Efficient Protein Recovery Research Articles

A techno-economic perspective on a microwave extraction process for efficient protein recovery from agri-food wastes

Different agri-food wastes (brewer´s spent grain (BSG), spent coffee ground (SCG) and kale stems) have been proposed as excellent sources of protein-enriched extracts with an antioxidant capacity. The optimization of the microwave-assisted hydrothermal and alkali extraction has been compared in this study. From a technical and economic point of view, the extraction of BSG under optimal conditions (110 ºC, 10 min and 0.5 M NaOH) provided the best extract with a content of 14.6 kg protein/100 kg BSG (dry matter), 13.8 g/L of total sugars and an antioxidant activity (DPPH method) of 17.1 mg trolox equivalents (TE)/g BSG. This extract had the lowest production cost (29.9 €/kg) and a minimum selling price of 51.7 €/kg, estimated for an extraction pilot plant of 15 kg/h of BSG. The microwave-assisted hydrothermal extraction of kale stems, a novel waste in the biorefinery context, also provides bioactive and green extracts of commercial interest. There is a need for specific research studies related to biorefining of agri-food wastes to produce proteins for food, contributing to the development of a future sustainable and climate-neutral agriculture. The proposed techno-economic assessment represents an important advance in research and scaling-up of microwave-assisted extraction processes for protein recovery from agri-food wastes.

Utilization of Laser Capture Microdissection Coupled to Mass Spectrometry to Uncover the Proteome of Cellular Protrusions.

Laser capture microdissection (LCM) provides a fast, specific, and versatile method to isolate and enrich cells in mixed populations and/or subcellular structures, for further proteomic study. Furthermore, mass spectrometry (MS) can quickly and accurately generate differential protein expression profiles from small amounts of samples. Although cellular protrusions-such as tunneling nanotubes, filopodia, growth cones, invadopodia, etc.-are involved in essential physiological and pathological actions such as phagocytosis or cancer-cell invasion, the study of their protein composition is progressing slowly due to their fragility and transient nature. The method described herein, combining LCM and MS, has been designed to identify the proteome of different cellular protrusions. First, cells are fixed with a novel fixative method to preserve the cellular protrusions, which are isolated by LCM. Next, the extraction of proteins from the enriched sample is optimized to de-crosslink the fixative agent to improve the identification of proteins by MS. The efficient protein recovery and high sample quality of this method enable the protein profiling of these small and diverse subcellular structures.

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.

Highly efficient recovery of nutritional proteins from Australian Rock Lobster heads (Jasus edwardsii) by integrating ultrasonic extraction and chitosan co-precipitation

Australian Rock lobster head by-products (LHBs) are underutilised, but they contain about 43.5% protein. An integrated process of ultrasonic intensification and chitosan co-precipitation was developed for the efficient protein recovery. This process efficiency is influenced by pH, the ratio of water/LHBs and chitosan concentration. Up to 99% lobster head proteins (LHPs) could be ultrasonically extracted within 5 min at a pH of 13 and water to LHBs ratio of 8 mL/g, of which 86.6% LHPs could be recovered by 250 mg/L chitosan co-precipitation. Nutritionally, 83.2% of LHPs are relatively intact proteins (molecular weights 50–150 kDa) enriched with umami, aromas and essential amino acids (EAAs – up to 38.9% - double the amount of adult requirements that is suggested by WHO/FAO), and low lysine/arginine ratio (1.0). The LHPs recovered have digestibility comparable to that of the egg protein and contain significantly lower toxic metals satisfying food safety standards, that makes this novel process and the extracted proteins very attractive for industrial applications.

A Novel Cell Fixation Method that Greatly Enhances Protein Identification in Microproteomic Studies Using Laser Capture Microdissection and Mass Spectrometry.

