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Development of fresh solid phase extractant is critical for selective separation and purification of special proteins. Herein, we demonstrated a recombinant Staphylococcal Protein G (rSPG) with a His-tag modified the novel single-metal organic framework (rSPG@Ni-MOF-74). The proposed solid-phase extraction material possessed a uniform spindle-shaped structure, large surface area (709.60 m2 g-1) and pore volume (0.08 m3 g-1), high metal content (22.57 wt%), which facilitated the interaction between host and guest. As results, the composite displayed outstanding selective recognition and adsorption of IgG, due to synergistic effect of the binding ability of rSPG with the Fc region of IgG, maintained through hydrogen bonding and electrostatic attraction, as well as hydrophobic interaction. The adsorption performance and mechanism of rSPG@Ni-MOF-74 have been fully investigated. Additionally, the rSPG@Ni-MOF-74 composite could effectively separate IgG from serum obtained from healthy humans, with the purity of the separated IgG verified through SDS-PAGE analysis. Furthermore, LC-MS/MS analysis identified a high content of IgG (55.3 %) in the eluate from rSPG@Ni-MOF-74, suggesting the great potential of rSPG@Ni-MOF-74 in IgG separation and enrichment from complex matrix.

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The accurate protein-protein separation is important but technically challenging. Achieving such a precise separation using membrane requires the selective channels with appropriate pore geometry structure and high anti-fouling property. In this study, polyethersulfone-b-poly(sulfobetaine methyl methacrylate) (PES-b-PSBMA) was synthesized and engineered onto polysulfone (PSF) ultrafiltration (UF) membrane to fabricate zwitterionic nanospheres engineered co-polymer (ZN-e-CoP) composite membrane via dynamic self-assembly micelle deposition. On the one hand, self-assembly zwitterionic nanospheres were used as blocks to construct hydrophilic layers with size-dependent sieving channels, endowing ZN-e-CoP composite membranes with enhanced permselectivity and protein-protein separation abilities, meanwhile zwitterionic groups from nanospheres reinforced the structure stability of nanospheres/nanospheres and nanospheres/membrane via multiple intermolecular interactions. On the other hand, zwitterionic nanospheres can induce to produce the hydration layer enveloping themselves by binding water molecules, where hydration layer acts as a protective barrier on the membrane surface, impeding the protein adhesion. Hence, ZN-e-CoP_1a composite membrane exhibited superior separation properties with Lysozyme/Bovine Serum Albumin (BSA) separation factor of 18.1 and 95.4 % rejection against BSA, 10.1 and 2.3 times, respectively, higher these of pristine PSF membrane (1.8 and 42.1 %), without obviously sacrificing water flux. Simultaneously, hydration layer enables the ZN-e-CoP_1a membrane with enhanced anti-fouling performance and durability during the long-term operations. The proposed approach opens new pathways to fabricate excellent anti-fouling membranes for precise protein-protein separation.

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The research used polyethersulfone (PES) as a membrane material, polyvinylpyrrolidone (PVP) k30 and polyethylene glycol 400 (PEG 400) as water-soluble additives, and dimethylacetamide (DMAc) as a solvent to prepare hollow-fiber ultrafiltration membranes through a nonsolvent-induced phase separation (NIPS) process. The hydrophilic nature of PVP-k30 and PEG caused them to accumulate on the membrane surface during phase separation. The morphology, chemical composition, surface charge, and pore size of the PES membranes were evaluated by SEM, FTIR, zeta potential, and dextran filtration experiments. The paper also investigated how different spinning solution compositions affected membrane morphology and performance. The separation efficiency of membranes with four different morphologies was tested in single-protein and double-protein mixed solutions. The protein separation effectiveness of the membrane was studied through molecular weight cutoff, zeta potential, and static protein adsorption tests. In addition, the operating pressure and pH value were adjusted to improve ultrafiltration process conditions. The PES membrane with an intact sponge-like structure showed the highest separation factor of 11, making it a prime candidate membrane for the separation of bovine serum albumin (BSA) and lysozyme (LYS). The membrane had a minimal static protein adsorption capacity of 48 mg/cm2 and had excellent anti-fouling properties. When pH = 4, the BSA retention rate was 93% and the LYS retention rate was 23%. Furthermore, it exhibited excellent stability over a pH range of 1-13, confirming its suitability for protein separation applications.

