Elution Of Proteins Research Articles

A batch screening technique for the calculation of chromatographic separability

This paper employs a high-throughput parallel batch (microtiter plate) adsorption screen with sequential salt step increases to rapidly generate protein elution profiles for multiple resins at different pHs using a protein library. The chromatographic set used in this work includes single mode, multimodal anion-exchange (MMA), and multimodal cation-exchange (MMC) resins. The protein library consists of proteins with isoelectric points ranging from 5.1 to 11.4 with varying hydrophobicities as determined by their retention on hydrophobic interaction chromatography. The batch sequential experiments are carried out using one protein at a time with a wide set of resins at multiple pH conditions, thus enabling simple microtiter plate detection. A mathematical formulation is then used to determine the first moment of the distributions from each chromatogram (sequential step elution) generated in the parallel batch experiments. Batch data first moments (expressed in salt concentration) are then compared to results obtained from column linear salt gradient elution, and the techniques are shown to be consistent. In addition, first moment data are used to calculate one-resin separability scores, which are a measure of a resin's ability, at a specified pH, to separate the entire set of proteins in the library from one another. Again, the results from the batch and column experiments are shown to be comparable. The first moment data sets were then employed to calculate the two-resin separability scores, which are a measure of the ability of two resins to synergistically separate the entire set of proteins in the library. Importantly, these results based on the two-resin separability performances derived from the batch and column experiments were again shown to be consistent. This approach for rapidly screening large numbers of chromatographic resins and mobile phase conditions for their elution behavior may prove useful for enabling the rapid discovery of new chromatographic ligands and resins.

Modeling multi-component separation in hydrophobic interaction chromatography with improved parameter-by-parameter estimation method

Mechanistic models are powerful tools for chromatographic process development and optimization. However, hydrophobic interaction chromatography (HIC) mechanistic models lack an effective and logical parameter estimation method, especially for multi-component system. In this study, a parameter-by-parameter method for multi-component system (called as mPbP-HIC) was derived based on the retention mechanism to estimate the six parameters of the Mollerup isotherm for HIC. The linear parameters (ks,i and keq,i) and nonlinear parameters (ni and qmax,i) of the isotherm can be estimated by the linear regression (LR) and the linear approximation (LA) steps, respectively. The remaining two parameters (kp,i and kkin,i) are obtained by the inverse method (IM). The proposed method was verified with a two-component model system. The results showed that the model could accurately predict the protein elution at a loading of 10 g/L. However, the elution curve fitting was unsatisfactory for high loadings (12 g/L and 14 g/L), which is mainly attributed to the demanding experimental conditions of the LA step and the potential large estimation error of the parameter qmax. Therefore, the inverse method was introduced to further calibrate the parameter qmax, thereby reducing the estimation error and improving the curve fitting. Moreover, the simplified linear approximation (SLA) was proposed by reasonable assumption, which provides the initial guess of qmax without solving any complex matrix and avoids the problem of matrix unsolvable. In the improved mPbP-HIC method, qmax would be initialized by the SLA and finally determined by the inverse method, and this strategy was named as SLA+IM. The experimental validation showed that the improved mPbP-HIC method has a better curve fitting, and the use of SLA+IM reduces the error accumulation effect. In process optimization, the parameters estimated by the improved mPbP-HIC method provided the model with excellent predictive ability and reasonable extrapolation. In conclusion, the SLA+IM strategy makes the improved mPbP-HIC method more rational and can be easily applied to the practical separation of protein mixture, which would accelerate the process development for HIC in downstream of biopharmaceuticals.

Online Mixed-Bed Ion Exchange Chromatography for Native Top-Down Proteomics of Complex Mixtures.

Native top-down mass spectrometry (nTDMS) allows characterization of protein structure and noncovalent interactions with simultaneous sequence mapping and proteoform characterization. The majority of nTDMS studies utilize purified recombinant proteins, with significant challenges hindering application to endogenous systems. To perform native top-down proteomics (nTDP), where endogenous proteins from complex biological systems are analyzed by nTDMS, it is essential to separate proteins under nondenaturing conditions. However, it remains difficult to achieve high resolution with MS-compatible online chromatography while preserving protein tertiary structure and noncovalent interactions. Herein, we report the use of online mixed-bed ion exchange chromatography (IEC) to enable separation of endogenous proteins from complex mixtures under nondenaturing conditions, preserving noncovalent interactions for nTDP analysis. We have successfully detected large proteins (>146 kDa) and identified endogenous metal-binding and oligomeric protein complexes in human heart tissue lysate. The use of a mixed-bed stationary phase allowed retention and elution of proteins over a wide range of isoelectric points without altering the sample or mobile phase pH. Overall, our method provides a simple online IEC-MS platform that can effectively separate proteins from complex mixtures under nondenaturing conditions and preserve higher-order structure for nTDP applications.