Microproteomic studies have improved our knowledge of cell biology. Yet, with mass spectrometry (MS) analysis, accuracy can be lost for protein identification and quantification when using heterogeneous samples. Laser capture microdissection (LCM) allows for the enrichment of specific subsets of cells to study their proteome; however, sample fixation is necessary. Unfortunately, fixation hampers MS results due to protein cross-linking. The aim of this study was to identify both a fixation protocol and an extraction method that returns the best yield of proteins for downstream MS analysis, while preserving cellular structures. We compared glutaraldehyde (GLU), a common fixative to preserve cells, to dithiobispropionimidate (DTBP), a cleavable cross-linker. Our DTBP fixation/extraction protocol greatly increased the protein recovery. In fact, while 1000 GLU fixed cells returned only 159 unique protein hits, from 1464 unique peptides of 1994 unique collected spectra, 1000 DTBP fixed cells resulted in 567 unique collected protein hits, from 7542 unique peptides, of 10,401 unique collected spectra. That is, a 3.57-fold increase in protein hits, 5.15-fold increase in unique peptides, and a 5.22-fold increase in unique collected spectra. Overall, the novel protocol introduced here allows for a very efficient protein recovery and good sample quality for MS after sample collection using LCM.

Poly(4-vinylpyridine): a polymeric ligand for mixed-mode protein chromatography

Poly(4-vinylpyridine) (P4VP) was proposed for use as a polymeric ligand of mixed-mode chromatography (MMC) of proteins. P4VP has linear hydrophobic chains with ionizable pyridyl groups in its backbone. The polymer was coupled onto Sepharose FF gel at a pyridyl group density of 190μmol/mL (FF-P4VP-190) by the substitution reaction of pyridyl amines with brominated Sepharose gel. Thereby the immobilized ligand possesses the intrinsic hydrophobic nature as well as the newly obtained electrostatic interaction properties endowed from the substituted positively charged pyridyl amines. The pore size distribution was measured by inverse size exclusion chromatography, and the results revealed that P4VP formed a three-dimensional layer on the matrix surface with a maximum layer depth of 4.2nm. The adsorption isotherms of γ-globulin and bovine serum albumin (BSA) to FF-P4VP-190 were determined under varying pH values and salt concentrations to provide insights into the adsorption properties of the medium. It was found that the adsorption capacity of γ-globulin and BSA both presented an increase with pH increasing from 8.0 to 9.0. Moreover, FF-P4VP-190 exhibited stronger adsorption for BSA than γ-globulin. The higher affinity for BSA might be attributed to its more net negative charges. Protein adsorption capacities to FF-P4VP-190 decreased with increasing NaCl concentration, but still manifested moderate levels at high salt concentration such as 75mg/mL for γ-globulin and 14mg/mL for BSA at 0.5mol/L NaCl and pH 9.0. The capacity decreases with increasing ionic strength, indicating the dominant role of electrostatic interactions, while the moderate capacity values at 0.5mol/L NaCl confirmed the presence of salt-tolerant feature of FF-P4VP-190, making it function as an MMC material. Column chromatography was conducted to investigate protein elution behavior. Efficient protein recovery was achieved at mild elution conditions such as pH 4.0 for γ-globulin and pH 4.5 for BSA. The results indicate that the P4VP-based adsorbent would provide new possibilities for protein purification by MMC.

Efficient recovery of proteins from multiple source samples after trizol® or trizol®LS RNA extraction and long-term storage