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Successive multiple ionic-polymer layers (SMILs) have long since proved their worth in capillary electrophoresis as they ensure stable electroosmotic flow (EOF) and relatively high separation efficiency. Recently, we demonstrated that plotting the plate height (H) against the solute migration velocity (u) enabled a reliable quantitative evaluation of the coating performances in terms of separation efficiency. In this work, various physicochemical and chemical parameters of the SMIL coating were studied and optimized in order to decrease the slope of the ascending part of the H vs u curve, which is known to be controlled by the homogeneity in charge of the coating surface and by the possible residual solute adsorption onto the coating surface. SMILs based on poly(diallyldimethylammonium chloride) (PDADMAC) and poly(sodium styrene sulfonate) (PSS) were formed and the effect of each polyelectrolyte molar mass and of the number of polyelectrolyte layers (up to 21 layers) was studied. The use of polyethylene imine as an anchoring first layer was considered. More polyelectrolyte couples based on PDADMAC, polybrene, PSS, poly(vinyl sulfate), and poly(acrylic acid) were tested. Finally, zwitterionic polymers based on the poly(α-l-lysine) scaffold were synthesized and used as the last layer of SMILs, illustrating their ability to finetune the EOF, while maintaining good separation efficiency.

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The need for excellent, affordable, rapid, reusable and biocompatible protein purification techniques is justified based on the roles of proteins as key biomacromolecules. Magnetic nanomaterials nowadays have become the subject of discussion in proteomics, drug delivery, and gene sensing due to their various abilities including rapid separation, superparamagnetism, and biocompatibility. These nanomaterials also referred to as magnetic nanoparticles (MNPs) serve as excellent options for traditional protein separation and analytical methods because they have a larger surface area per volume. From ionic metals to carbon-based materials, MNPs are easily functionalized by modifying their surface to precisely recognize and bind proteins. This review excavates state-of-the-art MNPs and their functionalizing agents, as efficient protein separation and purification techniques, including ionic metals, polymers, biomolecules, antibodies, and graphene. The MNPs could be reused and efficaciously manipulated with these nanomaterials leading to highly improved efficiency, adsorption, desorption, and purity rate. We also discuss the binding and selectivity parameters of the MNPs, as well as their future outlook. It is concluded that parameters like charge, size, core-shell, lipophilicity, lipophobicity, and surface energy of the MNPs are crucial when considering protein selectivity, chelation, separation, and purity.

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A process-simplified hard template approach was established to synthesize the monodisperse macroporous silica microspheres with homogeneous structures by twice alkali-thermal treatment and calcination routes. Porous vinyl-functionalized polysesquioxane microspheres (V-PMSQ) were synthesized through a hydrolyzation-polycondensation method and used as templates. The template particles with large aperture and high pore volume were obtained by adjusting the pH value and reaction time of the twice alkali-thermal reaction. After calcination, monodisperse silica microspheres with an average pore size of 30 nm, homogeneous pore structures, and narrow particle size distribution were fabricated, which can be directly used as chromatographic matrices without classification. After that, a new reversed-phase/strong anion-exchange (RP/SAX) mixed-mode stationary phase Sil-S-VOIM was prepared by bonding the 1-vinyl-3-octyl-imidazole ligands to the above silica microspheres through a "thiol-ene" click reaction. The performance of the Sil-S-VOIM column was evaluated by one acidic protein (transferrin) and two basic proteins (lysozyme, α-chymotrypsin) and compared to a single imidazole-modified Sil-S-VIM column and an octyl-modified Sil-C8 column, respectively. Due to the synergistic effect of electrostatic repulsion and hydrophobic interactions, baseline separations of the above proteins were observed only on the Sil-S-VOIM column, with resolutions of 2.55 and 2.01 between lysozyme and transferrin, and between transferrin and α-chymotrypsin, respectively, indicating good selectivity and separation ability compared with single-mode stationary phases. It was applied to the isolation of egg white samples with peaks identified by SDS-PAGE and MALDI-TOF-MS. The results showed that the selective retention and isolation of ovomucoid and ovotransferrin were successfully achieved, with yields of 78.8% and 67.2%, respectively. The protocol described in this work is simpler, faster, and has higher protein recovery. Overall, this new mixed-mode stationary phase provided a promising potential for the separation and determination of intact proteins.

