High-resolution Protein Separation Research Articles

DIGE Analysis Software and Protein Identification Approaches.

Two-dimensional difference gel electrophoresis (2D-DIGE) is a high-resolution protein separation technique, with the excellent dynamic range obtained by fluorescent tag labeling of protein samples. Scanned images of 2D-DIGE gels show thousands of protein spots, each spot representing a single or a group of protein isoforms. By using commercially available software, each protein spot is defined by an outline, which is digitized and correlated with the quantity of proteins present in each spot. Software packages include DeCyder, SameSpots, and Dymension 3. In addition, proteins of interest can be excised from post-stained gels and identified with conventional mass spectrometric techniques. High-throughput mass spectrometry is performed using sophisticated instrumentation, including matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), MALDI-TOF/TOF, and liquid chromatography tandem mass spectrometry (LC-MS/MS). Tandem MS (MALDI-TOF/TOF or LC-MS/MS) analyzes fragmented peptides, resulting in amino acid sequence information, which is especially useful when protein spots are low abundant or where a mixture of proteins is present.

Two-Dimensional Blue Native/SDS Polyacrylamide Gel Electrophoresis for Analysis of Brazilian Bothrops Snake Venoms.

Viperidae snakes are the most important agents of snakebites in Brazil. The protein composition of snake venoms has been frequently analyzed by means of electrophoretic techniques, but the interaction of proteins in venoms has barely been addressed. An electrophoretic technique that has gained prominence to study this type of interaction is blue native polyacrylamide gel electrophoresis (BN-PAGE), which allows for the high-resolution separation of proteins in their native form. These protein complexes can be further discriminated by a second-dimension gel electrophoresis (SDS-PAGE) from lanes cut from BN-PAGE. Once there is no study on the use of bidimensional BN/SDS-PAGE with snake venoms, this study initially standardized the BN/SDS-PAGE technique in order to evaluate protein interactions in Bothrops atrox, Bothrops erythromelas, and Bothrops jararaca snake venoms. Results of BN/SDS-PAGE showed that native protein complexes were present, and that snake venom metalloproteinases and venom serine proteinases maintained their enzymatic activity after BN/SDS-PAGE. C-type lectin-like proteins were identified by Western blotting. Therefore, bidimensional BN/SDS-PAGE proved to be an easy, practical, and efficient method for separating functional venom proteins according to their assemblage in complexes, as well as to analyze their biological activities in further details.

PEPPI-MS: Polyacrylamide-Gel-Based Prefractionation for Analysis of Intact Proteoforms and Protein Complexes by Mass Spectrometry

Prefractionation of complex mixtures of proteins derived from biological samples is indispensable for proteome analysis via top-down mass spectrometry (MS). Polyacrylamide gel electrophoresis (PAGE), which enables high-resolution protein separation based on molecular size, is a widely used technique in biochemical experiments and has the potential to be useful in sample fractionation for top-down MS analysis. However, the lack of a means to efficiently recover the separated proteins in-gel has always been a barrier to its use in sample prefractionation. In this study, we present a novel experimental workflow, called Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS ("PEPPI-MS"), which allows top-down MS of PAGE-separated proteins. The optimization of Coomassie brilliant blue staining followed by the passive extraction step in the PEPPI-MS workflow enabled the efficient recovery of proteins, separated on commercial precast gels, from a wide range of molecular weight regions in under 10 min. Two-dimensional separation combining offline PEPPI-MS with online reversed-phase liquid chromatographic separation resulted in identification of over 1000 proteoforms recovered from the target region of the gel (≤50 kDa). Given the widespread availability and relatively low cost of traditional sodium dodecyl sulfate (SDS)-PAGE equipment, the PEPPI-MS workflow will be a powerful prefractionation strategy for top-down proteomics.

Morphology optimization and assessment of the performance limits of high-porosity nanostructured polymer monolithic capillary columns for proteomics analysis

This study targets the synthesis of high external-porosity poly(styrene-co-divinylbenzene) monolithic support structures with macropore and globule sizes in the sub-micron range, aiming at the realization of high-speed and high-resolution gradient separations of intact proteins and peptides. The thermodynamic and kinetic aspects of the free-radical polymerization synthesis were adjusted by tuning the porogen to monomer ratio, the porogen ratio, the initiator content, and polymerization temperature. Next, column morphology was linked to eddy-dispersion and mobile-phase mass-transfer contributions and the chromatographic performance limits were benchmarked against conventional packed columns and silica monoliths. Polymer monolithic structures yielding a separation impedance as low as 976 were created allowing to generate N > 1,000,000 (for an unretained marker), albeit the expense of very long analysis times. Decreasing the macropore and globule sizes below a certain threshold led to significant increase in eddy dispersion, as globular entities agglomerate, and a small number of large flow-through pores permeate the overall fine interconnected polymer network with small diameter flow-through pores. The potential of monolith chromatography for proteomics application is demonstrated with a ballistic 6 s gradient separation of intact proteins and a high-resolution nanoLC-Orbitrap mass spectrometric analysis of a tryptic E. coli digest applying a coupled-column system.

