Protein Sample Preparation Research Articles

4D solid-state NMR for protein structure determination

Solid-state NMR offers the chance to extend structural studies to proteins that are otherwise difficult to study at atomic resolution, such as protein fibrils, membrane proteins or poorly diffracting crystals. As two-dimensional spatial correlation NMR spectra of proteins suffer from severe resonance overlap, we analyze in this perspective article the potential of higher-dimensional (3D and 4D) proton-detected experiments, which have an increased number of identifiable and assignable distance restraints for solid-state structural studies. We discuss practical considerations for the NMR measurements and the preparation of suitable protein samples and show results of structure calculations from 4D solid-state NMR spectra.

Method optimization for proteomic analysis of soybean leaf: improvements in identification of new and low-abundance proteins

The most critical step in any proteomic study is protein extraction and sample preparation. Better solubilization increases the separation and resolution of gels, allowing identification of a higher number of proteins and more accurate quantitation of differences in gene expression. Despite the existence of published results for the optimization of proteomic analyses of soybean seeds, no comparable data are available for proteomic studies of soybean leaf tissue. In this work we have tested the effects of modification of a TCA-acetone method on the resolution of 2-DE gels of leaves and roots of soybean. Better focusing was obtained when both mercaptoethanol and dithiothreitol were used in the extraction buffer simultaneously. Increasing the number of washes of TCA precipitated protein with acetone, using a final wash with 80% ethanol and using sonication to ressuspend the pellet increased the number of detected proteins as well the resolution of the 2-DE gels. Using this approach we have constructed a soybean protein map. The major group of identified proteins corresponded to genes of unknown function. The second and third most abundant groups of proteins were composed of photosynthesis and metabolism related genes. The resulting protocol improved protein solubility and gel resolution allowing the identification of 122 soybean leaf proteins, 72 of which were not detected in other published soybean leaf 2-DE gel datasets, including a transcription factor and several signaling proteins.

Fluorinated liquid-enabled protein handling and surfactant-aided crystallization for fully in situ digital microfluidic MALDI-MS analysis

A droplet (digital) microfluidic device has been developed that enables complete protein sample preparation for MALDI-MS analysis. Protein solution dispensing, disulfide bond reduction and alkylation, tryptic digestion, sample crystallization, and mass spectrometric analysis are all performed on a single device without the need for any ex situ sample purification. Fluorinated solvents are used as an alternative to surfactants to facilitate droplet movement and limit protein adsorption onto the device surface. The fluorinated solvent is removed by evaporation and so does not interfere with the MALDI-MS analysis. Adding a small amount of perfluorooctanoic acid to the MALDI matrix solution improves the yield, quality and consistency of the protein-matrix co-crystals, reducing the need for extensive 'sweet spot' searching and improving the spectral signal-to-noise ratio. These innovations are demonstrated in the complete processing and MALDI-MS analysis of lysozyme and cytochrome c. Because all of the sample processing steps and analysis can be performed on a single digital microfluidic device without the need for ex situ sample handling, higher throughput can be obtained in proteomics applications. More generally, the results presented here suggest that fluorinated liquids could also be used to minimize protein adsorption and improve crystallization in other types of lab-on-a-chip devices and applications.

Structural Insights for the Full Length Influenza A M2 Proton Channel in Native E. Coli Membranes

Membrane proteins are structurally characterized in membrane mimetic environments, environments that may or may not reflect the properties of native membrane proteins. This is critically important since the structure of membrane proteins is influenced by the protein's environment. Here we will show structural data from magic angle spinning solid state NMR of the full length M2 protein from Influenza A observed in E. coli membranes. This protein has never been exposed to the denaturing influence of detergents nor to the length isolation, purification and reconstitution protocols that typify membrane protein sample preparation for structural studies. The data will be compared to the purified full length protein in synthetic liposomes and to spectra of smaller constructs for which there is a high resolution structure in lipid bilayers.

