Improved detection of ‘suppressed’ peptides in enzymic digests analyzed by fab mass spectrometry

Abstract

positive charge for each part of the molecules on ionization. The data are presented with the NH2 groups being treated as single units. This allows for the fact that removal of electrons from the nitrogen is compensated for by increased polarization of the N-H bonds. For urea the oxygen gains only 0.17+ whereas the sulphur of thiourea gains 0.71+. By contrast each amino group in urea gains 0.48+ whereas each amino group in thiourea gains only 0.12+. This is in broad agreement with our earlier proposal that the charge is localized on N in ureas and on S in thioureas.The fact that both amino groups gain identical amounts of charge suggests a delocalized orbital extending over both nitrogens and the carbon. Ionization of urea is largely by removal of an electron from this orbital, weakening it and causing the N-C-N angle to reduce and the N-C bonds to lengthen. At the same time the carbonyl bond is strengthened as is shown by a small reduction in the C-0 bond length. Ionization of thiourea is largely by removal of a non-bonding electron from sulphur. This weakens the thiocarbonyl bond, which shows an increase in length, and allows the N-C-N orbital to increase its influence as the bond angle increases and the N-C bonds shorten.We plan to employ more complex calculations in further studies on this system, although we do not anticipate arriving at substantially different conclusions. We also intend extending this study to the methylated ureas and thioureas, and to amides and thioamides. AcknowledgementsWe are grateful for the use of the computer facilities at the University of London Computer Centre and the assistance of the advisory staff. We also acknowledge the assistance of Dr D. G. Peacock of the School of Pharmacy. We thank a referee for helpful comments. References1.

Continuous-flow fast atom bombardment mass spectrometry of oligonucleotides

“…During the analysis of peptide mixtures by positive ion FAB mass spectrometry, hydrophobic peptides have been shown to form more abundant protonated molecules relative to those from hydrophilic peptides in the mixture [9,10]. Caprioli and co-workers [9, 101 reported that continuous-flow FAB mass spectrometry reduced the ion suppression effects observed for mixtures of peptides during standard FAB mass spectrometry.…”

“…Recently, FAB mass spectrometry has been coupled with high-performance liquid chromatography (HPLC) by Ito and co-workers [5][6][7] in a system called frit-FAB liquid chromatography mass spectrometry (frit-FAB LC/MS). A similar technique, continuous-flow FAB mass spectrometry, was developed by Caprioli and co-workers first for flow injection analysis (8)(9)(10) and later for LC/MS [11).…”

“…Advantages of continuous-flow FAS and frit-FAB mass spectrometry over the standard FAB probe method of sample introduction include the ability to carry out on-line HPLC separations with detection by FAB mass spectrometry [5], reduced chemical noise from the FAS matrix [8], increased signal-to-noise ratio [8), and less suppression of sample ions when mixtures of samples are analyzed [9,10]. Examples of classes of compounds that have been analyzed by continuous-flow or frit-FAB LC/MS in clu de peptides [11][12][13], bile acids [6), and oligosaccharides [7).…”

Precolumn derivatization and capillary liquid chromatographic/frit-fast atom bombardment mass spectrometric analysis of cytokinins inArabidopsis thaliana

“…Chromatographic separation also minimizes suppression e †ects in biological extracts. 21 The use of B/E-linked mass spectrometric modes greatly reduced the chemical background originating from the extract and the FAB glycerol matrix. Quantitative analysis, performed by SRM with diagnostic transitions shown in Table 3 (bold entries), resulted in a sensitivity in the femtomole range ( Table 3).…”

Continuous flow fast atom bombardment with packed microcolumns: A comparison of precolumn versus coaxial matrix delivery

“…The combination of these microcolumns with "soft" ionization techniques such as FAB permits not only the sensitive detection (typically <10 ng) of the various protonated molecular ions, but also exploitation of the inherent advantages of microcolumns, namely, high plate numbers (>50,000 theoretical plates per meter), high mass sensitivity «10 pmol per injection), and lower optimum flow rates [10]. Numerous applications using such interfaces have been reported for the analysis of peptide mixtures [11,12], permethylated oligosaccharides [13], and enzymatic digests of proteins [14][15][16][17][18][19].…”