Modeling Chromatography Binding through Molecular Dynamics Simulations with Resin Fragments.

Accurate atomistic modeling of the interactions of a chromatography resin with a solute can inform the selection of purification conditions for a product, an important problem in the biotech and pharmaceutical industries. We present a molecular dynamics simulation-based approach for the qualitative prediction of interaction sites (specificity) and retention times (affinity) of a protein for a given chromatography resin. We mimicked the resin with an unrestrained ligand composed of the resin headgroup coupled with successively larger fragments of the agarose backbone. The interactions of the ligand with the protein are simulated in an explicit solvent using the Replica Exchange Molecular Dynamics enhanced sampling approach in conjunction with Hydrogen Mass Repartitioning (REMD-HMR). We computed the ligand interaction surface from the simulation trajectories and correlated the features of the interaction surface with experimentally determined retention times. The simulation and analysis protocol were first applied to a series of ubiquitin mutants for which retention times on Capto MMC resin are available. The ubiquitin simulations helped identify the optimal ligand that was used in subsequent simulations on six proteins for which Capto MMC elution times are available. For each of the six proteins, we computed the interaction surface and characterized it in terms of a range of simulation-averaged residue-level physicochemical descriptors. Modeling of the salt concentrations required for elution with respect to the descriptors resulted in a linear fit in terms of aromaphilicity and Kyte-Doolittle hydrophobicity that was robust to outliers, showed high correlation, and correctly ranked the protein elution order. The physics-based model building approach described here does not require a large experimental data set and can be readily applied to different resins and diverse biomolecules.

Characterization of vitamin D3 biotransformation by the cell lysate of Actinomyces hyovaginalis CCASU-A11-2

A former work conducted in our Lab, lead to in a effective scale up of vitamin D3 bioconversion into calcitriol by Actinomyces (A.) hyovaginalis isolate CCASU-A11-2 in Lab fermenter (14 L) resulting in 32.8 µg/100 mL of calcitriol. However, the time needed for such a bioconversion process was up to 5 days. Therefore, the objective of this study was to shorten the bioconversion time by using cell-free lysate and studying different factors influencing bioconversion. The crude cell lysate was prepared, freeze-dried, and primarily fractionated into nine fractions, of which, only three fractions, 50, 100, and 150 mM NaCl elution buffers showed 22, 12, and 2 µg/10 mL, calcitriol production, respectively. Ammonium sulfate was used for protein precipitation, and it did not affect the bioconversion process except at a concentration of 10%w/v. Secondary fractionation was carried out using 80 mL of the 50 mM NaCl elution buffer and the results showed the 80 mL eluent volume was enough for the complete elution of the active protein. The pH 7.8, temperature 28 °C, and 6 h reaction time were optimum for maximum calcitriol production (31 µg/10 mL). In conclusion, the transformation of vitamin D3 into calcitriol was successfully carried out within 6 h and at pH 7.8 and 28 °C using fractionated cell lysate. This process resulted in a 10-fold increase in calcitriol as compared to that produced in our previous study using a 14 L fermenter (32.8 µg/100 mL). Therefore, cell-free lysate should be considered for industrial and scaling up vitamin D3 bioconversion into calcitriol.