Post-collection processing of Schirmer strip-collected human tear fluid impacts protein content

We examine the impact of post-collection sample handling on the protein composition of human tear samples. In particular, we characterize diffusion-based protein extraction from Schirmer strips. These strips of filter paper membrane are the de facto standard for tear fluid collection and storage, with diffusion-based protein elution off the strip being the most widely reported protein extraction strategy. Nevertheless, the diffusion-based protein elution strategy remains uncharacterized regarding downstream functional protein assays. Here, the time-dependence, concentration-dependence, and repeatability of the diffusion-based protein recovery protocol are characterized. Levels of protein irrecoverable from the Schirmer strip and lost during sample handling are isolated and compared for several major tear proteins. Further, the impact of the Schirmer strip and sample handling on the downstream concentration of proteins ranging in molecular weight, surface charge, and surface hydropathicity is quantified. Diffusion-based protein extraction from Schirmer strips was observed to be protein-dependent. Schirmer strips retained tear proteins to varying extents: 14.2% of lysozyme, 9.5% of human serum albumin, 27.7% of secretory IgA, and 30.9% of mucin 4. Tear protein loss during sample handling ranged from 2% (lysozyme) to 41.2% (mucin 4). Strip retention of protein was observed to be associated with protein molecular weight and hydrophobic surface area. Greater sample handling loss was associated with increased hydrophobic surface area of model proteins. Surface charge or surface hydrophilicity was not significantly associated with protein loss. We therefore conclude that, although diffusion-based processing of Schirmer strip-collected tear samples is widely used, these protocols may result in total post-collection protein loss which is considerable, consistent, and protein-dependent. This loss alters the relative and absolute protein concentrations in the sample. A priori prediction of strip-losses for individual proteins does not appear to be facile, based on cursory knowledge of protein surface properties. Thus, we emphasize "spike and recover" control experiments to determine expected elution profiles for target proteins when using diffusion-based protein sample preparation for Schirmer strip-collected tear fluid.

A High-Throughput Microchromatography Platform for Quantitative Analytical Scale Protein Sample Preparation

Protein analysis (using either specific protein quantitation by methods such as HPLC and immunoassays or structural analysis by methods such as LC-MS) usually requires significant sample preparation, including quantitative purification of the target protein from complex sample matrices and potentially enzymatic treatment or labeling. We have developed platform for high-throughput microchromatography, capable of running 96 or more small volume samples in parallel, producing from 10 pg to 100 μg of purified protein from each sample. The platform is based on disposable cartridge devices with 5 μL packed bed of resin. The cartridges may be operated as spin columns or run on a modified 96-channel liquid handler with ultra-low dead volume syringes that directly connect to the cartridges, providing very precisely controlled positive-displacement flow control. A major application is quantitative purification of target proteins using affinity or physical chromatography. Using large diameter nonporous beads, standard microplate enzyme-linked immunosorbent assay reagents can be used to perform 30-min immunoassays. Enzymatic digestion methods have also been developed on the system for application in glycan profiling.

Backbone assignment of the OmpA-like domain of peptidoglycan-associated lipoprotein from Acinetobacter baumannii

Acinetobacter baumannii is a multidrug-resistant opportunistic pathogen which induces the cytotoxicity of host cells by nosocomial infections. Peptidoglycan-associated lipoprotein (Pal) is one component of Tol-Pal system, which is involved in maintaining the integrity and stability of the outer membrane (OM) as well as the virulence of bacterial pathogens. Pal is composed of N-terminal hydrophobic domain anchoring to the OM and C-terminal OmpA-like domain interacting with peptidoglycan. Herein, we report the preparation of protein sample and the backbone assignment of recombinant OmpA-like domain of Pal protein from Acinetobacter baumannii (AbPal-71). All the backbone chemical shifts of AbPal-71 (Cα, Cβ, CO, HN, and N) were completely assigned and the secondary structure was estimated from the assigned chemical shifts. This result shows that AbPal-71 is a typical OmpA-like fold, which consists of three α-helices and four β-strands. Based on this NMR spectroscopic results, the three dimensional structural study and the interaction study between AbPal-71 and peptidoglycan are in progress.