BackgroundSimultaneous isolation of nucleic acids and proteins from a single biological sample facilitates meaningful data interpretation and reduces time, cost and sampling errors. This is particularly relevant for rare human and animal specimens, often scarce, and/or irreplaceable. TRIzol® and TRIzol®LS are suitable for simultaneous isolation of RNA, DNA and proteins from the same biological sample. These reagents are widely used for RNA and/or DNA isolation, while reports on their use for protein extraction are limited, attributable to technical difficulties in protein solubilisation.ResultsTRIzol®LS was used for RNA isolation from 284 human colon cancer samples, including normal colon mucosa, tubulovillous adenomas, and colon carcinomas with proficient and deficient mismatch repair system. TRIzol® was used for RNA isolation from human colon cancer cells, from brains of transgenic Alzheimer’s disease mice model, and from cultured mouse cortical neurons. Following RNA extraction, the TRIzol®-chloroform fractions from human colon cancer samples and from mouse hippocampus and frontal cortex were stored for 2 years and 3 months, respectively, at −80°C until used for protein isolation.Simple modifications to the TRIzol® manufacturer’s protocol, including Urea:SDS solubilization and sonication, allowed improved protein recovery yield compared to the TRIzol® manufacturer’s protocol. Following SDS-PAGE and Ponceau and Coomassie staining, recovered proteins displayed wide molecular weight range and staining pattern comparable to those obtainable with commonly used protein extraction protocols. We also show that nuclear and cytosolic proteins can be easily extracted and detected by immunoblotting, and that posttranslational modifications, such as protein phosphorylation, are detectable in proteins recovered from TRIzol®-chloroform fractions stored for up to 2 years at −80°C.ConclusionsWe provide a novel approach to improve protein recovery from samples processed for nucleic acid extraction with TRIzol® and TRIzol®LS compared to the manufacturer`s protocol, allowing downstream immunoblotting and evaluation of steady-state relative protein expression levels. The method was validated in large sets of samples from multiple sources, including human colon cancer and brains of transgenic Alzheimer’s disease mice model, stored in TRIzol®-chloroform for up to two years. Collectively, we provide a faster and cheaper alternative to the TRIzol® manufacturer`s protein extraction protocol, illustrating the high relevance, and wide applicability, of the present protein isolation method for the immunoblot evaluation of steady-state relative protein expression levels in samples from multiple sources, and following prolonged storage.

A quantitative proteomic workflow for characterization of frozen clinical biopsies: laser capture microdissection coupled with label-free mass spectrometry.

This paper describes a simple, highly efficient and robust proteomic workflow for routine liquid-chromatography tandem mass spectrometry analysis of Laser Microdissection Pressure Catapulting (LMPC) isolates. Highly efficient protein recovery was achieved by optimization of a "one-pot" protein extraction and digestion allowing for reproducible proteomic analysis on as few as 500 LMPC isolated cells. The method was combined with label-free spectral count quantitation to characterize proteomic differences from 3000-10,000 LMPC isolated cells. Significance analysis of spectral count data was accomplished using the edgeR tag-count R package combined with hierarchical cluster analysis. To illustrate the capability of this robust workflow, two examples are presented: 1) analysis of keratinocytes from human punch biopsies of normal skin and a chronic diabetic wound and 2) comparison of glomeruli from needle biopsies of patients with kidney disease. Differentially expressed proteins were validated by use of immunohistochemistry. These examples illustrate that tissue proteomics carried out on limited clinical material can obtain informative proteomic signatures for disease pathogenesis and demonstrate the suitability of this approach for biomarker discovery.

IgG Affinity Capture of TAP-tagged Protein Complexes from Cell Extracts: Affinity Purification Step 1

INTRODUCTIONOne approach to identifying protein–protein interactions is the biochemical purification of a target protein from cells or tissues under nondenaturing conditions, followed by the mass spectrometric identification of the components of the purified protein complex. The combination of highly specific protein purification strategies and mass spectrometry has proven to be a very successful approach for identifying protein–protein interactions. The tandem affinity purification (TAP) affinity tag and purification method allows efficient recovery of proteins present at low cellular concentrations under native conditions. Expressing the target protein at its natural levels avoids the assembly of overexpressed proteins into nonphysiological complexes. This protocol describes the immunoglobulin G (IgG) affinity capture of TAP-tagged complexes. This procedure is followed by calmodulin affinity purification.