Histidine tag modified magnetic beads for analysis of arsenic binding proteins

BackgroundMany proteins with thiol groups can bind with trivalent arsenic which are termed as arsenic binding proteins, thus change their physiological functions. Therefore, it is vital to analyze the arsenic binding proteins in cells. The Pull-Down strategy based on biotinylated phenylarsenic acid (Bio-PAO(III)) probes is an effective way for analysis of arsenic binding proteins. In this strategy, streptavidin magnetic beads (SA-MBs) was applied to capture the arsenic binding proteins conjugating with Bio-PAO(III) probe. However, strong interaction between SA and biotin makes the elution of arsenic binding proteins not easy. ResultsWe developed a novel affinity separation strategy to address the challenge of eluting arsenic binding proteins, a key issue with the existing Bio-PAO(III) Pull-Down method. By employing magnetic beads modified with Nα-Bis(carboxymethyl)-l-lysine (NTA-Lys), polyhistidine-tag (His6-Tag), and SA (MB-NTA(Ni)-His6-SA), we established a more efficient purification process. This innovative approach enables selective capture of arsenic binding proteins in HepG2 cells labeled by Bio-PAO(III) probes, facilitating gentle digestion by trypsin for precise identification through capillary high performance liquid chromatography (Cap HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS). What is more, the magnetic beads can be regenerated by using imidazole as the eluent, and the obtained MB-NTA(Ni) can be reloaded with His6-SA for next use. Our method successfully identified 41 arsenic binding proteins, including those involved in cytoskeletal structure, heat shock response, transcriptional regulation, DNA damage repair, redox state regulation, mitochondrial dehydrogenase function, and protein synthesis and structure. SignificanceThis work contributes to a more comprehensive understanding of the toxic mechanisms of arsenic, potentially providing valuable insights for the prevention or treatment of arsenic-related diseases.

On-column refolding and off-column assembly of parvovirus B19 virus-like particles from bacteria-expressed protein

Virus-like particles (VLPs) are nanometric structures composed of structural components of virions, keeping most of the cellular recognition and internalization properties, but are non-infective as they are deprived of their genetic material. VLPs have been a versatile platform for developing vaccines by carrying their own or heterologous antigenic epitopes. Moreover, VLPs can also be used as nanovessels for encapsulating molecules with therapeutic applications, like enzymes, nucleic acids, and drugs. Parvovirus B19 (B19V) VLPs can be self-assembled in vitro from the denatured major viral particle protein VP2 by equilibrium dialysis. Despite its fair productivity, this process is currently a time-consuming task. Affinity chromatography is used as an efficient step for concentration and purification, but it is only sometimes seen as a method that facilitates the oligomerization of proteins. In this research, we report a novel approach for the in vitro assembly of B19V VLPs through the immobilization of the denatured VP2 into an immobilized metal affinity chromatography (IMAC) column, followed by the on-column folding and the final VLP assembly upon protein elution. This method is suitable for the fast production of B19V VLPs.Key points• Biotechnological applications for inclusion bodies• Efficient single-step purification and immobilization strategies• Rapid VLP assembly strategyGraphical abstract

Ultrasound-assisted alkaline hydrolysis of fish(Cyprinus carpio L.) scale gelatin: A possible curcumin delivery system with increased water solubility and sustained release

This study investigated the effect of ultrasound-assisted alkaline treatment on the structural characteristics and curcumin loading efficiency in fish scale gelatin (FSG). The results showed that FSG treated with a combination of ultrasound and alkaline hydrolysis outperformed non-treated FSG, as well as alkaline- or ultrasound-treated FSG in augmenting curcumin loading efficiency (from 3.02 to 18.36 μg/mL). In contrast to FSG exposed to single treatments (alkaline or ultrasound), the FSG subjected to combined treatment exhibited a notably higher ratio of hydrophobic amino acids (47.51 ± 2.27%) and protein elution amount (180.87 μg/mL). Meanwhile, single and combined treatments significantly modified the molecular conformation as well as the microstructure of FSG, transforming the α-helical conformation to the β-sheet structure and presenting an ordered structure with increased voids. The curcumin-loaded FSG subjected to ultrasound-assisted alkaline treatment revealed a sustained release profile under simulated gastrointestinal conditions. The elucidated outcomes underscore the significant potential of FSG subjected to ultrasound-assisted alkaline treatment as a promising avenue for facilitating the controlled delivery of bioactive hydrophobic nutrients.

Enhancing protein trapping efficiency of graphene oxide-polybutylene succinate nanofiber membrane via molecular imprinting

