Probing High-order Protein Complexes Using Native Mass Spectrometry and Hydrogen/Deuterium Exchange Mass Spectrometry: A Case Study Using Fresh and Commercial Hemoglobin Samples.

Native mass spectrometry (MS) analysis of protein complexes is highly susceptible to matrix effect, and addressing this predicament using buffer exchange is a common approach. Nevertheless, optimization of the buffer exchange protocol is not trivial. With the use of hemoglobin (Hb) as the model entity, it was discovered that the native mass spectrum of protein assembly is highly dependent on the buffer-exchange protocol. Given the dependence of native MS on the purification protocol, this work attempts to use hydrogen/deuterium exchange mass spectrometry (HDX-MS) for comparative studies of hemoglobin complexes in untreated fresh and commercial samples. The information obtained from the HDX study was found to correlate well with the native mass spectrometry analysis of the properly buffer-exchanged Hb samples. Both native MS and HDX-MS showed that the fresh Hb sample has retained the expected tetrameric structure, whereas the commercial Hb has largely been denatured to the dimeric form. These findings prove the complementarity of native MS and HDX-MS in the analysis of high-order protein complexes and stress the necessity to validate the integrity of the high-order structures of the proteins prior to the use of the protein samples for other biomedical studies.

A millimeter water-in-oil droplet as an alternative back exchange prevention strategy for hydrogen/deuterium exchange mass spectrometry of peptides/proteins.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a versatile bioanalytical technique for protein analysis. Since the reliability of HDX-MS analysis considerably depends on the retention of deuterium labels in the post-labeling workflow, deuterium/hydrogen (D/H) back exchange prevention strategies, including decreasing the pH, temperature, and exposure time to protic sources of the deuterated samples, are widely adopted in the conventional HDX-MS protocol. Herein, an alternative and effective back exchange prevention strategy based on the encapsulation of a millimeter droplet of a labeled peptide solution in a water-immiscible organic solvent (cyclohexane) is proposed. Cyclohexane was used to prevent the undesirable uptake of water by the droplet from the atmospheric vapor through the air-water interface. Using the pepsin digest of deuterated myoglobin, our results show that back exchange kinetics of deuterated peptides is retarded in a millimeter droplet as compared to that in the bulk solution. Performing pepsin digestion directly in a water-in-oil droplet at room temperature (18-21 °C) was found to preserve more deuterium labels than that in the bulk digestion with an ice-water bath. Based on the present findings, it is proposed that keeping deuterated peptides in the form of water-in-oil droplets during the post-labelling workflow will facilitate the preservation of deuterium labels on the peptide backbone and thereby enhance the reliability of the H/D exchange data.

Peptide Oxidation Induced by Liquid-Solid Contact Electrification as Revealed in Liquid Microjunction-Surface Sampling Probe Mass Spectrometry.

Reproducible peptide oxidation was observed using a homebuilt liquid microjunction-surface sampling probe (LMJ-SSP) platform for analyzing peptide standards. Although electrochemical oxidation and corona discharges have previously been associated with analyte oxidation in electrospray ionization (ESI) and ESI-related ambient ionization mass spectrometry (MS) methods, they were unlikely the causes for the peptide oxidation observed in the LMJ-SSP studies. A systematic investigation demonstrated that analyte oxidation was induced during the droplet drying on a solid surface through liquid-solid electrification processes. To minimize unwanted analyte oxidation, the water content in the sample solution should be decreased and the use of hydroxyl-functionalized substrates, such as glass slides, should be avoided. In addition, if water is an essential solvent component, adding an antioxidant, such as ascorbic acid, to the sample solution before droplet evaporation on the solid surface could lower the percentage of analyte oxidation. The present findings apply to all the MS methods that involve drying microliters of sample solution onto a suitable substrate in their sample preparation protocols.

CaptiveSpray Differential Ion Mobility Spectrometry Device with Enhanced Ion Transmission and Improved Resolving Power.

A performance enhanced CaptiveSpray differential ion mobility device was designed and constructed by incorporating a circular channel and a gas flow homogenizing channel (GFHC) between the CaptiveSpray ion source and planar differential ion mobility spectrometry (DMS). The GFHC was used to reduce gas flow heterogeneity prior to the entrance of the DMS device. The optimal flared entrance greatly reduces gas flow velocity at the inlet region owing to its relatively large gas inlet interface, which assists in reducing disparities between the minimum and maximum gas velocity along the x-axis. The circular electrode was machined with channels along the x- and y-axis for the passage of auxiliary gas and was applied with a potential to focus the incoming ions from the CaptiveSpray source into the DMS channel. Using reserpine as a reference standard, substantial signal enhancement was achieved with a concomitant reduction of the peak width in the ionogram.

Tandem Mass Spectrometry for Structural Characterization of Doubly-Charged N-Linked Glycopeptides.

Three dissociation methods, including collision-induced dissociation (CID), electron capture dissociation (ECD), and electronic excitation dissociation (EED), were systematically compared for structural characterization of doubly charged glycopeptide. CID produced distinctively different tandem mass spectra for glycopeptide adducted with different charge carriers. Protonated species produced mainly glycosidic cleavages in high abundance. CID of magnesiated glycopeptide formed more cross-ring cleavages, whereas doubly sodiated species produced cleavages at both glycan and peptide moieties. The effect of charge carriers on the fragmentation in ECD and EED was lower than that in CID. ECD produced mainly peptide backbone cleavages but limited cleavages at the glycan moiety, whereas EED of glycopeptide resulted in extensive fragmentation throughout the molecular ion regardless of the charge carriers. Magnesiated species gave, however, more cross-ring cleavages than other charge carriers did. These results demonstrated that EED of magnesiated species could be used as a one-step dissociation method for comprehensive structural analysis of glycopeptides.