A novel ion source based on the principle of sonic spray ionization has been built and used to optimize mass spectrometric conditions for generating amino acid clusters. The ion source employs a simple pneumatic spray operated at extremely high nebulizing gas flow rates. Several factors that affect the performance of the cluster source are identified, and information from these observations provides insights into the mechanisms of gas phase ion formation. Serine is used as a model system in optimizing instrumental and sample parameters to maximize cluster ion formation. The sonic spray results for this oligomer compare favorably with electrospray data, showing an order of magnitude better signal intensity and excellent signal-to-noise ratios. The performance of the system for the protonated serine octamer includes a limit of detection of 10 nM and a linear dynamic range of 4 orders of magnitude. Ion formation was observed to go into saturation above 1 mM. This result and data on pH, electrolyte concentration, and solvent composition are interpreted as supporting a charge residue model of sonic spray ionization. Other amino acids can be substituted for serine in the octamer, with a strong chiral preference in favor of homochiral cluster formation in the cases of threonine and cysteine. These amino acids show a preference for substitution of more than two serine molecules. Phenylalanine, asparagine, tryptophan, and tyrosine also substitute into the serine octamer; however, the process yields only two incorporations and only small chiral effects.
A new anionic surfactant (RapiGest SF) was successfully used for site-specific analysis of glycosylation in human alpha-1-acid glycoprotein (AGP). By means of this analytical approach combined with capillary HPLC-mass spectrometry (and tandem mass spectrometry), the N-linked glycosylation pattern of AGP was explored. On the basis of mass matching and MS/MS experiments ca 80 different AGP-derived glycopeptides were identified. Glycosylation shows a markedly different pattern for the various glycosylation sites. At sites I and II, triantennary complex-type oligosaccharides predominate and at sites III, IV and V, tetra-antennary complex-type oligosaccharides predominate. Sites IV and V show the presence of additional N-acetyl lactosamine (Gal-GlcNAc) units (even higher degree of branching and/or longer antennae are also present).
The recently discovered homochiral serine octamer has been a focus of interest because of its possible implications for the origin of homochirality in living systems. Electrospray ionization (ESI) and sonic spray ionization (SSI) tandem mass spectrometry have been used to generate this unusually stable magic number cluster. Several structures have been suggested for the serine octamer, based on tandem mass spectrometry, ion mobility measurements, and quantum mechanical calculations. We now report experimental hydrogen/deuterium (H/D) exchange data, which demonstrate the existence of two different structures for the serine octamer. These forms undergo exchange at significantly different rates. One form may correspond to solution-phase assembled clusters and the other to octamers formed during the ionization process. Experiments done at higher resolution confirm that the experimental observations made here apply to the serine octamer without interference from metaclusters, namely, higher order clusters (Ser(16) + 2H)(+2), etc., the (12)C isotopes of which have mass-to-charge ratios identical to the protonated octamers. H/D exchange of racemic serine shows predominantly the extensively exchanged ion population, as well as providing evidence that racemic serine generates abundant metaclusters. The evidence presented here shows that one type of serine octamer is responsible for the strong chiral effects associated with the formation of these magic number clusters. These slowly exchanging more fragile clusters are the octamers that might have played a role in homochirogenesis.
The association properties of natural and non-natural amino acids were studied in detail using electrospray ionization mass spectrometry. The results show a highly diverse cluster formation behavior of amino acids. There are differences regarding the degree of clustering (average cluster size), the presence or absence of one or several 'magic' clusters of special stability and the influence of chirality on cluster stability. Cluster formation does not show a good correlation with simple physico-chemical properties (such as solubility), indicating that it is a specific process and not only a simple aggregation during evaporation/ionization. A systematic study of cluster formation of serine derivatives reveals that all functional groups play a prominent role in the binding of the magic octamer. The results support the idea of the zwitterionic character of the octamer. Electrospray ionization of the side-chain acetylated serine shows the formation of a very stable tetramer with a strong preference for homochirality. The results suggest that Ser8 is made up of two tetramer subunits, held together by hydrogen bonds of the side-chain.
Internal energy changes during the electrospray process have been studied. Our results suggest that, contrary to conventional wisdom, ions that are quite hot are formed in electrospray, even without acceleration in the cone-skimmer region. The main role of the curtain gas seems to be not to break up existing clusters but, rather, to cool down the nascent ions and to prevent cluster formation by shielding them from solvent vapors.
Tandem mass spectrometry is an important tool for structure elucidation of natural and synthetic organic products. Fragmentation of odd electron ions (OE+) generated by electron ionization (EI) was extensively studied in the last few decades, however there are only a few systematic reviews available concerning the fragmentation of even-electron ions (EE+/EE−) produced by the currently most common ionization techniques, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). This review summarizes the most important features of tandem mass spectra generated by collision-induced dissociation fragmentation and presents didactic examples for the unexperienced users.
Pathogenic bacteria excrete a variety of virulence factors into extracellular medium and to the cell surface which have essential roles in the colonization and insurrection of the host cells, and thus reflect the degree of bacterial pathogenicity. For the exploration of virulence factors expressed in the secreted proteome fraction, different Staphylococcus aureus strains were analyzed using gel-based bottom-up proteomic approach. A total of 119 distinct proteins were identified for the enterotoxin gene cluster (egc) negative and seb gene positive S. aureus American Type Culture Collection (ATCC) 14458 strain by the use of one- and 2-DE based proteomics. Detailed analysis of enterotoxin region of the 2-D map confirmed, beside the highly expressed staphylococcal enterotoxin B (SEB), the presence of enterotoxin-like proteins SElK and SElQ previously predicted by genotyping (Sergeev et al.., J. Clin. Microbiol. 2004, 42, 2134-2143). Exoprotein patterns at the late-exponential (7 h) and stationary (24 h) phases of cellular growth show a high-level similarity in this region. Comparative analysis of enterotoxin region of five S. aureus strains including two clinical isolates (RIMD 31092 and A900322), a food derived strain (AB-8802) with highly prevalent egc positive operon and a nonenterotoxigenic reference strain (ROS) revealed the presence of different known enterotoxins and other virulence factors along with a number of core exoproteins. In addition, production of SElL (RIMD 31092) and SElP (A900322) was demonstrated for the first time at the protein level. Under the experimental conditions applied none of the enterotoxins encoded by the genes of egc operon was identified.
Abstract. The similarity between two tandem mass spectra, which were measured on different instruments, was compared quantitatively using the similarity index (SI), defined as the dot product of the square root of peak intensities in the respective spectra. This function was found to be useful for comparing energy-dependent tandem mass spectra obtained on various instruments. Spectral comparisons show the similarity index in a 2D Bheat map^, indicating which collision energy combinations result in similar spectra, and how good this agreement is. The results and methodology can be used in the pharma industry to design experiments and equipment well suited for good reproducibility. We suggest that to get good long-term reproducibility, it is best to adjust the collision energy to yield a spectrum very similar to a reference spectrum. It is likely to yield better results than using the same tuning file, which, for example, does not take into account that contamination of the ion source due to extended use may influence instrument tuning. The methodology may be used to characterize energy dependence on various instrument types, to optimize instrumentation, and to study the influence or correlation between various experimental parameters.
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