Filtration of biological liquids has been widely employed in biological, medical, and environmental investigations due to its convenience; many could be performed without energy and on-site, particularly protein separation. However, most available membranes are universal protein absorption or sub-fractionation due to molecule sizes or properties. SPMA, or syringe-push membrane absorption, is a quick and easy way to prepare biofluids for protein evaluation. The idea of initiating SPMA was to filter proteins from human urine for subsequent proteomic analysis. In our previous study, we developed nanofiber membranes made from polybutylene succinate (PBS) composed of graphene oxide (GO) for SPMA. In this study, we combined molecular imprinting with our developed PBS fiber membranes mixed with graphene oxide to improve protein capture selectivity in a lock-and-key fashion and thereby increase the efficacy of protein capture. As a model, we selected albumin from human serum (ABH), a clinically significant urine biomarker, for proteomic application. The nanofibrous membrane was generated utilizing the electrospinning technique with PBS/GO composite. The PBS/GO solution mixed with ABH was injected from a syringe and transformed into nanofibers by an electric voltage, which led the fibers to a rotating collector spinning for fiber collection. The imprinting process was carried out by removing the albumin protein template from the membrane through immersion of the membrane in a 60% acetonitrile solution for 4 h to generate a molecular imprint on the membrane. Protein trapping ability, high surface area, the potential for producing affinity with proteins, and molecular-level memory were all evaluated using the fabricated membrane morphology, protein binding capacity, and quantitative protein measurement. This study revealed that GO is a controlling factor, increasing electrical conductivity and reducing fiber sizes and membrane pore areas in PBS-GO-composites. On the other hand, the molecular imprinting did not influence membrane shape, nanofiber size, or density. Human albumin imprinted membrane could increase the PBS-GO membrane’s ABH binding capacity from 50 to 83%. It can be indicated that applying the imprinting technique in combination with the graphene oxide composite technique resulted in enhanced ABH binding capabilities than using either technique individually in membrane fabrication. The suitable protein elution solution is at 60% acetonitrile with an immersion time of 4 h. Our approach has resulted in the possibility of improving filter membranes for protein enrichment and storage in a variety of biological fluids.

Liquid-chromatography mass spectrometry describes post-translational modification of Shewanella outer membrane proteins

Electrogenic bacteria deliver excess respiratory electrons to externally located metal oxide particles and electrodes. The biochemical basis for this process is arguably best understood for species of Shewanella where the integral membrane complex termed MtrCAB is key to electron transfer across the bacterial outer membranes. A crystal structure was recently resolved for MtrCAB from S. baltica OS185. However, X-ray diffraction did not resolve the N-terminal residues so that the lipidation status of proteins in the mature complex was poorly described. Here we report liquid chromatography mass spectrometry revealing the intact mass values for all three proteins in the MtrCAB complexes purified from Shewanella oneidensis MR-1 and S. baltica OS185. The masses of MtrA and MtrB are consistent with both proteins being processed by Signal Peptidase I and covalent attachment of ten c-type hemes to MtrA. The mass of MtrC is most reasonably interpreted as arising from protein processed by Signal Peptidase II to produce a diacylated lipoprotein containing ten c-type hemes. Our two-step protocol for liquid-chromatography mass spectrometry used a reverse phase column to achieve on-column detergent removal prior to gradient protein resolution and elution. We envisage the method will be capable of simultaneously resolving the intact mass values for multiple proteins in other membrane protein complexes.

A highly carboxylated sponge-like material: preparation, characterization and protein adsorption

Producing of high-purity protein products using separation materials is critical to the biopharmaceutical industry. Efforts to achieve protein adsorption materials with large adsorption capacity and high processing efficiency have been challenging. Herein, a highly carboxylated sponge-like monolithic adsorbent material was prepared by using SiO2 nanofibers as a rigid support framework, and esterification crosslinking reactants with polyvinyl alcohol (PVA)/polymeric carboxylic acid (polyacrylic acid, PAA) as a functional organic binder, which were modified in situ on the nanofiber framework. The obtained PVA/PAA sponge-like monolithic adsorbent material (PVA/PAA SMAM) exhibits a porous structure and possesses mechanical stability and recoverability of underwater compression. The high porosity provides more sites for carboxyl groups, making PVA/PAA SMAM rich in carboxyl groups. Experiments showed that PVA/PAA SMAM selectively adsorbes proteins with high isoelectric points. Using Cytochrome C (Cyt-C) as the main model protein with a high isoelectric point, the maximum adsorption of Cyt-C by the PVA/PAA SMAM at pH = 9 was 3079.3 mg/g with a rapid equilibration time (40 min), which is clearly superior to the currently reported Metal-organic frame structure (MOF) composites for Cyt-C adsorption. The isothermal adsorption model for the Cyt-C adsorption of the PVA/PAA SMAM material agrees well with the Freundlich adsorption model and the quasi-first kinetic model. Excellent mechanical and chemical structural stability endow the PVA/PAA SMAM material with protein elution capacity and good reusability, and secondary structure of the protein after elution was unchanged. Subsequently, selective adsorption and separation of Cyt-C from porcine heart supernatant was successfully achieved with PVA/PAASMAM material. This work provides new materials with excellent performance for various bio-separation applications.