Simulation and experimental study of the transport of protein bands through cuboid packed-bed devices during chromatographic separations

Recent studies have demonstrated that box-shaped or cuboid packed-bed chromatographic devices represent an efficient alternative to conventional cylindrical columns for high-resolution preparative protein separations. This has been attributed to the greater uniformity of flow within these devices. However, for a more complete explanation, it is important to understand how the system hydrodynamics affects band broadening during the transport of proteins through these devices. In this study, we present first principle mathematical models to capture this interplay. These models were validated by flow-through and bind-and-elute experiments carried out using a colored protein as tracer. Control experiments were also carried out using equivalent commercial columns, i.e. having same bed height and cross-sectional area, and packed with same media. The trends observed in the experiments matched those predicted by the models, though there were deviations in the absolute values. These deviations are explained in terms of non-idealities that exist in the experimental set-up, as well as in terms of factors that were not considered in the model. The models discussed in this paper are not only useful for understanding the workings of the cuboid packed-bed device, but are also useful tools for designing, optimizing and scaling-up such devices.

Instrument makers advance spectrometry

Advances in ion separation technology are behind a new instrument from Waters being called the first cyclic ion mobility mass spectrometer and a $15 million series A fundraising for the scientific instrumentation start-up MOBILion Systems. Agilent Technologies, a maker of mass spectrometers with which MOBILion partnered in September, led the fundraising round. Agilent plans to begin offering MOBILion’s ion separation technology on its Q-TOF mass spectrometers in 2020. MOBILion’s technology, known as structures for lossless ion manipulation (SLIM), was invented by Richard D. Smith at Pacific Northwest National Laboratory. By moving ions through a compact serpentine path, SLIM enables fast, high-resolution separation of glycans, proteins, and metabolites for drug research. MOBILion expects to eventually offer SLIM units for mass specs from instrument makers other than Agilent. MOBILion plans to use the funds to hire more people, advance product development, and generate fee-for-service revenue through a new...

An annular-flow, hollow-fiber membrane chromatography device for fast, high-resolution protein separation at low pressure

High-resolution protein separation is currently carried out using columns packed with ultrafine particulate chromatographic media which can only be operated at very high pressures. This restricts the flow rate and thereby the speed of separation. A membrane with ultrafine pores shares the same mass transport advantages as a bed of ultrafine particles. However, application of membrane chromatography has largely been restricted to low-resolution separation applications, primarily due to limitations associated with poorly designed devices. In this paper, we describe a novel annular-flow hollow-fiber membrane chromatography (AHMC) device, suitable for fast, high-resolution protein separation. The AHMC device was designed primarily from a fluidics perspective to ensure a narrow solute residence time distribution, a low dead-volume, and minimal fluid back-mixing. Using this device, it was possible to separate a ternary protein mixture in less than a minute, at less than 0.3 MPa backpressure. The high-resolution separation capability of AHMC was further demonstrated through its use for the separation of bovine serum albumin (BSA) dimer from the monomeric form of the protein. This was achieved in less than 3 min which was considerably faster than other reported techniques for protein aggregates separation.

Fast, low-pressure chromatographic separation of proteins using hydroxyapatite nanoparticles

Columns packed with ultrafine particles (e.g. sub-2 µm porous particles) are suitable for high-resolution, high-speed analytical separation of proteins. However, they require very expensive chromatography systems to provide the ultra-high pressure required for carrying out separations using such columns. Also, frictional heating at high pressure could result in peak broadening and on-column protein degradation. In this paper, we discuss the use of nanoparticles, packed in a box-shaped or cuboid packed-bed device having 50 mm length, 5 mm width, and 3 mm bed-height for fast, high-resolution separation of proteins at low pressure. The low bed height allows the separation to be carried out at low pressure while the cuboid device reduces dispersion effects and thereby keeps the resolution high. Two different types of hydroxyapatite nanoparticles, i.e., needle-shaped (about 20 nm × 150 nm) and spherical (<200 nm) ones, were examined. The experimental results showed that while the needle-shaped nanoparticles were suitable for the separation of small proteins such as lysozyme, the spherical nanoparticles were better suited for separation of larger proteins such as bovine serum albumin and monoclonal antibody. The separation of the two proteins could be carried out in less than 2 min at a pressure lower than 0.8 MPa, using inexpensive chromatography devices a7nd systems, and without high pressure related problems such as frictional heating and on-column protein denaturation.

