American Chemical Society (ACS)

The gas-phase deprotonation reactions of multiply protonated bovine ubiquitin, insulin chain B, and renin substrate tetradecapeptide ions have been studied in a Fourier transform ion cyclotron resonance mass spectrometer coupled with an external electrospray source. Rate constants were measured for the reactions of these peptide ions with a series of reference compounds of known gas-phase basicities ranging from 195.6 to 232.6 kcal/mol. The apparent gas-phase acidities (GAapp) of the multiply protonated peptide ions [M + nH]n+ were determined with deprotonation reactions. The deduced values of GAapp show a strong dependence on the charge states of the multiply protonated peptide ions. In general, the values decrease as the charge states of the peptide ions increase. For ubiquitin ions, the determined GAapps values decrease from >232.6 to 205.0 kcal/mol for n=4–13; for insulin B ions, the GAapps decrease from >232.6 to 198.2 kcal/mol for n=2–5; for renin substrate ions, the GAapps decrease from 221.6 to <195.6 kcal/mol for n=2–4. Interestingly, at a given mass-to-charge ratio, the GAapps of these peptide ions agree within 10 kcal/mol despite large differences in their mass and charge. The ubiquitin and insulin B ions generated under the present conditions reveal multiple isomers at certain charge states, n=4, 5, 6, 12 for ubiquitin and n=4, 5 for insulin B, as evidenced by the fact that the isomers display distinctively different deprotonation reaction rates with certain reference compounds.

Mass spectrometry-based methods have made significant progress in characterizing post-translational modifications in peptides and proteins; however, certain aspects regarding fragmentation methods must still be improved. A good technique is expected to provide excellent sequence information, locate PTM sites, and retain the labile PTM groups. To address these issues, we investigate 10.6 μm IRMPD, 213 nm UVPD, and combined UV and IR photodissociation, known as HiLoPD (high-low photodissociation), for phospho-, sulfo-, and glyco-peptide cations. IRMPD shows excellent backbone fragmentation and produces equal numbers of N- and C-terminal ions. The results reveal that 213 nm UVPD and HiLoPD methods can provide diverse backbone fragmentation producing a/x, b/y, and c/z ions with excellent sequence coverage, locate PTM sites, and offer reasonable retention efficiency for phospho- and glyco-peptides. Excellent sequence coverage is achieved for sulfo-peptides and the position of the SO3 group can be pinpointed; however, widespread SO3 losses are detected irrespective of the methods used herein. Based on the overall performance achieved, we believe that 213 nm UVPD and HiLoPD can serve as alternative options to collision activation and electron transfer dissociations for phospho- and glyco-proteomics.

We report the introduction and use of an atmospheric pressure chemical ionization liquid chromatography-mass spectrometry instrument that has been designed specifically for use by the synthetic chemist on an open access, walk-in basis. This instrument has been configured with an easy-to-use sample log-in terminal that requires the user to provide only a sample identification number and a user name. Sample analysis takes approximately 4 min and provides the synthetic and medicinal chemist with rapid and reliable mass spectrometry analysis. Since installation of the system, it has analyzed an average of about 80 samples per day and has the capacity to run over 100 samples per day without the intervention of a specialist operator. This capability has eliminated the need for an operator to analyze routine samples and allows the mass spectroscopist more time to deal with problem solving.

Supramolecular assemblies that are formed between amines and trifluoroacetic acid were studied using electrospray ionization mass spectrometry. Distinctive association behavior of primary, secondary, and tertiary amines with trifluoroacetic acid upon identical experimental conditions is observed and indicates that steric effects dominate in the formation of these protonated clusters. Extraordinary complexation behavior is observed in the case of R−(+)-α-methylbenzylamine and 4-tert-butyl-cyclohexylamine that form high-order clusters. The strong relation between stereochemistry and assembly results in the specific association characteristics of trans 4-tert-butyl-1-phenylcyclohexylamine when compared with the cis isomer. The cis isomer gives rise to a highly abundant [M4TFA3+H]+ ion (M=amine molecule, TFA=trifluoroacetic acid), as observed for other primary amines. However, the trans isomer generates higher [MnTFAm+H]+ cluster ions, the largest and most abundant being an [M7TFA6+H]+ ion. Collision induced dissociation spectra that were recorded for several [MnTFAm+H]+ cluster ions typically show the consecutive losses of M·TFA moieties. Density functional theory calculations indicate that the highly abundant [M4TFA3+H]+ clusters are macrocycles and support the formation of these structures with TFA and not with acetic acid.

Gas-phase reactions of W-, Si-, P-, Br-, and I-containing ions with the target molecule perfluorohexane at low collision energies (<15 eV) parallel known ion/surface reactions of the same projectile ions at fluorinated self-assembled monolayer surfaces. Charge exchange, dissociative charge exchange, and fluorine atom abstraction are observed and the majority of the projectile ions also undergo reactive charge exchange to produce specific fluorocarbon fragment ions of the target molecule in distinctive relative abundances. Abstraction of up to five fluorine atoms is observed upon collision of W+ with gaseous perfluorohexane, while similar experiments with CI+, SiCl+, and PCl+· show abstraction of one or two fluorine atoms. Other projectiles, including Si+·, PCl 2 + , Br+, CBr+, and I+, abstract only a single fluorine atom. These patterns of fluorine atom abstraction are similar to those observed in ion/surface collisions. Also paralleling the ion/surface reactions, halogen exchange (Cl-for-F) reactions occur between the Cl-containing projectile ions and perfluorohexane to produce C6F12Cl+, a product of chemical modification of the target. Collisions of PCl+· and PCl 2 + also result in production of C6F 12 +· , indicating that the corresponding surface modification reaction involving molecular defluorination should be sought. Implications for previously proposed mechanisms, new ion/surface reactions, and for the use of gas-phase studies to guide investigations of the ion/surface reactions are discussed.

The structural characterization of glycans by mass spectrometry is particularly challenging. This is because of the high degree of isomerism in which glycans of the same mass can differ in their stereochemistry, attachment points, and degree of branching. Here we show that the addition of cryogenic vibrational spectroscopy to mass and mobility measurements allows one to uniquely identify and characterize these complex biopolymers. We investigate six disaccharide isomers that differ in their stereochemistry, attachment point of the glycosidic bond, and monosaccharide content, and demonstrate that we can identify each one unambiguously. Even disaccharides that differ by a single stereogenic center or in the monosaccharide sequence order show distinct vibrational fingerprints that would clearly allow their identification in a mixture, which is not possible by ion mobility spectrometry/mass spectrometry alone. Moreover, this technique can be applied to larger glycans, which we demonstrate by distinguishing isomeric branched and linear pentasaccharides. The creation of a database containing mass, collision cross section, and vibrational fingerprint measurements for glycan standards should allow unambiguous identification and characterization of these biopolymers in mixtures, providing an enabling technology for all fields of glycoscience.