Effect of the Competing Ligand on Ni-NTA/Histag Strength Revealed by Click Chemistry-Based Force Spectroscopy.

The specific interaction between Ni-nitrilotriacetic acid and the six-histidine tag may be one of the most important coordination bonds utilized in biological research because of its wide application for recombinant protein purification. The complex stability is critical for target protein binding. Thus, measurement of the mechanical stability of the system was attempted soon after the invention of atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) two decades ago. Moreover, the competing ligand imidazole and protons are the two critical factors for target protein elution. However, the mechanochemistry between the system and the imidazole/proton has not been determined. Here, an AFM-SMFS system using strain-promoted alkyne-azide cycloaddition and Cu-free click chemistry was used to characterize the system. Consequently, the destabilizing effect of the imidazole and proton on the interaction was revealed quantitatively, leading to a 3-fold increase in the bond dissociation rate.

Purification of a monoclonal antibody using a novel high-capacity multimodal cation exchange nonwoven membrane

A high-capacity, multimodal cation exchange (MMC) chromatographic membrane was developed by conjugating a multimodal ligand – 2-mercaptopyridine-3-carboxylic acid (MPCA) – on a polybutylene terepthalate (PBT) nonwoven fabric. The membrane features an equilibrium binding capacity of ≈ 1000 mg of human polyclonal IgG (IgG) per g of membrane and dynamic binding capacities (DBC10%) ranging from 77.5 to 115.1 mg/mL (residence times of 1 and 5 min, respectively); these values are 2-to-3-fold higher than those of commercial MMC adsorbents. The effects of buffer composition, pH, conductivity on the binding behavior of the MMC-MPCA membrane were investigated in detail. As a moderate cation exchange binder, MPCA enables effective protein elution using buffers with mild pH (8.0–9.0) and conductivity (≈13 mS/cm), thus circumventing the harsh conditions often needed in multimodal chromatography. The MMC-MPCA membrane was evaluated for product capture in bind-and-elute mode on a Chinese hamster ovary (CHO) cell culture harvest containing therapeutic monoclonal antibodies, using commercial multimodal (Capto MMC and MX-Trp-650M) and affinity (AF-rProtein A HC-650F) resins as controls. The MMC-MPCA membrane outperformed the multimodal resins in terms of binding capacity as well as clearance of host cell proteins (HCPs) and aggregates. The membrane was then evaluated by polishing the mAb from a Protein A eluate in bind-and-elute mode. The MMC-MPCA membrane reduced the level of high molecular weight components from 11% to 4% and the HCP content from 1319.7 ppm to 48.7 ppm (LRV of 1.4). Most notably, proteomics analysis of the product demonstrated the clearance of a significant fraction of persistent, high-risk HCPs from the Protein A eluate.

Accurate determination of the milk protein allergen β-lactoglobulin by on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry

An on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry (AA-SPE-CE-MS) method was developed to purify, preconcentrate, separate, and characterize the milk allergenic protein β-lactoglobulin (β-LG) in food samples. The sorbent to pack into the SPE microcartidges was prepared by immobilizing an aptamer against β-LG onto magnetic bead particles. After optimizing the SPE-CE-MS method, the sample (ca. 75 μL) was loaded in separation background electrolyte (BGE, 2 M acetic acid pH 2.2), while a solution of 100 mM NH4OH (pH 11.2) (ca. 100 nL) was used for the protein elution. The linearity of the method ranged between 0.1 and 20 μg mL−1 and the limit of detection (LOD) was 0.05 μg mL−1, which was 200 times lower than by CE-MS. The method was repeatable in terms of relative standard deviation (RSD) for migration times and peak areas (<0.5% and 2.4%, respectively) and microcartridge lifetime was more than 25 analyses. The applicability of the method for the determination of low levels of β-LG was shown by analyzing milk-free foods (i.e. a 100% cocoa dark chocolate, a hypoallergenic formula for infants, and a dairy-free white bread) and milk-containing white breads. Results were satisfactory in all cases, thus demonstrating the great potential of the developed method for accurate food safety and quality control.