Two-Dimensional Gel Electrophoresis with Immobilized pH Gradients.

Isoelectric focusing (IEF) is a technique in protein research that has been used since 1975 to separate proteins based on their isoelectric point. When combined with sodium dodecyl sulfate polyacrylamide gel electrophoresis, this procedure allows for high-resolution separation of cellular proteins for analytical purposes. Laboratories perform IEF by (a) using carrier ampholytes that migrate through a gel to create the pH gradient or (b) using immobilized pH gradients (IPG) that contain ampholytes bound covalently to a gel. Here we describe an IEF system that uses immobilized pH gradient (IPG) strips that undergo the desired current and voltage setting to separate proteins based on its charge in the first dimension followed by SDS PAGE to generate a two-dimensional map of serum proteins.

Top-down/Bottom-up Mass Spectrometry Workflow Using Dissolvable Polyacrylamide Gels.

Biologists' preeminent toolbox for separating, analyzing, and visualizing proteins is SDS-PAGE, yet recovering the proteins embedded in these polyacrylamide media as intact species is a long-standing challenge for mass spectrometry. In conventional workflows, protein mixtures from crude biological samples are electrophoretically separated at high-resolution within N,N'-methylene-bis-acrylamide cross-linked polyacrylamide gels to reduce sample complexity and facilitate sensitive characterization. However, low protein recoveries, especially for high molecular weight proteins, often hinder characterization by mass spectrometry. We describe a workflow for top-down/bottom-up mass spectrometric analyses of proteins in polyacrylamide slab gels using dissolvable, bis-acryloylcystamine-cross-linked polyacrylamide, enabling high-resolution protein separations while recovering intact proteins over a broad size range efficiently. The inferior electrophoretic resolution long associated with reducible gels has been overcome, as demonstrated by SDS-PAGE of crude tissue extracts. This workflow elutes intact proteins efficiently, supporting MS and MS/MS from proteins resolved on biologists' preferred separation platform.

Comparative Skeletal Muscle Proteomics Using Two-Dimensional Gel Electrophoresis.

The pioneering work by Patrick H. O’Farrell established two-dimensional gel electrophoresis as one of the most important high-resolution protein separation techniques of modern biochemistry (Journal of Biological Chemistry 1975, 250, 4007–4021). The application of two-dimensional gel electrophoresis has played a key role in the systematic identification and detailed characterization of the protein constituents of skeletal muscles. Protein changes during myogenesis, muscle maturation, fibre type specification, physiological muscle adaptations and natural muscle aging were studied in depth by the original O’Farrell method or slightly modified gel electrophoretic techniques. Over the last 40 years, the combined usage of isoelectric focusing in the first dimension and sodium dodecyl sulfate polyacrylamide slab gel electrophoresis in the second dimension has been successfully employed in several hundred published studies on gel-based skeletal muscle biochemistry. This review focuses on normal and physiologically challenged skeletal muscle tissues and outlines key findings from mass spectrometry-based muscle proteomics, which was instrumental in the identification of several thousand individual protein isoforms following gel electrophoretic separation. These muscle-associated protein species belong to the diverse group of regulatory and contractile proteins of the acto-myosin apparatus that forms the sarcomere, cytoskeletal proteins, metabolic enzymes and transporters, signaling proteins, ion-handling proteins, molecular chaperones and extracellular matrix proteins.

On the workings of laterally-fed membrane chromatography

Membrane chromatography has significant potential for use in biopharmaceutical purification processes. However, currently available membrane chromatography devices give poor resolution and therefore their use is restricted to polishing applications, typically carried out in the flow-through mode. Laterally-fed membrane chromatography (or LFMC) was developed specifically for carrying high-resolution multi-component protein separations. Recent studies have shown that resolution obtained with LFMC is comparable to that obtained with equivalent resin-based packed bed chromatography. Therefore, LFMC combines high-resolution with high-productivity, a highly desirable attribute in any purification technique. In this paper we explain the workings of LFMC based on theoretical and experimental analysis of hydraulic flow-path and residence time distribution. Factors likely to affect efficiency of LFMC are discussed using a simple electrical circuit analogy.

Online Hydrophobic Interaction Chromatography-Mass Spectrometry for Top-Down Proteomics.

