Selective enrichment of glycopeptides for mass spectrometry analysis using C18 fractionation and titanium dioxide chromatography

Comprehensive glycoprotein characterization based on mass spectrometry (MS) is challenging because of low concentration of glycopeptides and suppression effect of abundant non-glycosylated peptides in MS. Therefore, it is vital to enrich glycopeptides before MS analysis. A new method was developed to selectively enrich glycopeptides from complex sample by coupling C18 fractionation with titanium dioxide (TiO(2)) enrichment. The new method allows to selectively enrich N-linked glycopeptides with various glycan forms and different sequence lengths. Compared with single TiO(2) method, the established method demonstrated higher glycopeptide selectivity and higher glycosylation heterogeneity coverage. Further application of this method to mixture of non-glycosylated protein and glycoprotein digests at different levels reveals the feasibility of enrichment of tryptic glycopeptides from simple proteomics samples.     

Mass diffusion-based separation of sugars in a microfluidic contactor with nanofiltration membranes

Processes such as chromatographic separation and nanofiltration can remove low molecular weight sugars from liquid mixtures of oligosaccharides. As an alternative for the separation of such liquid mixtures, we studied mass diffusion separation of such sugars in a microfluidic device with incorporated nanofiltration membranes. This separation method is based on differences between diffusivities of components and does not require high transmembrane pressures. The effects of channel depth and flow rate were studied in experiments. The key parameters selectivity and rejection increased with increasing channel depth due to increased external mass transfer limitations. Among the studied membranes, the obtained selectivities and rejections correlated to the specified retention values by the manufacturers. Compared to more conventional nanofiltration where high pressure forces solutes through membranes, we obtained corresponding selectivities and fluxes of only an order of magnitude smaller. Simulated results indicated that with optimized microchannel and membrane dimensions, the presented separation process can compete with currently available separation technologies.     

The boundary layers of an unsteady incompressible stagnation-point flow with mass transfer

In the current work, the boundary layers of an unsteady incompressible stagnation-point flow with mass transfer were further investigated. Similarity transformation technique was used and the similarity equation group was solved using numerical methods. Interesting observation is that there are multiple solutions seen for negative unsteadiness parameters, β. The influences of mass transfer, unsteadiness parameter, and Prandtl numbers on velocity and temperature profiles, wall drag, and wall heat fluxes were investigated and analyzed. The asymptotic behaviors for the similarity equations in limiting situations were theoretically analyzed. It is found that solutions exist for all mass transfer parameters for β≥−1. For a certain mass transfer parameter, there are two solutions when β_c<β<0; there is one solution for (β=β_c)∪(β≥0); there is no solution for β<β_c, where β_c is a critical unsteadiness parameter dependent on mass transfer parameter.     

Double-beta decay Q values of Cd and Te

The Q values of the ¹¹⁶Cd and ¹³⁰Te double-beta decaying nuclei were determined by using a Penning trap mass spectrometer. The new atomic mass difference between ¹¹⁶Cd and ¹¹⁶Sn of 2813.50(13) keV differs by 4.5 keV and is 30 times more precise than the previous value of 2809(4) keV. The new value for ¹³⁰Te, 2526.97(23) keV is close to the Canadian Penning trap value of 2527.01 ± 0.32 keV (Scielzo et al., 2009) [1], but differs from the Florida State University trap value of 2527.518 ± 0.013 keV (Redshaw et al., 2009) [2] by 0.55 keV (2σ). These values are sufficiently precise for ongoing neutrinoless double-beta decay searches in ¹¹⁶Cd and ¹³⁰Te. Hence, our Q values were used to compute accurate phase-space integrals for these double-beta decay nuclei. In addition, experimental two-neutrino double-beta decay nuclear matrix elements were determined and compared with the theoretical values. The neutrinoless double-beta decay half-lives for these nuclei were estimated using our precise phase-space integrals and considering the range of the best available matrix elements values.     

Peak clustering in two-dimensional gas chromatography with mass spectrometric detection based on theoretical calculation of two-dimensional peak shapes: the 2DAid approach

A method is presented to facilitate the non-target analysis of data obtained in temperature-programmed comprehensive two-dimensional (2D) gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-ToF-MS). One main difficulty of GC×GC data analysis is that each peak is usually modulated several times and therefore appears as a series of peaks (or peaklets) in the one-dimensionally recorded data. The proposed method, 2DAid, uses basic chromatographic laws to calculate the theoretical shape of a 2D peak (a cluster of peaklets originating from the same analyte) in order to define the area in which the peaklets of each individual compound can be expected to show up. Based on analyte-identity information obtained by means of mass spectral library searching, the individual peaklets are then combined into a single 2D peak. The method is applied, amongst others, to a complex mixture containing 362 analytes. It is demonstrated that the 2D peak shapes can be accurately predicted and that clustering and further processing can reduce the final peak list to a manageable size.     

Membrane-assisted capillary isoelectric focusing coupling with matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry for neuropeptide analysis

Herein we report a highly efficient and reliable membrane-assisted capillary isoelectric focusing (MA-CIEF) system being coupled with MALDI-FTMS for the analysis of complex neuropeptide mixtures. The new interface consists of two membrane-coated joints made near each end of the capillary for applying high voltage, while the capillary ends were placed in the two reservoirs which were filled with anolyte (acid) and catholyte (base) to provide pH difference. Optimizations of CIEF conditions and comparison with conventional CIEF were carried out by using bovine serum albumin (BSA) tryptic peptides. It was shown that the MA-CIEF could provide more efficient, reliable and faster separation with improved sequence coverage when coupled to MALDI-FTMS. Analyses of orcokinin family neuropeptides from crabs Cancer borealis and Callinectes sapidus brain extracts have been conducted using the established MA-CIEF/MALDI-FTMS platform. Increased number of neuropeptides was observed with significantly enhanced MS signal in comparison with direct analysis by MALDI-FTMS. The results highlighted the potential of MA-CIEF as an efficient fractionation tool for coupling to MALDI MS for neuropeptide analysis.     

Antibody capture by mixed-mode chromatography: a comprehensive study from determination of optimal purification conditions to identification of contaminating host cell proteins

We evaluated mixed mode chromatography for the capture of recombinant antibodies from CHO cell culture supernatants. We studied PPA HyperCel, HEA HyperCel, MEP HyperCel and Capto adhere resins, which all contain hydrophobic and cationic groups. A microplate approach combined with DoE modeling allowed the exploration of the complex behaviors of these mixed mode resins. Optimal conditions for antibody purification and host cell proteins (HCPs) elimination were determined and then directly up-scaled to laboratory columns. Then we used mass spectrometry to identify the major HCPs potentially coeluted with the antibody. Differences between the four resins in terms of amount, complexity and identity of the HCPs present in the elution fractions were investigated.