Optimization and evaluation of tear protein elution from Schirmer's strips in dry eye disease.

Extraction of tear protein from Schirmer's strip is a prerequisite for the proper identification and screening of biomarkers in dry eye disease. The study compares different methods of extraction of tear proteins from the Schirmer's strip. Reflex tear was collected from healthy controls (HC; n = 12), Stevens-Johnson syndrome (SJS; n = 3) and dry eye disease (DED; n = 3) patients using capillary tube. This tear was used to measure the volume absorbed by Schirmer's strip per microliter. Different buffers (6) were used to compare the protein yield from the Schirmer's strip in four different conditions. The tear proteins extracted using the highest protein yield buffer were analyzed by mass spectrometry. A linear relationship between the tear volume and wetting length was observed (r = 0.0.997, n = 6). The highest yield was observed after incubation of the Schirmer's strip in 100 mM ammonium bicarbonate (ABC) with 0.25% Nonidet P-40(NP-40) at 4°C for an hour (P < 0.00005). The in-solution digestion of tear eluted in the above condition 100 Mm ABC + 0.25% NP-40 with one-hour incubation yielded a total of 2119 proteins in HC, SJS, and DED. The unique protein observed in SJS and DED was 0.6% and 17.9%, respectively. The significantly expressed proteins are associated with innate immune response, proteolysis, wound healing, and defense response. A method for extraction of protein from Schirmer's strip was optimized for increase in protein yield from the tear sample. SJS and DED tear samples have unique protein signature. The study will aid in better design of tear protein-based experimental study.

DNA aptamer-based affinity chromatography system for purification of recombinant proteins tagged with lysine tag

Affinity chromatography (AC) is one of the techniques widely used for the purification of recombinant proteins. In our previous study, we presented a successful application of the Argi system [1] for the purification of recombinant proteins, based on the specific interaction between an arginine tag and a DNA aptamer. Exploring the possible application of positively charged peptide tags in the purification of recombinant proteins, in this study we developed and characterized an AC system based on the specific and reversible interaction between a DNA aptamer and a lysine tag (Lys-tag) comprising five lysine residues (5 K). We optimized the length of both the selected DNA aptamer and Lys-tag which were named B5K aptamer and 5K-tag, respectively. The results showed that the stability of the B5K aptamer and 5K-tag was dependent on the presence of potassium ions. The conditions for mild elution of 5K-tagged protein from B5K aptamer were determined. Our study proved that the developed system can be used for the purification of recombinant proteins from Escherichia coli total protein extracts.

Native Size-Exclusion Chromatography–Based Mass Spectrometry Reveals New Components of the Early Heat Shock Protein 90 Inhibition Response Among Limited Global Changes

The molecular chaperone heat shock protein 90 (HSP90) works in concert with co-chaperones to stabilize its client proteins, which include multiple drivers of oncogenesis and malignant progression. Pharmacologic inhibitors of HSP90 have been observed to exert a wide range of effects on the proteome, including depletion of client proteins, induction of heat shock proteins, dissociation of co-chaperones from HSP90, disruption of client protein signaling networks, and recruitment of the protein ubiquitylation and degradation machinery—suggesting widespread remodeling of cellular protein complexes. However, proteomics studies to date have focused on inhibitor-induced changes in total protein levels, often overlooking protein complex alterations. Here, we use size-exclusion chromatography in combination with mass spectrometry (SEC-MS) to characterize the early changes in native protein complexes following treatment with the HSP90 inhibitor tanespimycin (17-AAG) for 8 h in the HT29 colon adenocarcinoma cell line. After confirming the signature cellular response to HSP90 inhibition (e.g., induction of heat shock proteins, decreased total levels of client proteins), we were surprised to find only modest perturbations to the global distribution of protein elution profiles in inhibitor-treated HT29 cells at this relatively early time-point. Similarly, co-chaperones that co-eluted with HSP90 displayed no clear difference between control and treated conditions. However, two distinct analysis strategies identified multiple inhibitor-induced changes, including known and unknown components of the HSP90-dependent proteome. We validate two of these—the actin-binding protein Anillin and the mitochondrial isocitrate dehydrogenase 3 complex—as novel HSP90 inhibitor-modulated proteins. We present this dataset as a resource for the HSP90, proteostasis, and cancer communities (https://www.bioinformatics.babraham.ac.uk/shiny/HSP90/SEC-MS/), laying the groundwork for future mechanistic and therapeutic studies related to HSP90 pharmacology. Data are available via ProteomeXchange with identifier PXD033459.