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Liquid chromatography/mass spectrometry (LC/MS) has become a routine powerful technology in clinical proteomic studies for protein identification, protein characterization, and the discovery of biomarkers. In this chapter, we describe two protocol methods to analyze clinical patient samples using a resin-based depletion column followed by either protein In-Gel enzymatic digestion or protein In-Solution enzymatic digestion using a simple kit-based approach (i.e., using the PreOmics iST sample preparation kit), followed by analysis using one-dimensional reverse-phase chromatography (RPC) or high pH reversed-phase peptide fractionation.

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Proteins are essential for various functions such as brain activity and muscle contraction in humans. Even though food is a source of proteins, the bioavailability of proteins in most foods is usually limited due to matrix interaction with other biomolecules. Thus, it is essential to extract these proteins and provide them as a nutraceutical supplement to maintain protein levels and avoid protein deficiency. Hence, protein purification and extraction from natural sources are highly significant in biomedical applications. Chromatography, crude mechanical disruption, use of extractive chemicals, and electrophoresis are some of the methods applied to isolate specific proteins. Even though these methods possess several advantages, they are unable to extract specific proteins with high purity. A suitable alternative is the use of nanoparticles, which can be beneficial in protein purification and extraction. Notably, magnetic iron and iron-based nanoparticles have been employed in protein extraction processes and can be reused via demagnetization due to their magnetic property, smaller size, morphology, high surface-to-volume ratio, and surface charge-mediated property. This chapter is a summary of various magnetic nanoparticles (MNPs) that can be used for the biomolecular separation of proteins.

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Differential precipitation of proteins (DiffPOP) is a simple technique for fractionating complex protein mixtures. Using stepwise addition of acidified methanol, ten distinct subsets of proteins can be selectively precipitated by centrifugation and identified by mass spectrometry-based proteomics. We have previously shown that the ability of a protein to resist precipitation can be altered by drug binding, which enabled us to identify a novel drug-target interaction. Here, we show that the addition of DiffPOP to a standard LC-MS proteomics workflow results in a three-dimensional separation of peptides that increases protein coverage and peptide identifications. Importantly, DiffPOP reveals solubility differences between proteoforms, potentially providing valuable insights that are typically lost in bottom-up proteomics.

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In food manufacturing and particular biomedical products selected proteins are often required. Obtaining the desired proteins in a pure form from natural resources is therefore important, but often very challenging. Herein, we design a sequential coacervation process that allows to efficiently isolate and purify proteins with different isoelectric points (pIs) from a mixed solution, namely Bovine Serum Albumin (BSA, pI = 4.9) and Peroxidase from Horseradish (HRP, pI = 7.2). The key to separation is introducing a suitable polyelectrolyte that causes selective complex coacervation at appropriate pH and ionic strength. Specifically, polyethyleneimine (PEI), when added into the mixture at pH 6.0, produces a coacervation which exclusively contains BSA, leading to a supernatant solution containing 100 % HRP with a purity of 91 %. After separating the dilute and dense phases, BSA is recovered by adding poly(acrylic acid) (PAA) to the concentrated phase, which displaces BSA from the complex because it interacts more strongly with PEI. The supernatant phase after this step contains approximately 75 % of the initial amount of BSA with a purity of 99 %. Our results confirm that coacervation under well-defined conditions can be selective, enabling separation of proteins with adequate purity. Therefore, the established approach demonstrates a facile and sustainable strategy with potential for protein separation at industrial scale.