Urea free and more efficient sample preparation method for mass spectrometry based protein identification via combining the formic acid-assisted chemical cleavage and trypsin digestion

A formic acid (FA)-assisted sample preparation method was presented for protein identification via mass spectrometry (MS). Detailedly, an aqueous solution containing 2% FA and dithiothreitol was selected to perform protein denaturation, aspartic acid (D) sites cleavage and disulfide linkages reduction simultaneously at 108°C for 2h. Subsequently, FA wiped off via vacuum concentration. Finally, iodoacetamide (IAA) alkylation and trypsin digestion could be performed ordinally. A series of model proteins (BSA, β-lactoglobulin and apo-Transferrin) were treated respectively using such method, followed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis. The identified peptide number was increased by ∼80% in comparison with the conventional urea-assisted sample preparation method. Moreover, BSA identification was achieved efficiently down to femtomole (25±0 sequence coverage and 16±1 peptides) via such method. In contrast, there were not peptides identified confidently via the urea-assisted method before desalination via the C18 zip tip. The absence of urea in this sample preparation method was an advantage for the more favorable digestion and MALDI-TOF MS analysis. The performances of two methods for the real sample (rat liver proteome) were also compared, followed by a nanoflow reversed-phase liquid chromatography with electrospray ionization tandem mass spectrometry system analysis. As a result, 1335±43 peptides were identified confidently (false discovery rate <1%) via FA-assisted method, corresponding to 295±12 proteins (of top match=1 and requiring 2 unique peptides at least). In contrast, there were only 1107±16 peptides (corresponding to 231±10 proteins) obtained from the conventional urea-assisted method. It was serving as a more efficient protein sample preparation method for researching specific proteomes better, and providing assistance to develop other proteomics analysis methods, such as, peptide quantitative analysis.

Influence of one- and two-dimensional gel electrophoresis procedure on metal–protein bindings examined by electrospray ionization mass spectrometry, inductively coupled plasma mass spectrometry, and ultrafiltration

Three independent methods, (i) electrospray ionization mass spectrometry (ESI-MS), (ii) carrying out the complete protein preparation procedure required for protein gel electrophoresis (GE) including extraction, precipitation, washing, and desalting with subsequent microwave digestion of the produced protein fractions for metal content quantification, and (iii) ultrafiltration for separating protein-bound and unbound metal fractions, were employed to elucidate the influences of protein sample preparation and GE running conditions on metal–protein bindings. A treatment of the protein solution with acetone instead of trichloroacetic acid or ammonium sulfate for precipitate formation led to a strongly enhanced metal binding capacity. The desalting step of the resolubilized protein sample caused a metal loss between 10 and 35%. The omission of some extraction buffer additives led to a diminished metal binding capacity of protein fractions obtained from the sample preparation procedure for GE, whereas a tenside addition to the protein solution inhibited metal–protein bindings. The binding stoichiometry of Cu and Zn–protein complexes determined by ESI-MS was influenced by the type of the metal salt which was applied to the protein solution. A higher pH value of the sample solution promoted the metal ion complexation by the proteins. Ultrafiltration experiments revealed a higher Cu- and Zn-binding capacity of the model protein lysozyme in both resolubilization buffers for 1D- and 2D-GE compared to the protein extraction buffer. Strongly diminished metal binding capacities of lysozyme were recorded in the running buffer of 1D-GE and in the gel staining solutions.

