally labile substances such as peptides, proteins, nucleosides, sugars, and so on, which can be ionized without degrading their. structures. 

As a rule of thumb, one can say that the efficiency of separation of mixtures and the simplicity of operating and maintaining apparatus are much greater for GC than for LC. Hence, other things being equal, GC is most often the technique of first choice and can be used with a very wide variety of compound types. However, for nonvolatile or thermally labile substances like peptides, proteins, nucleotides, sugars, carbohydrates, and many organometallics, GC may be ruled out completely... 

In contrast to the lability of certain dN adducts formed by the BHT metabolite above, amino acid and protein adducts formed by this metabolite were relatively stable.28,29 The thiol of cysteine reacted most rapidly in accord with its nucleophilic strength and was followed in reactivity by the a-amine common to all amino acids. This type of amine even reacted preferentially over the e-amine of lysine.28 In proteins, however, the e-amine of lysine and thiol of cysteine dominate reaction since the vast majority of a-amino groups are involved in peptide bonds. Other nucleophilic side chains such as the carboxylate of aspartate and glutamate and the imidazole of histidine may react as well, but their adducts are likely to be too labile to detect as suggested by the relative stability of QMs and the leaving group ability of the carboxylate and imidazole groups (see Section 9.2.3). 

Sheppard RC, Williams BJ. Acid-labile resin linkage agents for use in solid phase peptide synthesis. Int J Peptide Protein Res 1982 20 451 454. 

Sulfur-bound L-Met, as opposed to S,N-chelated L-Met, is more reactive as a ligand on Pt(II) and can be slowly replaced by N7 of G (95, 96). Transfer of Pt onto DNA via Met-containing peptides or proteins may therefore be possible. Monofunctional adducts of the type [Pt(en)(G)(L-Met-S)] appear to be very stable (97) and so methionine may play a role in trapping these adducts. Also, the high trans influence of S as a Pt(II) ligand can lead to the facile labilization of trans-am(m)ine ligands and this allows cisplatin to react with GMP faster in the presence of L-Met then in its absence (98), which introduces another route to DNA platination. 

Low expression levels and the lability of the HslVU complex make work with proteins from wild-type strains difficult. Gratifyingly, the active protease can be reconstituted in vitro from over-expressed and purified components (Rohrwild et al. 1996). It requires ATP for the degradation of folded substrates and ATP or some of its analogs for the purification of small chromogenic peptides. As expected, ATP-hydrolysis and proteolysis activities are mutually dependent (Seol et al. 1997). In addition, the peptidase activity was found to depend in complex ways on the presence of various cations, especially K in the buffers (Huang and Goldberg 1997). 

ESI has become the most commonly used interface for LC/MS. It was recognized by John Fenn and co-workers as an important interface for LC/MS immediately after they developed it as an ionization technique for MS. ESI transforms ions in solution to ions in the gas phase and may be used to analyze any polar molecule that makes a preformed ion in solution. The technique has facilitated the ionization of heat-labile compounds and high-molecular-weight molecules such as proteins and peptides. ESI is a continuous ionization method that is particularly suitable for use as an interface with FiPLC. It is the most widely accepted soft-ionization technique for the determination of molecular weights of a wide variety of analytes and, has made a significant impact on drug discovery and development since the late 1980s. 

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