Recent progress in top-down proteomics has led to a demand for mass spectrometry (MS)-compatible chromatography techniques to separate intact proteins using volatile mobile phases. Conventional hydrophobic interaction chromatography (HIC) provides high-resolution separation of proteins under nondenaturing conditions but requires high concentrations of nonvolatile salts. Herein, we introduce a series of more-hydrophobic HIC materials that can retain proteins using MS-compatible concentrations of ammonium acetate. The new HIC materials appear to function as a hybrid form of conventional HIC and reverse phase chromatography. The function of the salt seems to be preserving protein structure rather than promoting retention. Online HIC-MS is feasible for both qualitative and quantitative analysis. This is demonstrated with standard proteins and a complex cell lysate. The mass spectra of proteins from the online HIC-MS exhibit low charge-state distributions, consistent with those commonly observed in native MS. Furthermore, HIC-MS can chromatographically separate proteoforms differing by minor modifications. Hence, this new HIC-MS combination is promising for top-down proteomics.

High-resolution protein separation using a laterally-fed membrane chromatography device

Radial-flow membrane chromatography devices which are used for flow-through separation are generally unsuitable for bind-and-elute chromatography, particularly where multiple components need to be separated. We discuss a laterally-fed membrane chromatography device, suitable for high-resolution, multi-component protein separation in the bind-and-elute mode. In the current study, a stack of cation exchange membranes was housed within the membrane device and its performance was compared with an equivalent commercial radial-flow device having the same membrane bed volume and bed height. Tracer experiments were carried out using sodium chloride solution to compare their residence time distributions. The laterally-fed device showed superior flow distribution characteristics, which could be attributed to a lower variability in solute-flow path-lengths, and a smaller dead volume. Single protein bind-and-elute experiments carried out using lysozyme showed that the peaks obtained with the laterally-fed device were significantly sharper and more symmetrical. Excellent separation of three model proteins ovalbumin, conalbumin and lysozyme demonstrated that the laterally-fed membrane chromatography device was indeed suitable for carrying out high-resolution multi-component protein purification. These proteins could be fractionated in about 10 membrane bed volumes using the laterally-fed device as opposed to 25 bed volumes with the radial-flow device. The design of the laterally-fed device is simple and its flat shape gives it significantly lower footprint and offers additional advantages such as stackability and ease of multiplexing.

Proteomic profiling of pig heart and lung samples using 2-dimensional gel electrophoresis

Two-dimensional gel electrophoresis is a common method used for proteome profiling to separate, visualize and identify new proteins. A high resolution protein separation on 2-D gel is important to obtain good reliable protein spots that do not overlap. Pig is an important animal species used for meat production; its proteomics studies have covered both physiological and biomedical applications. In this study, proteome profiling of heart and lung samples of Pig by cup loading two-dimensional gel electrophoresis resulted in high resolution 2-D gels with clear protein spots, and conditions were optimized for identifying maximum number of protein spots.

Effective protein separation by coupling hydrophobic interaction and reverse phase chromatography for top-down proteomics.

One of the challenges in proteomics is the proteome’s complexity, which necessitates the fractionation of proteins prior to the mass spectrometry (MS) analysis. Despite recent advances in top-down proteomics, separation of intact proteins remains challenging. Hydrophobic interaction chromatography (HIC) appears to be a promising method that provides high-resolution separation of intact proteins, but unfortunately the salts conventionally used for HIC are incompatible with MS. In this study, we have identified ammonium tartrate as a MS-compatible salt for HIC with comparable separation performance as the conventionally used ammonium sulfate. Furthermore, we found that the selectivity obtained with ammonium tartrate in the HIC mobile phases is orthogonal to that of reverse phase chromatography (RPC). By coupling HIC and RPC as a novel two-dimensional chromatographic method, we have achieved effective high-resolution intact protein separation as demonstrated with standard protein mixtures and a complex cell lysate. Subsequently, the separated intact proteins were identified by high-resolution top-down MS. For the first time, these results have shown the high potential of HIC as a high-resolution protein separation method for top-down proteomics.

