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Characterisation of mass distributions of solvent-fractionated lignins using analytical ultracentrifugation and size exclusion chromatography methods.

ABSTRACT: Lignins are valuable renewable resources for the potential production of a large array of biofuels, aromatic chemicals and biopolymers. Yet native and industrial lignins are complex, highly branched and heterogenous macromolecules, properties that have to date often undermined their use as starting materials in lignin valorisation strategies. Reliable knowledge of weight average molar mass, conformation and polydispersity of lignin starting materials can be proven to be crucial to and improve the prospects for the success of such strategies. Here we evaluated the use of commonly-used size exclusion chromatography (SEC)-calibrated with polystyrene sulphonate standards-and under-used analytical ultracentrifugation-which does not require calibration-to characterise a series of lignin fractions sequentially extracted from soda and Kraft alkaline lignins using ethyl acetate, methyl ethyl ketone (MEK), methanol and acetone:water (fractions F01-F04, respectively). Absolute values of weight average molar mass (M_w) determined using sedimentation equilibrium in the analytical ultracentrifuge of (3.0 ± 0.1) kDa and (4.2 ± 0.2) kDa for soda and Kraft lignins respectively, agreed closely with previous SEC-determined M_ws and reasonably with the size exclusion chromatography measurements employed here, confirming the appropriateness of the standards (with the possible exceptions of fraction F05 for soda P1000 and F03 for Indulin). Both methods revealed the presence of low (~ 1 kDa) M_w material in F01 and F02 fractions followed by progressively higher M_w in subsequent fractions. Compositional analysis confirmed > 90% (by weight) total lignins successively extracted from both lignins using MEK, methanol and acetone:water (F02 to F04). Considerable heterogeneity of both unfractionated and fractionated lignins was revealed through determinations of both sedimentation coefficient distributions and polydispersity indices. The study also demonstrates the advantages of using analytical ultracentrifugation, both alongside SEC as well as in its own right, for determining absolute M_w, heterogeneity and conformation information for characterising industrial lignins.

SUBMITTER: Lu Y

PROVIDER: S-EPMC8260654 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundCirculating small extracellular vesicles (sEVs) and its associated proteins are of great interest in the early detection of many diseases. However, there is no gold standard for plasma sEVs isolation, especially for proteomic profiling which could be largely affected by contamination such as lipoproteins and plasma proteins. Previous studies suggested combinations of different sEVs isolation methods could improve the yield and purity of the isolated fractions. Nevertheless, there is no systematic evaluation of size-exclusion chromatography (SEC), ultracentrifugation (UC), and their combination in a proteomic perspective.ResultsPlasma samples were collected from healthy individuals, and sEVs were separated by one-step SEC, one-step UC, and combining SEC with UC, respectively. Here we exhibited that the purity of sEVs was improved by SEC in contrast to traditional UC. Furthermore, by conducting a SEC procedure followed by UC, we separated sEVs with the highest purity. In the proteomic analysis, 992 protein species were identified in the plasma sEVs isolated by our novel separation method, of which several proteins are sEVs-associated proteins but hitherto never been identified in the previous studies and database, much more than plasma sEVs isolated by UC (453) or SEC (682) alone. As compared to Vesiclepedia and Exocarta databases, plasma sEVs isolated by the new procedure kept 584 previously identified sEVs-associated proteins and 360 other proteins that have not been detected before. Detailed analysis suggested that more kinds of sEVs biomarkers, such as CD9, ALIX, and FLOT1, could be identified in plasma sEVs isolated by the novel isolation method as compared to one-step UC/SEC. Furthermore, the lower abundance ranks of common contaminants, such as lipoproteins and IgG chains, in the sEVs fractions obtained by our new method as compared to one-step UC/SEC also demonstrated the purity of sEVs had been improved.ConclusionsCombining SEC with UC could significantly improve the performance of mass spectrometry-based proteomic profiling in analyzing plasma-derived sEVs.

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Characterisation of mass distributions of solvent-fractionated lignins using analytical ultracentrifugation and size exclusion chromatography methods.

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