Elevated Pressure Improves the Extraction and Identification of Proteins Recovered from Formalin-Fixed, Paraffin-Embedded Tissue Surrogates

BackgroundProteomic studies of formalin-fixed paraffin-embedded (FFPE) tissues are frustrated by the inability to extract proteins from archival tissue in a form suitable for analysis by 2-D gel electrophoresis or mass spectrometry. This inability arises from the difficulty of reversing formaldehyde-induced protein adducts and cross-links within FFPE tissues. We previously reported the use of elevated hydrostatic pressure as a method for efficient protein recovery from a hen egg-white lysozyme tissue surrogate, a model system developed to study formalin fixation and histochemical processing.Principal FindingsIn this study, we demonstrate the utility of elevated hydrostatic pressure as a method for efficient protein recovery from FFPE mouse liver tissue and a complex multi-protein FFPE tissue surrogate comprised of hen egg-white lysozyme, bovine carbonic anhydrase, bovine ribonuclease A, bovine serum albumin, and equine myoglobin (55∶15∶15∶10∶5 wt%). Mass spectrometry of the FFPE tissue surrogates retrieved under elevated pressure showed that both the low and high-abundance proteins were identified with sequence coverage comparable to that of the surrogate mixture prior to formaldehyde treatment. In contrast, non-pressure-extracted tissue surrogate samples yielded few positive and many false peptide identifications. Studies with soluble formalin-treated bovine ribonuclease A demonstrated that pressure modestly inhibited the rate of reversal (hydrolysis) of formaldehyde-induced protein cross-links. Dynamic light scattering studies suggest that elevated hydrostatic pressure and heat facilitate the recovery of proteins free of formaldehyde adducts and cross-links by promoting protein unfolding and hydration with a concomitant reduction in the average size of the protein aggregates.ConclusionsThese studies demonstrate that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues in a form suitable for proteomic analysis.

Elevated hydrostatic pressure promotes protein recovery from formalin-fixed, paraffin-embedded tissue surrogates

High-throughput proteomic studies on formalin-fixed, paraffin-embedded (FFPE) tissues have been hampered by inefficient methods to extract proteins from archival tissue and by an incomplete knowledge of formaldehyde-induced modifications to proteins. We previously reported a method for the formation of ‘tissue surrogates' as a model to study formalin fixation, histochemical processing, and protein retrieval from FFPE tissues. In this study, we demonstrate the use of high hydrostatic pressure as a method for efficient protein recovery from FFPE tissue surrogates. Reversal of formaldehyde-induced protein adducts and crosslinks was observed when lysozyme tissue surrogates were extracted at 45 000 psi and 80–100°C in Tris buffers containing 2% sodium dodecyl sulfate and 0.2 M glycine at pH 4. These conditions also produced peptides resulting from acid-catalyzed aspartic acid cleavage. Additives such as trimethylamine N-oxide or copper (II) chloride decreased the total percentage of these aspartic acid cleavage products, while maintaining efficient reversal of intermolecular crosslinks in the FFPE tissue surrogates. Mass spectrometry analysis of the recovered lysozyme yielded 70% sequence coverage, correctly identified all formaldehyde-reactive amino acids, and demonstrated hydrolysis at all of the expected trypsin cleavage sites. This study demonstrates that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues for proteomic analysis.

Z basic—A novel purification tag for efficient protein recovery

A positively charged protein domain, Z basic, can be used as a general purification tag to achieve efficient recovery of recombinantly produced target proteins using cation-exchange chromatography. To construct a protein domain usable for ion-exchange chromatography, the surface of protein Z was engineered to be highly charged, which allowed for selective capture of target proteins on a cation-exchanger at physiological pH values. Interestingly, the novel domain, denoted Z basic, was shown to be selective also under denaturing conditions and could preferably be used for purification of proteins solubilised from inclusion bodies. Moreover, a flexible process for solid-phase refolding was developed, using Z basic as a reversible linker to the cation-exchanger resin. This procedure has the inherited advantage of combining purification and refolding into a single step and still enabling elution of a concentrated product in a suitable buffer. This article summarizes development and use of the Z basic tag in small and pilot-plant-scale downstream processing.