Optimization of a phenol extraction‐based protein preparation method amenable to downstream 2DE and MALDI‐MS based analysis of bacterial proteomes

2DE is one of the most efficient and widely used methods for resolving complex protein mixtures. For efficient analysis of complex samples, high-resolution separation of proteins on 2D gel is essential, and for that purpose good sample preparation is crucial. In this study, we have improvized a method for preparing bacterial total cellular proteome, from a strategy applied earlier to recalcitrant plant tissues, which gave high-quality resolution on 2DE. The method involving phenol extraction followed by methanol/ammonium acetate precipitation was first optimized for the chemolithotrophic proteobacteria Tetrathiobacter kashmirensis WT001 and Pseudaminobacter salicylatoxidans KCT001 that did not yield quality protein preps in conventional trichloroacetic acid/acetone precipitation method. Subsequently, to validate its general applicability, the method was evaluated against the trichloroacetic acid/acetone precipitation method for two other model bacteria, i.e. Escherichia coli DH5α and Mycobacterium smegmatis mc(2) 6. Identification of at least four proteins each from the outer membrane, periplasm, and cytoplasm of T. kashmirensis by MALDI-MS not only proved the efficiency of the method in extracting proteins from the different cellular compartments but also the amenability of the obtained protein spots toward MALDI-MS based identification.

Ultrahigh pressure fast size exclusion chromatography for top-down proteomics

Top-down MS-based proteomics has gained a solid growth over the past few years but still faces significant challenges in the LC separation of intact proteins. In top-down proteomics, it is essential to separate the high mass proteins from the low mass species due to the exponential decay in S/N as a function of increasing molecular mass. SEC is a favored LC method for size-based separation of proteins but suffers from notoriously low resolution and detrimental dilution. Herein, we reported the use of ultrahigh pressure (UHP) SEC for rapid and high-resolution separation of intact proteins for top-down proteomics. Fast separation of intact proteins (6-669 kDa) was achieved in < 7 min with high resolution and high efficiency. More importantly, we have shown that this UHP-SEC provides high-resolution separation of intact proteins using a MS-friendly volatile solvent system, allowing the direct top-down MS analysis of SEC-eluted proteins without an additional desalting step. Taken together, we have demonstrated that UHP-SEC is an attractive LC strategy for the size separation of proteins with great potential for top-down proteomics.

Analysis of protein species differentiation among mycobacterial low-Mr-secreted proteins by narrow pH range Immobiline gel 2-DE-MALDI-MS

Secreted proteins of bacteria are preferentially capable of interacting with host cells and are therefore of special biological and medical interest. Narrow pH range 2-DE and MALDI-TOFTOF-MS combine high-resolution protein separation with highly sensitive identification of proteins. Secreted proteins of Mycobacterium tuberculosis were separated at the protein species level, distinguishing different protein species of one protein. We focused on the pI range 4.0-4.7 and the Mr range 6-20kDa of the 2-DE pattern. Out of 128 analyzed spots, 121 were identified resulting in 33 different proteins with 277 different protein species, accumulating in a mean of 8.4 protein species per protein. Overrepresentation was found for the protein classes "virulence, detoxification, adaption", "information pathways", "cell wall and cell processes" and "intermediary metabolism and respiration". Thus far, 15 protein species of the ESX-1 family are characterized with 100% sequence coverage. More automated 2-DE procedures and more sensitive identification techniques are required for complete characterization of all of the protein species even in highly enriched samples, such as culture filtrates. Only then the functional level of proteomics will be achieved and potential biomarkers can be postulated at the molecular level. Proteomics is dominated by bottom-up approaches largely ignoring protein speciation. A prerequisite to reach the protein species level is to obtain 100% sequence coverage, which is a major challenge in proteomics. Here we show complete sequence information with a 2-DE-MS approach for 15 protein species. Acetylation of the N-terminus of ESAT-6 inhibits interaction with CFP-10, with direct consequences for pathogen-host interaction. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.

Developing a reproducible method for the high-resolution separation of peritoneal dialysate proteins on 2-D gels

PurposeDespite recent progress in the proteomic analysis of peritoneal dialysate effluent (PDE), there remains unresolved problems in the development of an optimal sample preparation method. Experimental designWe examined five protocols for concentrating PDE proteins and the effects of immobilized pH gradient (IPG) strips with different pH ranges and sample loading techniques. In addition, we examined three kits for depleting high abundance proteins by SDS–PAGE and two-dimensional gel electrophoresis (2-DE). ResultsPDE proteins precipitated with 75% acetonitrile (ACN) showed the greatest number of protein spots by 2-DE, with over 800 distinct spots. Higher-resolution images were obtained using IPG strips with a pH range of 4–7. The ProteoPrep immunoaffinity albumin and IgG depletion kit removed high abundance proteins with higher efficiency and more compatibility with isoelectric focusing (IEF). Removing high abundance proteins also increased the resolution and improved the intensity of low abundance proteins. Conclusion and clinical relevanceHigh-resolution 2-DE images of PDE proteins were obtained by concentrating samples with 75% ACN, using pH 4–7 IPG strips, and depleting high abundance proteins. This optimized method will enable future studies to discover predictive biomarkers of disease in patients on dialysis.