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Organic polymeric ultrafiltration (UF) membranes have been widely used in protein separation due to their advantages of high flux and simple manufacturing process. However, due to the hydrophobic nature of the polymer, pure polymeric UF membranes need to be modified or hybrid to increase their flux and anti-fouling performance. In this work, tetrabutyl titanate (TBT) and graphene oxide (GO) were simultaneously added to the polyacrylonitrile (PAN) casting solution to prepare a TiO2@GO/PAN hybrid ultrafiltration membrane using a non-solvent induced phase separation (NIPS). During the phase separation process, TBT underwent a sol-gel reaction to generate hydrophilic TiO2 nanoparticles in situ. Some of the generated TiO2 nanoparticles reacted with the GO through a chelation interaction to form TiO2@GO nanocomposites. The resulting TiO2@GO nanocomposites had higher hydrophilicity than the GO. They could selectively segregate towards the membrane surface and pore walls through the solvent and non-solvent exchange during the NIPS, significantly improving the membrane's hydrophilicity. The remaining TiO2 nanoparticles were segregated from the membrane matrix to increase the membrane's porosity. Furthermore, the interaction between the GO and TiO2 also restricted the excessive segregation of the TiO2 nanoparticles and reduced their losing. The resulting TiO2@GO/PAN membrane had a water flux of 1487.6 L·m-2·h-1 and a bovine serum albumin (BSA) rejection rate of 99.5%, which were much higher than those of the currently available UF membranes. It also exhibited excellent anti-protein fouling performance. Therefore, the prepared TiO2@GO/PAN membrane has important practical applications in the field of protein separation.

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Mobile phase composition is an important factor for a further improvement of ion exchange chromatography steps of proteins. In this work, the effects of mixed salts on the retention factors of the two model proteins lysozyme (LYZ) and bovine serum albumin (BSA) in cation exchange chromatography (CEC) were investigated and compared to effects previously observed in hydrophobic interaction chromatography (HIC). The model equation describing the effects in HIC was adjusted for linear gradient elution experiments in CEC. The investigated salts were sodium chloride, sodium sulfate, ammonium chloride and ammonium sulfate. By varying binary salt mixtures as well as using pure salts, model parameters were determined. The normalized root mean square error (NRMSE) of the predicted retention factors for the calibration runs was 4.1% for BSA and 3.1% for LYZ. Additional validation experiments proved the ability of the model to describe and predict retention behavior of the proteins for further salt compositions. Hereby, the NRMSE values for BSA and LYZ were 2.0% and 1.5%, respectively. While the retention factors of LYZ changed linearly with the salt composition, non-linearities in the impact of the anion composition were found for BSA. This was contributed to an overlay of a synergetic salt effect on a protein-specific effect by sulfate on BSA with non-specific effects of the ions for CEC. However, the impact of the synergetic effects on protein separation is lower for CEC than for HIC, as mixed salts do not increase the separation of these proteins. The best salt composition for separating BSA and LYZ is pure ammonium sulfate. Thus, synergetic salt effects can also occur in CEC, but they have a lower impact than in HIC.

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Membrane fouling during the filtration process is a perennial issue and could lead to reduced separation efficiency. In this work, poly(citric acid)-grafted graphene oxide (PGO) was incorporated into a matrix of single-layer hollow fibre (SLHF) and dual-layer hollow fibrr (DLHF) membranes, respectively, aiming to improve membrane antifouling properties during water treatment. Different loadings of PGO ranging from 0 to 1 wt% were first introduced into the SLHF to identify the best PGO loading for the DLHF preparation with its outer layer modified by nanomaterials. The findings showed that at the optimized PGO loading of 0.7 wt%, the resultant SLHF membrane could achieve higher water permeability and bovine serum albumin rejection compared to the neat SLHF membrane. This is due to the improved surface hydrophilicity and increased structural porosity upon incorporation of optimized PGO loading. When 0.7 wt% PGO was introduced only to the outer layer of DLHF, the cross-sectional matrix of the membrane was altered, forming microvoids and spongy-like structures (more porous). Nevertheless, the BSA rejection of the membrane was improved to 97.7% owing to an inner selectivity layer produced from a different dope solution (without the PGO). The DLHF membrane also demonstrated significantly higher antifouling properties than the neat SLHF membrane. Its flux recovery rate is 85%, i.e. 37% better than that of a neat membrane. By incorporating hydrophilic PGO into the membrane, the interaction of the hydrophobic foulants with the membrane surface is greatly reduced.