Strategy for identifying protein–protein interactions of gel-separated proteins and complexes by mass spectrometry

A strategy for identifying and characterizing protein interactions among gel-separated proteins and complexes has been developed and tested. The method involves the efficient recovery of proteins or complexes from native gels without affecting their conformational integrity. The use of limited proteolysis of protein complexes, isolated from the gel or formed from the interaction of gel-recovered proteins with potential binding partners, has enabled local binding domains to be efficiently identified using a combination of microfiltration and mass spectrometric analysis. The application of mass spectrometry affords high detection sensitivities, enabling the strategy to be applied to low levels of protein and protein mixtures. The approach is demonstrated for both antigen–antibody and peptide–protein complexes for which protein-binding regions are characterized among simple peptide mixtures and proteolytic digests. The strategy can be easily adapted to achieve high sample throughput and automation using gel-excision robotics and provides a means to study protein interactions in complex biological mixtures and extracts.

Charged polymer brush grafted onto porous hollow-fiber membrane improves separation and reaction in biotechnology

An epoxy-group-containing polymer brush anchored onto the pore surface of a porous hollow-fiber membrane, followed by the introduction of ionizable groups, extends due to mutual electrostatic repulsion, and holds proteins in multilayers. This phenomenon provides novel insight into the static structure and dynamic behavior of the charged polymer brush, and enables highly efficient protein recovery, chiral separation, and enzymatic reaction. Various proteins were recovered using the porous hollow-fiber membranes immobilizing the charged polymer brush at a higher rate and higher capacity compared to bead-packed column because of convection of the protein solution through the pores and multilayering of the proteins in the polymer brush.

Radiation-induced graft polymerization is the key to develop high-performance functional materials for protein purification

We have described a preparation scheme for immobilizing polymer chains at a uniformly high density onto a microfiltration membrane. Highly efficient protein recovery was demonstrated by the results of the determination of breakthrough and elution curves. The three requirements of high rate, high capacity, and repeated use for the protein recovery were satisfied by ensuring the occurrence of convection, multilayer binding, and hydrophilization, respectively. In addition, easy scale-up to fabrication of a membrane module was verified on a small scale.

10] Specificity mapping of bacterial lectins by inhibition of hemagglutination using deoxy and deoxyfluoro analogs of receptor-active saccharides

Publisher Summary This chapter discusses the specificity mapping of bacterial lectins by the inhibition of hemagglutination using deoxy and deoxyfluoro analogs of receptor–active saccharides. The lectin-saccharide interaction makes it possible to interfere with bacterial binding and provide an opportunity to develop antiadhesive agents for treating or preventing infections. Information on the molecular details of the interactions is obtained by the inhibition of lectin–saccharide binding using either whole bacteria or the purified lectins. The structural information obtained from receptor mapping with deoxy and deoxyfluoro analogs of saccharides is potentially useful for the design of antiadhesive drugs against bacterial infection. Such analogs have improved metabolic stability and uptake characteristics, which are important for their use as drugs. In addition, the saccharide analogs may be used as haptens on affinity chromatography gels for lectin purification. Attenuated binding between the protein and the saccharide hapten can be useful in cases where the protein binds its natural counterpart too strongly to permit efficient protein recovery. Such affinity chromatography gels are used for the preparation of the PapG/PapD complex, which is currently being crystallized for X-ray analysis.

Recovery of functional proteins from sodium dodecyl sulfate-polyacrylamide gels

A simple method and apparatus are described for the efficient recovery of proteins from sodium dodecyl sulfate-polyacrylamide gel systems after electrophoretic resolution. This procedure provides for high yields of proteins which are free of sodium dodecyl sulfate and in certain cases, exhibit significant levels of biological activity.