Immunoglobulins M (IgM) are key natural antibodies produced initially in humoral immune response. Due to their large molecular weights and extensive glycosylation loads, IgMs represent a challenging target for conventional mass analysis. Charge detection mass spectrometry (CDMS) may provide a unique approach to tackle heterogeneous IgM assemblies, although this technique can be quite laborious and technically challenging. Here, we describe the use of online size exclusion chromatography (SEC) to automate buffer exchange and sample introduction, and demonstrate its adaptability with Orbitrap-based CDMS. We discuss optimal experimental parameters for online SEC-CDMS experiments, including ion activation, choice of column, and resolution. Using this approach, CDMS histograms containing hundreds of individual ion signals can be obtained in as little as 5 min from single injections of <1 μg of sample. To demonstrate the unique utility of online SEC-CDMS, we performed real-time kinetic monitoring of pentameric IgM digestion by the protease IgMBRAZOR, which cleaves specifically in the hinge region of IgM. Several digestion intermediates corresponding to processive losses of F(ab')2 subunits could be mass-resolved and identified by SEC-CDMS. Interestingly, we find that for the J-chain linked IgM pentamer, cleavage of one of the F(ab')2 subunits is much slower than the other four F(ab')2 subunits, which we attribute to the symmetry-breaking interactions of the J-chain within the pentameric IgM structure. The online SEC-CDMS methodologies described here open new avenues into the higher throughput automated analysis of heterogeneous, high-mass protein assemblies by CDMS.
α -Lactalbumin, an abundant protein present in the milk of most mammals, is associated with biological, nutritional and technological functionality. Its sequence presents N-glycosylation motifs, the occupancy of which is species-specific, ranging from no to full occupancy. Here, we investigated the N-glycosylation of bovine α-lactalbumin in colostrum and milk sampled from four individual cows, each at 9 time points starting from the day of calving up to 28.0 d post-partum. Using a glycopeptide-centric mass spectrometry-based glycoproteomics approach, we identified N-glycosylation at both Asn residues found in the canonical Asn-Xxx-Ser/Thr motif, i.e. Asn45 and Asn74 of the secreted protein. We found similar glycan profiles in all four cows, with partial site occupancies, averaging at 35% and 4% for Asn45 and Asn74, respectively. No substantial changes in occupancy occurred over lactation at either site. Fucosylation, sialylation, primarily with N-acetylneuraminic acid (Neu5Ac), and a high ratio of N,N'-diacetyllactosamine (LacdiNAc)/N-acetyllactosamine (LacNAc) motifs were characteristic features of the identified N-glycans. While no substantial changes occurred in site occupancy at either site during lactation, the glycoproteoform (i.e. glycosylated form of the protein) profile revealed dynamic changes; the maturation of the α-lactalbumin glycoproteoform repertoire from colostrum to mature milk was marked by substantial increases in neutral glycans and the number of LacNAc motifs per glycan, at the expense of LacdiNAc motifs. While the implications of α-lactalbumin N-glycosylation on functionality are still unclear, we speculate that N-glycosylation at Asn74 results in a structurally and functionally different protein, due to competition with the formation of its two intra-molecular disulphide bridges.
Viscosity is an important property in the crystallisation process of lactose from supersaturated solutions during lactose production. Viscosity, however, is difficult to measure for supersaturated solutions by conventional, invasive, rheological techniques. To overcome this issue, dynamic light scattering (DLS) was used, whereby latex particles were added to the sample as a tracer. From the known size and measured diffusion coefficient of the latex particles, viscosity of lactose solutions could be determined as a function of temperature (20–80 °C), lactose concentration (10–50 g α-lactose monohydrate 100 g−1 solution) and degree of supersaturation (−40 to +25 g α-lactose 100 g−1). When viscosity was expressed as a function of degree of supersaturation of α-lactose, curves at different temperatures collapsed onto a single master-curve. The results highlight the potential of DLS with latex tracer particles as a convenient and reliable tool for measuring viscosity of even strongly supersaturated solutions of lactose.
The chemical synthesis of new molecules with biological activity is very interesting and opens new fields of research, important in molecular biology and pharmacology. The biological activity of some substituted dipyrido-quinolino-phenazine heptacycles derived from 2-ethoxy-6,9- diaminoacridine was investigated. The antifungal and antibacterial effects of the tested compounds depend on their molecular structure and doses.
We investigated protein glycation in a complex milk system under controlled conditions representative of real-life consumer products, analysing intermediate and final products from skim milk powder production, and aged powder samples. We combined protein-centric LC-MS(/MS) with peptide-centric multi-protease LC-MS/MS focusing on the six most abundant bovine milk proteins. This strategy resulted in the identification of glycated proteoforms and of the extent of glycation per protein, high protein sequence coverage, and identification and relative occupancy of the glycation sites. We identified new glycation hot-spots additionally to the ones already described in literature. Primary sequence motif analysis revealed that glycation hot-spots were preceded N-terminally by a stretch rich in basic amino acids, and followed C-terminally by a stretch enriched in aliphatic and hydrophobic amino acids. Our study considerably extends the current understanding of milk protein glycation, discussing glycation hot-spots and their localisation in relation to the primary sequences and higher-order protein structures.
Caseins play a central role in the dairy sector, as it is their controlled coagulation that allows the conversion of milk into cheese, yoghurt and caseinates, but also the creation of high-quality co-products from whey. Understanding caseins, their association into casein micelles and the properties of casein micelles is thus very important. Providing a complete and holistic understanding requires integration of findings from many fields of study, including organic and inorganic chemistry, biochemistry and physics, biology, lactational physiology, and food science. Combining such insights has gained insights over the past decades how 4 gene products and their genetic variants and post-translational modification can lead to casein associates which further grow to sterically-stabilized casein micelles through combination with calcium phosphate. No two cows produce the exact same casein micelles, and such differences can be explored and utilized, to provide both further insights into casein micelle properties and for industrial utilization.
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