Identification of Isomeric Biomolecules by Infrared Spectroscopy of Solvent-Tagged Ions.

The difference in functionality of many isomeric biomolecules requires their analytical identification for life science studies. We present a universal approach for quantitative identification of different small- to medium-sized isomeric biomolecules that can be brought to the gas phase from solution by electrospray ionization (ESI). The method involves infrared (IR) fragment cold ion spectroscopy of analyte molecules that are incompletely desolvated by soft ESI. The use of solvent molecules as natural tags removes a need for adding to solutions any special compounds, which may interfere with liquid chromatography or mass spectrometric measurements. The tested peptides and especially monosaccharides and lipids exhibit highly isomer-specific IR fragment spectra of such noncovalent complexes, which were produced from water, methanol, acetonitrile, and 2-butanol solutions. The relative concentrations in solution mixtures of, for instance, two isomeric dipeptides can be quantified with the accuracy of 1.6% and 2.9% for the acquisition time of 25 min and, potentially, 5 s, respectively; for three isomeric phospho-octapeptides, the accuracy becomes 4.1% and 11% for 17 min and, potentially, 10 s measurements, respectively.

Identification of Isomeric Lipids by UV Spectroscopy of Noncovalent Complexes with Aromatic Molecules.

The tremendous structural and isomeric diversity of lipids enables a wide range of their functions in nature but makes the identification of these biomolecules challenging. We distinguish and quantify isomeric lipids using cold ion UV fragmentation spectroscopy of their noncovalent complexes with aromatic amino acids and dipeptides. On the basis of structural simulations, specific isomer-sensitive aromatic "sensors" have been preselected for lipids of each studied class. Tyrosine appeared to be a good "sensor" to distinguish steroids and prostaglandins, which are rich in functional groups, while diphenylalanine is a better choice for sensing largely hydrophobic phospholipids. With this sensor, the relative concentrations of two isomeric glycerophospholipids mixed in solution have been determined with 3.3% accuracy, which should degrade only to 3.7% for a 14 s express measurement.

Accelerating photofragmentation UV Spectroscopy-Mass spectrometry fingerprinting for quantification of isomeric peptides.

Identification of isomeric biomolecules remains a challenging analytical problem. A recently developed spectroscopic method that combines UV photofragmentation and mass spectrometry for fingerprinting of cold ions (2D UV-MS), has already demonstrated its high performance in the library-based identification and quantification of different types of biomolecular isomers. The practical use of the method has been hindered by a slow rate of data acquisition, which makes the fingerprinting incompatible with high-throughput analysis and online liquid chromatography (LC) separation. Herein we demonstrate how the use of a few pre-selected wavelengths can accelerate the method by two orders of magnitude without a significant loss of accuracy. As a proof of principle, 2D UV-MS fingerprinting was coupled to online LC separation and tested for quantification of isomeric peptides containing either Asp or isoAsp residues. The relative concentrations of the peptides mixed in solution have been determined, on average, with better than 4% and 6% accuracy for resolving and non-resolving gradients of LC separation, respectively.

Microhydration of Biomolecules: Revealing the Native Structures by Cold Ion IR Spectroscopy.

The native-like structures of protonated glycine and peptide Gly3H+ were elucidated using cold ion IR spectroscopy of these biomolecules hydrated by a controlled number of water molecules. The complexes were generated directly from an aqueous solution using gentle electrospray ionization. Already with a single retained water molecule, GlyH+ exhibits the native-like structure characterized by a lack of intramolecular hydrogen bonds. We use our spectra to calibrate the available data for the same complexes, which are produced by cryogenic condensation of water onto the gas-phase glycine. In some conformers of these complexes, GlyH+ adopts the native-like structure, while in the others, it remains "kinetically" trapped in the intrinsic state. Upon condensation of 4-5 water molecules, the embedded amino acid fully adopts its native-like structure. Similarly, condensation of one water molecule onto the tripeptide is insufficient to fully eliminate its kinetically trapped intrinsic states.

Identification and Quantification of Any Isoforms of Carbohydrates by 2D UV-MS Fingerprinting of Cold Ions.

Biological functionality of isomeric carbohydrates may differ drastically, making their identifications indispensable in many applications of life science. Because of the large number of isoforms, structural assignment of saccharides is challenging and often requires a use of different orthogonal analytical techniques. We demonstrate that isomeric carbohydrates of any isoforms can be distinguished and quantified using solely the library-based method of 2D ultraviolet fragmentation spectroscopy-mass spectrometry (2D UV-MS) of cold ions. The two-dimensional "fingerprint" identities of UV transparent saccharides were revealed by photofragmentation of their noncovalent complexes with aromatic molecules. We assess the accuracy of the method by comparing the known relative concentrations of isomeric carbohydrates mixed in solution with the concentrations that were mathematically determined from the measured in the gas-phase fingerprints of the complexes. For the tested sets with up to five isomers of di- to heptasaccharides, the root-mean-square deviation of 3-5% was typically achieved. This indicates the expected level of accuracy in analysis of unknown mixtures for isomeric carbohydrates of similar complexity.

Revealing Single-Bond Anomeric Selectivity in Carbohydrate-Protein Interactions.

The noncovalent binding of proteins to glycans is amazingly selective to the isoforms of carbohydrates, including α/ß anomers that coexist in solution. We isolate in the gas phase and study at the atomic level the simplest model system: noncovalent complexes of monosaccharide α/ß-GalNAc and protonated aromatic molecule tyramine. IR/UV cold ion spectroscopy and quantum chemistry calculations jointly solve the structures of the two complexes. Although the onsets of the measured UV absorptions of the complexes differ significantly, the networks of H bonds in both complexes appear identical and do not include the anomeric hydroxyl. The detailed analysis reveals that, through inductive polarization, the α- to ß-reorientation of this group nevertheless reduces the length of one remote short intermolecular H-bond by 0.03 Å. Although small, this change substantially strengthens the bond, thus contributing to the anomeric selectivity of the binding.

Spectroscopic Evidence for Peptide-Bond-Selective Ultraviolet Photodissociation.

We study the photodissociation induced by ultraviolet excitation of amide bonds in gas-phase protonated peptides. Jointly, mass spectrometry and cold ion spectroscopy provide evidence for a selective nonstatistical dissociation of specific peptide bonds in the spectral region of the formally forbidden n → π* transition of amide groups. Structural analysis reveals that the activation of this transition, peaked at 226 nm, originates from the nonplanar geometry of the bond. In contrast, the statistical dissociation in the electronic ground state appears to be the main outcome of the π → π* excitation of the peptide bonds at 193 nm. We propose a tentative model that explains the difference in the fragmentation mechanisms by the difference in localization of the electronic transitions and the higher amount of vibrational energy released in the electronic excited state upon absorption at 193 nm.

Interplay of H-Bonds with Aromatics in Isolated Complexes Identifies Isomeric Carbohydrates.

The tremendous isomeric diversity of carbohydrates enables a wide range of their biological functions but makes the identification and study of these molecules difficult. We investigated the ability of intermolecular interactions to communicate structural specificity of carbohydrates to protonated aromatic molecules in non-covalent complexes, isolated and cooled in the gas phase. Our study revealed that small structural differences between carbohydrate isomers of any type, including enantiomers, are accurately communicated by these interactions to aromatic molecules as detectable changes in their electronic excitation spectra. The specific response of the aromatics to the isomers of carbohydrates is fine-tuned by the interplay of the various involved non-covalent bonds. These findings enable the gas-phase identification and relative quantification of any isomers of oligosaccharides in their solution mixtures using the 2D UV-MS fingerprinting technique.