Integrated micro/nano-fluidic system for mixing and preconcentration of dissolved proteins

An integrated micro/nano-fluidic system is presented for protein analysis. It is comprised of an integrated micromixer (IMM) and a preconcentrator with a separation column. The passive and planar type of IMM is based on an unbalanced split and the cross collision of the fluidic streams. The IMM can be easily fabricated and integrated to the microfluidic system. The preconcentrator has nanochannels formed by the electrical breakdown of polydimethylsiloxane (PDMS) membrane by applying a high electrical shock, but without any nano-lithography. The integrated microdevice was used for sample preparation (mixing with tagging molecules) and subsequent concentration of proteins. Proteins were electrokinetically trapped near the junction of the micro/nanochannels. We show a conceptual design and a simple microfluidic system for purposes of mixing and preconcentration.

Effects of Acupuncture at Taixi Acupoint (KI3) on Kidney Proteome

This research explored and identified the protein composition of rat kidneys after acupuncture at the Taixi acupoint (KI3). Twelve adult male Wistar rats were randomly divided into a control group (n = 6) and an acupuncture group (n = 6). Rats in the acupuncture group received electroacupuncture on the bilateral KI3 for seven days. The kidneys were perfused with ice-cold saline and all kidney proteins were isolated. After protein sample preparation, two-dimensional gel electrophoresis (2-DE) was performed. The interesting spots were analyzed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). There were nine protein spots with three-fold up-regulation in the kidney after the acupuncture. NAD-dependent isocitrate dehydrogenase and quinone reductase, the proteins involved in energy metabolism, the reduction of endogenous quinones, chemoprotection, and electrophilic stress, were identified. The data indicated that acupuncture at the KI3 of the kidney meridian of the foot shaoyin was able to increase NAD-dependent isocitrate dehydrogenase and quinone reductase expression in the kidney, and supported the relationship between the kidney and KI3.

Automating Protein Sample Preparation

Automating Protein Sample Preparation

Tandem mass spectrometry identification and LC–MS quantification of intact cytokinin nucleotides in K-562 human leukemia cells

We describe here a new reversed-phase high-performance liquid chromatography with mass spectrometry detection method for quantifying intact cytokinin nucleotides in human K-562 leukemia cells. Tandem mass spectrometry was used to identify the intracellular metabolites (cytokinin monophosphorylated, diphosphorylated, and triphosphorylated nucleotides) in riboside-treated cells. For the protein precipitation and sample preparation, a trichloroacetic acid extraction method is used. Samples are then back-extracted with diethyl ether, lyophilized, reconstituted, and injected into the LC system. Analytes were quantified in negative selected ion monitoring mode using a single quadrupole mass spectrometer. The method was validated in terms of retention time stabilities, limits of detection, linearity, recovery, and analytical accuracy. The developed method was linear in the range of 1-1,000 pmol for all studied compounds. The limits of detection for the analytes vary from 0.2 to 0.6 pmol.

Parallel sample preparation of proteins, from crude samples to crystals ready for MALDI-MS, in an integrated microfluidic system

A microfluidic structure is presented where selective capture of proteins in complex samples, followed by clean-up, enzymatic processing, and MALDI-MS sample preparation of peptides generated, can be performed. The structure uses an affinity column to capture the protein while all other components in the sample are disposed of. The protein of interest is then eluted from the affinity column and captured on a second column on which the enzymatic processing is performed. Salts and hydrophilic contaminants are then removed before the products from the enzymatic reaction are eluted together with a suitable MALDI matrix and the solvent evaporated in a designated MALDI target structure. All steps can be performed automatically in 54 parallel microstructures on a microfluidic compact disc. The process is demonstrated by the selective capture and tryptic digest of recombinant IgG molecules from samples containing other proteins: an excess of bovine serum albumin or spent cell culture media.