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Since the introduction of polyelectrolyte multilayers to protein separation in capillary electrophoresis (CE), some progress has been made to improve separation efficiency by varying different parameters, such as buffer ionic strength and pH, polyelectrolyte nature and number of deposited layers. However, CE is often overlooked as it lacks robustness compared to other separation techniques. In this work, critical parameters for the construction of efficient and reproducible Successive multiple ionic-polymer layers (SMIL) coatings were investigated, focusing on experimental conditions, such as vial preparation and sample conservation which were shown to have a significant impact on separation performances. In addition to repeatability, intra- and inter-capillary precision were assessed, demonstrating the improved capability of poly(diallyldimethylammonium chloride) / poly(sodium styrene sulfonate) (PDADMAC / PSS) coated capillaries to separate model proteins in a 2 M acetic acid background electrolyte when all the correct precautions are put in place (with run to run%RSD(tm) < 1.8%, day to day%RSD(tm) < 3.2% and cap to cap%RSD(tm) < 4.6%). The approach recently introduced to calculate retention factors was used to quantify residual protein adsorption onto the capillary wall and to assess capillary coating performances. 5-layer PDADAMAC / PSS coatings led to average retention factors for the five model proteins of ∼4×10-2. These values suggest a relatively low residual protein adsorption leading to reasonably flat plate height vs linear velocity curves, obtained by performing electrophoretic separations at different electrical voltages (-10 to -25 kV).

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Upon recently studying the use of pressure gradients during liquid chromatography (LC), it was noted that pressure differentials across a column can have a significant impact on peak shape, not just retention as has been noted several times before. Theoretical models and thought experiments were performed here to more carefully study these effects. Two situations have been elucidated. The first is one that reflects a protein reversed phase separation wherein solute retention increases with pressure. In this condition, it has been found that a positive pressure gradient will result in band broadening while a negative pressure gradient will help yield sharper peaks. The second case that has come to be better appreciated is when solute retention decreases with pressure, which can occur in protein ion exchange (IEX) and hydrophobic interaction chromatography (HIC). In this situation, a positive pressure gradient will conversely result in peak sharpening, and a negative pressure gradient will introduce band broadening. These observations have facilitated making new fundamental understandings on pressurized separations which has in turn made it possible to begin envisioning new ways of and reasons for applying pressure enhanced LC methods.

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Separation of nucleic acids and proteins using gels has always been a crucial part of molecular biology research. For low-molecular-weight nucleic acids and proteins, low- and medium-concentration agarose gels cannot achieve the high resolution as polyacrylamide gels. We found that 6 %-14 % high-concentration agarose gels (HAGs) could be easily dissolved in an autoclave and the vertical gel cast can be effortlessly filled using an easy-made plastic box. Coupled with the improved buffer condition, HAG electrophoresis resulted in a good resolution of DNA and protein bands. With conventional TBE buffer plus 0.2 % NaCl, DNA fragments that differ by 2-5-bp within the 50-200-bp size range can be resolved on 6 %-8 % HAGs. By using TBE without NaCl, DNA fragments that differ by 2-bp or 2-nt within the 10-100-bp size range can be well resolved on >8 % HAGs. Using a buffer system comprising 1 M Tris-Cl for gel preparation, 0.2 M Tris-Cl/0.2 % SDS as upper tank buffer, and 0.2 M Tris-Cl as the lower tank buffer, HAGs achieved good molecular weight separation of total bacterial and plant proteins in the 10-200 kDa range. In conclusion, we developed a method for HAG preparation and electrophoresis of low-molecular-weight nucleic acids and proteins.

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In this study, composite monoliths with porous structures were prepared using quaternized chitosan and diatom earth for protein separation. Quaternized chitosan (N-[(2-hydroxy-3-trimethylammonium)propyl] chitosan chloride) dissolved in water was mixed with diatom earth and crosslinked with glutaraldehyde under low-temperature conditions to form a cryogel. Interconnected porous monoliths were obtained after removing ice crystals from the cryogel. The monoliths adsorbed bovine serum albumin selectively from the solution mixture of bovine serum albumin and bovine ɤ-globulin, and bovine ɤ-globulin was recovered in the flow-through fraction. The adsorption selectivity was enhanced by changing the solution pH from 6.8 to 5.5. The adsorption of bovine serum albumin by the monolith was replicated at least five times following its washing with a buffer containing 400 mM NaCl and subsequent regeneration with a 10 mM acetate buffer. The composited monolith is a promising adsorbent for the removal of acidic proteins, such as serum albumin contamination in neutral proteins, for example, ɤ-globulins, in bioproduction processes.

