Thermodynamics and Reaction Mechanisms for Decomposition of a Simple Protonated Tripeptide, H+GGA: From H+GGG to H+GAG to H+GGA.

We present a thorough characterization of fragmentations observed in threshold collision-induced dissociation (TCID) experiments of protonated glycylglycylalanine (H+GGA) with Xe using a guided ion beam tandem mass spectrometer. Kinetic energy dependent cross sections for nine ionic products were obtained and analyzed to provide 0 K barriers for the five primary products, [b2]+, [y1 + 2H]+, [b3]+, [y2 + 2H]+, and [a1]+; and four secondary products, [a2]+, [a3]+, high-energy [y1 + 2H]+, and CH3CHNH2+, after accounting for multiple ion-molecule collisions, the internal energy of reactant ions, unimolecular decay rates, competition between channels, and sequential dissociations. Relaxed potential energy surface scans performed at the B3LYP-GD3BJ/6-311+G(d,p) level of theory are used to identify transition states (TSs) and intermediates of the five primary and three secondary products (with the mechanism of the other secondary product previously established). Geometry optimizations and single point energy calculations of reactants, products, intermediates, and TSs were performed at several levels of theory. These theoretical energies are compared with experimental threshold energies and found to give reasonable agreement, with B3LYP-GD3BJ and M06-2X levels of theory performing slightly better than MP2 and better than B3LYP. The results obtained here are compared with previous results for decomposition of H+GGG and H+GAG to probe the effect of changing the amino acid sequence. Methylation in H+GGA has a significant effect on the competition between the primary sequence products, [b2]+ and [y1 + 2H]+, suppressing the [b2]+ cross section by raising its threshold energy, while enhancing that of [y1 + 2H]+ by lowering its threshold energy.

Linkage Memory in Underivatized Protonated Carbohydrates.

Carbohydrates are among the most complex class of biomolecules, and even subtle variations in their structures are attributed to diverse biological functions. Mass spectrometry has been essential for large scale glycomics and glycoproteomics studies, but the gas-phase structures and sometimes anomalous fragmentation properties of carbohydrates present long-standing challenges. Here we investigate the gas-phase properties of a panel of isomeric protonated disaccharides differing in their linkage configurations. Multiple conformations were evident for most of the structures based on their fragment ion abundances by tandem mass spectrometry, their ion mobilities in several gases, and their deuterium uptake kinetics by gas-phase hydrogen-deuterium exchange. Most notably, we find that the properties of the Y-ion fragments are characteristically influenced by the precursor carbohydrate's linkage configuration. This study reveals how protonated carbohydrate fragment ions can retain "linkage memory" that provides structural insight into their intact precursor.

High-Precision, Gas-Phase Hydrogen/Deuterium-Exchange Kinetics by Mass Spectrometry Enabled by Exchange Standards.

Mass spectrometry (MS) has become a primary tool for identifying and quantifying biological molecules. In combination with other orthogonal techniques, such as gas-phase hydrogen/deuterium exchange (gHDX), MS is also capable of probing the structure of ions. However, gHDX kinetics can depend strongly on many factors, including laboratory temperature, instrumental conditions, and instrument platform selection. These effects can lead to high variability with gHDX measurements, which has hindered the broader adoption of gHDX for structural MS. Here we introduce an approach for standardizing gHDX measurements using cosampled standards. Quantifying the exchange kinetics for analytes relative to the exchange kinetics of the standards results in greater accuracy and precision than the underlying absolute measurements. The standardization was found to be effective for several types of analytes including small molecules and intact proteins. A subset of analytes showed deviations in their standardized exchange profiles that are attributed to field heating and the concomitant conformational isomerization. Inclusion of helium during the gHDX process for collisional cooling helps mitigate such variations in exchange kinetics related to ion heating. We anticipate that the outcomes of this research will enable the broader use of gHDX in MS-based workflows for molecular identification and isomer differentiation.

Dissection of Fragmentation Pathways in Protonated N-Acetylhexosamines.