Global landscape of protein complexes in postprandial-state livers from diet-induced obese and lean mice

Obesity is associated with a spectrum of nonalcoholic fatty liver disease (NAFLD) which is characterized by steatosis. Prolonged fat deposition aggravates liver dysfunctions leading to an advanced form of NAFLD such as steatohepatitis and cirrhosis. As liver function in the postprandial state is critical for macronutrient metabolism and energy homeostasis, we sought to determine the differences in protein complex profiles in lean and fatty livers in the postprandial state. Protein complex profiling is of interest as proteins often do not function alone and the information on the interactions may reveal novel etiology of NAFLD, which is currently limited compared with proteome profiles or RNA-sequencing profiles. To this end, we fractionated liver lysates using size-exclusion chromatography (SEC) and analyzed each fraction using untargeted LC-MS/MS. We identified 1172 proteins that were discovered in lean and fatty livers, and their elution profiles were compared. We found that the majority of liver proteins were present as putative complexes. Also, the fatty liver protein elution profile showed great conservations as lean liver despite the metabolic disease state. Yet, we discovered a few proteins that showed different elution patterns in the fatty liver, including Acadm, Aldh1a7, Aldh1a1, Akr1a1, Eif3l, Fkbp2, G6pdx, Gm20441, Hao1, Pcna, Pkm, Ppif, Prdx4, Stmn1, Tagln, Tubb4b, Ubqln2, and Usp14, which may be involved in high fat diet-induced alterations of protein oligomerization and hepatic functions. Overall, our protein complex profiling could expand our understanding of hepatic abnormalities that cannot be uncovered by simple quantitation of protein expression.

Comparative analysis of trilobal capillary-channeled polymer fiber columns with superficially porous and monolithic phases toward reversed-phase protein separations.

A trilobal capillary-channeled polymer fiber stationary phase is evaluated for its performance for intact protein separations under reversed-phase high-performance liquid chromatography conditions. The separation quality, operational characteristics, and protein dynamic loading capacity on the fiber phases are compared to commercially-available superficially porous and monolithic columns. The trilobal or "y-shaped" polypropylene fiber phase was employed to separate a synthetic mixture of five proteins (having diverse chemistries and molecular weights). The separation quality was evaluated based on the resolution, peak heights/recoveries, peak widths, and peak areas. The present work illustrates the unique ability to operate at higher linear velocities (47.5mm/s) while maintaining lower back pressures (∼4 MPa), faster separation times (<8 min), and faster gradient rates using the fiber columns while yielding comparable chromatographic performance to the commercial columns. The separations employing the commercial stationary phases operate at lower linear velocities (∼3.0 mm/s), higher back pressures (∼9 MPa), require longer separation times (10 min), and require slightly higher compositions of organic mobile phase to effect protein elution. Likewise, based on breakthrough loading analysis of lysozyme and bovine serum albumin, the trilobal, polypropylene C-CP fiber column stationary phases demonstrate 3-9X greater binding capacities on a bed volume basis versus the commercial columns.

Application of Quantum Cascade Laser-Infrared Spectroscopy and Chemometrics for In-Line Discrimination of Coeluting Proteins from Preparative Size Exclusion Chromatography.

An external-cavity quantum cascade laser (EC-QCL)-based flow-through mid-infrared (IR) spectrometer was placed in line with a preparative size exclusion chromatography system to demonstrate real-time analysis of protein elutions with strongly overlapping chromatographic peaks. Two different case studies involving three and four model proteins were performed under typical lab-scale purification conditions. The large optical path length (25 μm), high signal-to-noise ratios, and wide spectral coverage (1350 to 1750 cm–1) of the QCL-IR spectrometer allow for robust spectra acquisition across both the amide I and II bands. Chemometric analysis by self-modeling mixture analysis and multivariate curve resolution enabled accurate quantitation and structural fingerprinting across the protein elution transient. The acquired concentration profiles were found to be in excellent agreement with the off-line high-performance liquid chromatography reference analytics performed on the collected effluent fractions. These results demonstrate that QCL-IR detectors can be used effectively for in-line, real-time analysis of protein elutions, providing critical quality attribute data that are typically only accessible through time-consuming and resource-intensive off-line methods.