Gas phase hydrogen / deuterium exchange in electrospray ionization mass spectrometry as a practical tool for structure elucidation.

Two methods for gas phase hydrogen/deuterium exchange have been developed for the analysis of small molecules. Hydrogen/deuterium exchange has been implemented by making simple modifications to the plumbing for the nebulizer and curtain gases on a nebulization-assisted electrospray ion source. The nebulizer gas exchange method has demonstrated deuterium exchange levels of 84-97% for a variety of molecules representing a wide range of structural classes containing up to 51 potentially exchangeable hydrogens; this allowed determination of the number of exchangeable hydrogens for all of the molecules studied containing ≤ 25 labile hydrogens (M r ≤ 3000). ND3 gas consumption is minimized in the nebulizer method by toggling the nebulizer from air to ND3 for only a few scans of the total sample elution period. The curtain gas exchange method is more variable, yielding exchange levels of 32-98% for the same set of molecules; this was still sufficient to allow determination of > 70% of the molecules studied containing ≤ 25 labile hydrogens. Gas consumption is minimized in the curtain method by replacing ≤ 10% of the curtain gas flow with ND3. Neither the nebulizer nor curtain exchange method requires the use of deuterated or aprotic solvents at typical 2 µL/min flow rates.

High-performance anion-exchange chromatography coupled with mass spectrometry for the determination of carbohydrates.

Methodology has been developed to couple high-performance anion-exchange chromatography (HPAE) with mass spectrometry utilizing the ion spray liquid chromatography/mass spectrometry (LC/MS) interface. Anion micro-membrane suppression (AMMS) has been used to remove the high concentrations of NaOH and NaOAc (10-400 mM total [Na+]) necessary for the separation of mixtures of monosaccharides and oligosaccharides. Post-suppressor addition of CH3CN/H2O solutions containing NH4OAc or LiOAc provided low-nanomole detection of the monosaccharides by selected ion monitoring of the cationized adducts. Maltooligosaccharide mixtures (three to seven residues) were separated and detected by the HPAE/AMMS LC/MS system in the full-scan mode. Low declustering potentials (35 V) in the LC/MS API source afforded intact singly and doubly charge ammoniated and diammoniated adducts of the sugars. Higher declustering potentials (65 V) produced abundant fragmentation of the ammoniated adducts. The corresponding lithiated and dilithiated species produced intact molecule ion species at higher declustering potentials. The endo H-released oligomannose species from RNase B were determined by the HPAE/AMMS LC/MS system as ammoniated adducts and resulting fragment ions with a high declustering potential (95 V) in the full-scan mode.

The determination of glycopeptides by liquid chromatography/mass spectrometry with collision-induced dissociation.

Glycopeptides derived from ribonuclease B and ovomucoid have been subjected to collisioninduced dissociation (CID) in the second quadrupole of a triple quadrupole mass spectrometer. Doubly charged parent ions gave predictable fragmentation that yielded partial sequence information of the attached oligosaccharide as Hex and HexNAc units. Common oxonium ions are observed in the product ion mass spectra of the glycopeptides that correspond to HexNAc(+) (m/z 204) and HexHexNAc(+) (m/z 366). A strategy for locating the glycopeptides in the proteolytic digest mixtures of glycoproteins by ions spray liquid chromatography mass spectrometry (LC/MS) is described by utilizing CID in the declustering region of the atmospheric pressure ionization mass spectrometer to produce these characteristic oxonium ions. This LC/CID/MS approach is used to identify glycopeptides in proteolytic digest mixtures of ovomucoid, asialofetuin, and fetuin. LC/CID/MS in the selected ion monitoring mode may be used to identify putative glycopeptides from the proteolytic digest of fetuin.

Nitrite-cured meats as a source of endogenously and exogenously formed N-nitrosoproline in the ferret.

Nitrite-cured meat containing 120 mg Na15NO2/kg was fed to male ferrets (Mustela putorius furo). During consumption of the meat, the animals were dosed orally with 0.87 mmol [2-2H]proline. All urine was collected throughout the study and analysed for total N-nitrosoproline (NPRO) and isotopic enrichment of NPRO by mass spectrometry. The cured-meat diet increased the urinary excretion of NPRO 14-fold. Isotope analyses indicated that approximately 70% of the NPRO came from the cured meat, the majority of which was analytically unavailable or 'bound' NPRO in the meat. A small portion of the excreted NPRO appeared to be formed in the stomach as a result of ingesting the cured meat. A minor amount of the excreted NPRO did not contain any isotopically labelled atoms. The administration of ascorbic acid did not significantly alter NPRO excretion. Animals dosed orally with 11.4 mumol of a peptide in which the N-terminal proline was nitrosated increased their excretion of NPRO by 385 nmol over the following 48 hr. These data indicate that nitrite-cured meat contains bound NPRO which contributes to the total amount of NPRO in the urine.

Photolytic interface for high-performance liquid chromatography--chemiluminescence detection of non-volatile N-nitroso compounds.

A photolytic interface between high-performance liquid chromatography (HPLC) and a chemiluminescence detector has been developed for the trace detection of non-volatile N-nitroso compounds in biological matrices. A chromatographic effluent containing separated N-nitrosoamino acids and N-nitrosamides is introduced into a glass coil with a purge stream of He and irradiated with ultraviolet light. Nitrogen oxide, cleaved by photolysis, is separated rapidly from the solvent through a series of cold traps and carried by the He into the reaction chamber of a chemiluminescence detector. The method is compatible with most types of HPLC, especially reversed-phase, and yields low-nanogram sensitivity for underivatised N-nitrosoamino acids and N-nitrosamides. The detection of a model N-nitrosamide, trimethylnitrosourea, in spiked porcine gastric fluid (42 micrograms l-1), and of N-nitrosoproline and N-nitroso-1,3-thiazolidine-4-carboxylic acid, in spiked human urine (7-8 micrograms l-1), is demonstrated.