Of methods for dissociation of multiply charged peptide and protein ions, electron capture dissociation (ECD) has the advantages of cleaving between a high proportion of amino acids, without loss of such posttranslational modifications as glycosylation and carboxylation. Here this capability is successfully extended to phosphorylation, for which collisionally activated dissociation (CAD) can cause extensive loss of H3PO4 and HPO3. As shown here, these losses are minimal in ECD spectra, an advantage for measuring the degree of phosphorylation. For phosphorylated peptides, ECD and CAD spectra give complementary backbone cleavages for identifying modification sites. For a 24-kDa heterogeneous phosphoprotein, bovine beta-casein, activated ion ECD cleaved 87 of 208 backbone bonds that identified a phosphorylation site at Ser-15, and localized three more among Ser-17,-18, -19, and -22 and Thr-24, and the last among four other sites. This is the first direct site-specific characterization of this key post-translational modification on a protein without its prior degradation, such as proteolysis.

A series of synthetic peptides (3-15 residues), C-terminally derivatized with 4-aminonaphthalenesulfonic acid (ansa), have been analyzed on a hybrid magnetic sector-orthogonal acceleration time-of-flight tandem mass spectrometer, fitted with a nano-electrospray (nano-ES) interface. Deprotonated molecules generated by negative-ion ES were subjected to collision-induced dissociation (CID) using either methane or xenon as the collision gas, at a collision energy of 400 eV (laboratory frame of reference). As a consequence of charge localization on the sulfonate group, only C-terminal fragment ions were formed, presumably by charge-remote fragmentation mechanisms. Interpretable CID spectra were obtained from fmol amounts of the small peptides (up to 6 residues), whereas low pmol amounts were required for the larger peptides. CID spectra were also recorded of derivatized, previously noncharacterised peptides obtained by proteolysis of cytosolic hamster liver aldehyde dehydrogenase. Interpretation of these CID spectra was based on rules established for the fragmentation of the synthetic peptides. This study shows that derivatization with ansa may be useful in the de novo sequencing of peptides.

The detection limits of a series of small peptides derivatized with 4-aminonaphthalenesulphonic acid have been determined using a magnetic sector mass spectrometer operated in the negative-ion mode. The instrument (Autospec-QFPD) is equipped with an electrospray ion source and both array and point detectors. Detection limits when using the array detector were on the sub-femtomole level at a mass resolution of 2000 (full width half maximum height). Cone-voltage and linked-scan collision-induced dissociation spectra were recorded using both the array and point detectors. When using the array detector, amino acid sequence information could be obtained from as little as 50 fmol of sample consumed.The development of the electrospray (ES) ion source' has had a revolutionary impact on the study of proteins and peptides by mass spectrometry. Up to the early 1990s most electrospray studies had been performed on quadrupole mass spectrometers.* However, magnetic sector instruments are now being equipped with reliable electrospray source^.^ Difficulties have arisen in the interfacing of ES sources to magnetic sector instruments because of the requirements of a high source voltage and a high vacuum. These problems have now been largely overcome with the use of effective pumping and with the advent of high-transmission ion optics within the ion transport r e g i~n .~As new types of ion sources have been developed in the 1980s and 1990s, different methods for ion detection have also been investigated. The array detector is now being used on modern magnetic sector mass spectrometers. These detectors provide a great increase in sensitivity compared with point photo-multiplier or electron-multiplier detectors.Although an array detector will provide an increase in sensitivity over a point detector, this advantage will not be seen when ions are generated by sputtering methods such as static fast-atom (or ion) bombardment (FAB), as both the intensity of the ion of interest and of the chemical background are increased. To obtain the best results with an array detector it is necessary to employ an ion source which does not give a copious amount of chemical noise. Impressive results have been 'obtained by Kenny and Ornaldo' using continuous flow-FAB (CF-FAB) on a four-sector instrument. On two-sector and hybrid instruments, the electrospray ion source may be more appropriate as it provides less background chemical noise than CF-FAB .Recent work in this laboratory has shown the benefit of derivatizing lipids with aminosulphonic acids for analysis by negative-ion FAB mass s p e c t r~m e t r y .~~ Series of bile acids, fatty acids and hydroxy acids have been derivatized with aminosulphonic acids in a peptide linkage at the carboxylic acid group. The effect is to increase the yield of pseudomolecular [M -HIions. Furthermore, high translational energy collisioninduced dissociation (CID) reactions of sulphonated lipids result in charge-remote fragmentations (CRF).

A series of small peptides has been studied by negative-ion fast-atom bombardment mass spectrometry with collision-induced dissociation. It has been found that by derivatizing peptides with 4-aminonaphthalenesulphonic acid in a peptide linkage at the C-terminus, negative-ion formation can be enhanced and fragmentation in collision-induced dissociation reactions controlled. The peptide-naphthalenesulphonates show charge-remote fragmentations and the resultant spectra give sequence information.

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