No abstract

The phosphorylation sites in a model phosphoprotein, alpha s1-casein from bovine milk, have been identified by tryptic peptide mapping (Gibson and Cohen, Methods Enzymol. vol. 193, p. 480 (1990)) employing reversed-phase high performance liquid chromatography (RPHPLC)/electrospray ionization mass spectrometry (ES-MS); by infusion tandem mass spectrometry (MS/MS) and LC/MS/MS in neutral loss mode of tryptic digests of alpha s1-casein, in which the characteristic neutral loss of phosphoric acid by phosphopeptides under collision-induced dissociation (CID) conditions is exploited to highlight phosphopeptides in a tryptic digest (Covey et al., in Methods in Protein Sequence Analysis, Jörnvall et al. (Eds), Birkhäuser Verlag, Basel 1991), and by a novel method, termed LC/CID-MS, in which phosphopeptides are located in mixtures of peptides by the generation and detection of phosphate-specific fragment ions during LC/ES-MS (Huddleston et al., J. Am. Soc. Mass Spectrom. vol. 4, p. 710 (1993)). An appraisal of the efficiency, sensitivity and practicality of each of these methods in the identification of phosphorylation sites in post-translationally modified proteins is given.

No abstract

Capillary electrochromatography/mass spectrometry (CEC/MS) has so far only been performed using electrospray or microspray ionization. CEC has, to date, not been reported coupled online to nanospray ionization, due to practical difficulties in coupling the CEC column to the nanospray emitter. However this combination is ideally suited, as the flow rates for CEC (100nL/min, approximately) are directly compatible with larger orifice (10mm) nanospray emitters.A CEC unit has been designed and engineered in-house, to enable the use of short columns and high field strengths, facilitating fast electro-osmotic flow and short retention times. This device was initially coupled via a 50 mm  37 cm C 18 /SCX column, and then by a C 6 /SCX 50 mm  45 cm column, to microspray and nanospray interfaces, also designed and built in-house. CEC/microspray-MS and CEC/nanospray-MS were successfully evaluated on an ion-trap mass spectrometer using five corticosteriods with the addition of thiourea as a flow marker. Limits of detection (S/N = 3) were found to be 500 fg (1-2 mol) for CEC/microspray-MS and initial studies using the nanospray interface have predicted detection limits in the region of 50 fg ($100 amol).

No abstract

Glycosylation sites in bovine alpha 1-acid glycoprotein (AGP) have been identified, and the inherent heterogeneity evaluated, by capillary electrophoretic and reversed-phase liquid chromatography/electrospray-mass spectrometric analyses of proteolytic digests of this glycoprotein. The success of these methods in locating glycopeptides relied on significant heterogeneity within each glycosylation site. In order to rapidly locate sites in glycoproteins of any degree of heterogeneity, a novel mass spectrometric method was applied to selectively identify the glycopeptides in a proteolytic digest of bovine alpha 1-AGP. The glycopeptides were selectively located by the generation and detection of characteristic oxonium ions from the carbohydrate moieties by collision-induced dissociation (CID) during liquid chromatography/electrospray-tandem mass spectrometry, and liquid chromatography/CID mass spectrometry, in which fragmentation was induced in the supersonic expansion region of the electrospray source.

The hepta-, hexa- and penta-carboxylic porphyrins found in the faeces of rats poisoned with hexachlorobenzene have been separated by high-pressure liquid chromatography and characterized largely by spectroscopie methods. Their structures were confirmed by total synthesis, as part of a programme in which eleven of the fourteen hepta-, hexa- and penta-carboxylic porphyrins derived from uroporphyrin III have now been synthesized as their methyl esters. The four isomeric heptacarboxylic and three of the pentacarboxylic porphyrinogens have been incubated with haemolysates of chicken erythrocytes, and they are all converted into protoporphyrin IX but at different rates. On the basis of this and other evidence we conclude that the decarboxylation of uroporphyrinogen III to coproporphyrinogen III is a stepwise process taking place by a preferred pathway (both in normal and abnormal metabolism); the acetic acid groups are decarboxylated in a sequential clockwise fashion starting with that on the D ring and followed by those on the A, B and C rings. In the poisoned rats the uroporphyrinogen decarboxylase enzyme (or group of enzymes) is probably partially inhibited and the pentacarboxylic porphyrinogen with an acetic acid group on ring C accumulates. The latter is then transformed by a side pathway into dehydroisocoproporphyrinogen and thence into dehydroisocoproporphyrin and its congeners.

The Argonne set of coals cover the rank range from lignite to semi-anthracite; these samples have been studied by matrix-assisted laser desorption mass spectrometry (MALDI-MS) in a time-of-flight mass spectrometer equipped with a nitrogen laser at 337 nm, using sinapinic acid as matrix. The coal particle size was less than 5 microns. The characteristics of the MALDI-MS spectra of the set of coals were found to be rank-related; desorption from highrank coals was found to take place with greater relative ease than from low-rank coals. Two major features were found in all spectra: a homologous series of peaks in the 200-500 u mass range and an intense peak between lo00 and SOOOu, the particular shape of the peak depending on coal rank. A continuum of lower intensity peaks extending to very large molecular masses was found in all spectra, the upper limit of molecular masses increasing with coal rank at the same laser fluence. The effect of changes in laser power on spectra was investigated: upper mass limits were found to increase with power up to the detection limit of the instrument but low-mass parts of spectra were found to distort, possibly due to detector overloading. A maximum laser fluence value acceptable over the coal-rank range represented by these samples could therefore not be easily defined. None of the mass spectra showed evidence of the presence of either carbon clusters or fullerene formation, indicating that laser fluences did not reach intensities high enough to induce substantial secondary reactions. Comparing molecular mass distributions detected by MALDI of coal pyrolysis tars and directly from coals suggests the MALDI and pyrolytic mechanisms of volatile release to be structurally different; in particular, the preferential evaporation of lighter species which occurs during pyrolytic tar evolution (and during field-ionization mass spectroscopy) appears to evolve material with a more restricted range of molecular masses compared to laser desorption mechanisms.