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The global analysis of the proteome is an important tool in cell biology. Comparative proteomic evaluations can identify and compare the composition, dynamics, and modifications between different samples. Comparing tissue proteomes under different conditions is crucial for advancing the biomedical field. Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) is a sensitive and robust biochemical method that can compare multiple protein samples over a broad dynamic range on the same analytical gel and can be used to establish differentially expressed protein profiles between different sample groups. 2D-DIGE involves fluorescently labeling protein samples with CyDye flours, via a two-dye or a three-dye system, pre-separation by isoelectric point, and molecular weight. DIGE circumvents gel-to-gel variability by multiplexing samples to a single gel and through the use of a pooled internal standard for normalization, thus enabling accurate high-resolution analysis of differences in protein abundance between samples. This chapter discusses 2D-DIGE as a comparative tissue proteomic technique and describes in detail the experimental steps required for comparative proteomic analysis employing both options of two-dye and three-dye DIGE minimal labeling.

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In proteomics, the need to precisely examine the protein compounds of small samples, requires sensitive analytical methods which can separate and enrich compounds with high precision. Current techniques require a minimal analysis time to obtain satisfactory compound separation where longer analysis time means better separation of compounds. But, molecular diffusion will create broadening of the separated compound bands over time, increasing the peak width, and thus reducing the resolution and the enrichment. Electric field gradient focusing (EFGF) is a separation technique, in which proteins are simultaneously separated and enriched by balancing a gradient electrostatic force with a constant hydrodynamic drag force. Because of this balance, analytes are continuously pushed back to their focusing point, limiting the time-dependent peak broadening due to molecular diffusion. Current EFGF techniques are however still suffering from peak broadening because of flow-profile inhomogeneities. In this paper, we propose to use AC electro-osmotic flow (AC EOF) to create a homogeneous flow in EFGF. The interference between the electric field gradient and the AC EOF was thoroughly analysed and the concept was validated using numerical simulations. The results show that a plug flow is obtained on top of a small, distorted boundary layer. While applying different DC electric fields in the electrolyte, a constant flow velocity can be obtained by including a DC offset to the electrodes generating the AC EOF. The plug flow is then maintained over the whole separation channel length, while an electric field gradient is applied. This way, the flow-induced contribution to peak broadening can be minimized in EFGF devices. By modelling the separation of green fluorescent protein (GFP) and R-Phycoerythrin (R-PE), it was shown that the peak width of separated compounds can be reduced and that the separation resolution can be improved, compared to current EFGF methods.

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The performance of the current bottom-up liquid chromatography hyphenated with mass spectrometry (LC-MS) analyses has undoubtedly been fueled by spectacular progress in mass spectrometry. It is thus not surprising that the MS instrument attracts the most attention during LC-MS method development, whereas optimizing conditions for peptide separation using reversed-phase liquid chromatography (RPLC) remains somewhat in its shadow. Consequently, the wisdom of the fundaments of chromatography is slowly vanishing from some laboratories. However, the full potential of advanced MS instruments cannot be achieved without highly efficient RPLC. This is impossible to attain without understanding fundamental processes in the chromatographic system and the properties of peptides important for their chromatographic behavior. We wrote this tutorial intending to give practitioners an overview of critical aspects of peptide separation using RPLC to facilitate setting the LC parameters so that they can leverage the full capabilities of their MS instruments. After briefly introducing the gradient separation of peptides, we discuss their properties that affect the quality of LC-MS chromatograms the most. Next, we address the in-column and extra-column broadening. The last section is devoted to key parameters of LC-MS methods. We also extracted trends in practice from recent bottom-up proteomics studies and correlated them with the current knowledge on peptide RPLC separation.

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