Structural characterization of carbohydrates by mass spectrometry necessitates a detailed understanding of their gas phase behavior, particularly for protonated carbohydrates that can undergo complex structural rearrangements during fragmentation. Here we utilize tandem mass spectrometry, isotopic labeling, gas-phase hydrogen/deuterium exchange, and ion mobility measurements to characterize structures of the various product ions of protonated N-acetylhexosamines. Following the facile loss of the reducing end hydroxyl group, we identify two primary fragmentation pathways. Detailed mapping of each step in the fragmentation pathway provides new insight into the mechanisms that drive collision-induced dissociation of protonated carbohydrates. Several of the smaller fragment ions are mixtures of structural isomers, and the relative distributions of these structures reveals information about the stereochemistry of the precursor molecule.

Bridging the structural gap of glycoproteomics with ion mobility spectrometry.

Over the past two decades mass spectrometry (MS) has enabled high throughput studies on the level of a complex proteome. While it has similarly advanced glycobiology, the level of progress has been more restrained. This is in large part due to the diversity and complexity of carbohydrate structures. While MS is now routinely used for glycobiology, it suffers from a critical limitation, that is the inability to resolve isobaric structures. Since so many structurally and functionally distinct carbohydrates are indistinguishable by MS, additional techniques are needed for detailed structural analysis. Ion mobility spectrometry (IMS), with its ability to resolve closely related isobaric structures, presents such a tool for alleviating the current limitations of MS. In the past few years studies have demonstrated the utility and immense potential of combining IMS with MS (IM-MS) for glycomics and it is now approaching the point of enabling comprehensive structural studies on the level of the glycoproteome.

Thermodynamics and Reaction Mechanisms of Decomposition of the Simplest Protonated Tripeptide, Triglycine: A Guided Ion Beam and Computational Study.

We present a thorough characterization of fragmentations observed in threshold collision-induced dissociation (TCID) experiments of protonated triglycine (H+GGG) with Xe using a guided ion beam tandem mass spectrometer (GIBMS). Kinetic energy-dependent cross-sections for 10 ionic products are observed and analyzed to provide 0 K barriers for six primary products: [b2]+, [y1 + 2H]+, [b3]+, CO loss, [y2 + 2H]+, and [a1]+; three secondary products: [a2]+, [a3]+, and [y2 + 2H - CO]+; and two tertiary products: high energy [y1 + 2H]+ and [a2 - CO]+ after accounting for multiple ion-molecule collisions, internal energy of reactant ions, unimolecular decay rates, competition between channels, and sequential dissociations. Relaxed potential energy surface scans performed at the B3LYP-D3/6-311+G(d,p) level of theory are used to identify transition states (TSs) and intermediates of the six primary and one secondary products. Geometry optimizations and single point energy calculations were performed at several levels of theory. These theoretical energies are compared with experimental energies and are found to give reasonably good agreement, in particular for the M06-2X level of theory. This good agreement between experiment and theory validates the reaction mechanisms explored computationally here and elsewhere and allows identification of the product structures formed at threshold energies. The present work presents the first measurement of absolute experimental threshold energies of important sequence ions and non-sequence ions: [y1 + 2H]+, [b3]+, CO loss, [a1]+, and [a3]+, and refines those for [b2]+ and [y2 + 2H]+ previously measured. Graphical Abstract ᅟ.

Kinetic determination of potassium affinities by IRMPD: elucidation of precursor ion structures.

Dissociation kinetics of the K(+) loss reaction of three potassiated tertiary amino acids (Scheme 1) were studied by infrared multiple photon dissociation (IRMPD) in a Fourier transform ion cyclotron resonance (FT ICR)-MS instrument. The aim of the study was to probe if a kinetic study by IRMPD can yield useful information on the ion structure of the precursor ion species. The measured activation energy values determined by IRMPD are related to the potassium affinity, DeltaH(K(+)), of N-methyl proline determined by threshold collision-induced dissociation experiments. By appropriate scaling with this reference value, the experimentally determined activation energy values for the K(+) loss are transformed into respective potassium affinities, DeltaH(K(+))(IRMPD). These values match the calculated potassium affinity values for salt bridge (SB) structures, DeltaH(K(+))(SB), substantially better than those for canonical structures with a single formal charge site (charge solvation (CS)), thereby allowing structure identification. This conclusion is consistent with other spectroscopic data, which yielded unambiguous evidence of these tertiary amino acids adopting SB structures in the gas phase. This study demonstrates that IRMPD can be applied to determine individual ion structures in the gas phase, given that adequate reference values are available for proper scaling.