Characterization of the Semiquinone Radical Stabilized by the Cytochrome aa3-600 Menaquinol Oxidase of Bacillus subtilis

Cytochrome aa(3)-600 is one of the principle respiratory oxidases from Bacillus subtilis and is a member of the heme-copper superfamily of oxygen reductases. This enzyme catalyzes the two-electron oxidation of menaquinol and the four-electron reduction of O(2) to 2H(2)O. Cytochrome aa(3)-600 is of interest because it is a very close homologue of the cytochrome bo(3) ubiquinol oxidase from Escherichia coli, except that it uses menaquinol instead of ubiquinol as a substrate. One question of interest is how the proteins differ in response to the differences in structure and electrochemical properties between ubiquinol and menaquinol. Cytochrome bo(3) has a high affinity binding site for ubiquinol that stabilizes a ubi-semiquinone. This has permitted the use of pulsed EPR techniques to investigate the protein interaction with the ubiquinone. The current work initiates studies to characterize the equivalent site in cytochrome aa(3)-600. Cytochrome aa(3)-600 has been cloned and expressed in a His-tagged form in B. subtilis. After isolation of the enzyme in dodecylmaltoside, it is shown that the pure enzyme contains 1 eq of menaquinone-7 and that the enzyme stabilizes a mena-semiquinone. Pulsed EPR studies have shown that there are both similarities as well as significant differences in the interactions of the mena-semiquinone with cytochrome aa(3)-600 in comparison with the ubi-semiquinone in cytochrome bo(3). Our data indicate weaker hydrogen bonds of the menaquinone in cytochrome aa(3)-600 in comparison with ubiquinone in cytochrome bo(3). In addition, the electronic structure of the semiquinone cyt aa(3)-600 is more shifted toward the anionic form from the neutral state in cyt bo(3).

Top-Down and Bottom-Up Proteomics of SDS-Containing Solutions Following Mass-Based Separation

SDS has recognized benefits for protein sample preparation, including solubilization and imparting molecular weight separation (e.g., SDS-PAGE). Here, we compare two proteome workflows which incorporate SDS for protein separation, namely, SDS-PAGE coupled to LC/MS (GeLC MS), along with a solution separation platform, GELFrEE, for intact proteome prefractionation and identification. Despite the clear importance of SDS in these and other proteome analysis workflows, the affect of SDS on an LC/MS proteome experiment has not been quantified. We first examined the influence of SDS on both a bottom-up as well as a top-down (intact protein) MS workflow. Surprisingly, at levels up to 0.01% SDS in the injected sample, reliable MS characterization is obtained. We subsequently explored organic precipitation protocols (chloroform/methanol/water and acetone) as a means of lowering SDS, and present a simple modified acetone precipitation protocol which consistently enables MS proteome characterizations from samples initially containing 2% SDS. With this effective strategy for SDS reduction, the GELFrEE MS workflow for bottom-up proteome analysis was characterized relative to GeLC MS. Remarkable agreement in the number and type of identified proteins was obtained from these two separation platforms, validating the use of SDS in solution-phase proteome analysis.

High-yield expression and purification of the transmembrane region of ion channel-forming amyloid-β protein for NMR structural studies

Numerous studies of APP in the literature to date have focused on its processing at the plasma membrane, its membrane-bound oligomeric state, and the calcium-permeable ion channel formation of non-fibrillar Aβ in the cell membrane. Despite these studies, little is known structurally about the transmembrane region of APP beyond a theoretical model. This is due to challenges in the expression, purification, and sample preparation of eukaryotic membrane proteins. To determine the three-dimensional structures of the intact transmembrane domain from human APP (hAPP-TM) and to elucidate the structure and formation mechanisms of the hAPP channel, the isotopically labeled protein must be produced and purified in sufficient quantity. Here, we describe a procedure whereby the hAPP-TM peptide, comprising residues 692–723 of hAPP, was successfully expressed and purified sufficiently to perform NMR analysis. To increase expression levels of the target protein, we designed a construct containing two tandem repeats of the target gene. The fusion protein was expressed in the form of inclusion bodies, purified on immobilized nickel affinity chromatography, and chemically cleaved by cyanogen bromide. Final purification of hAPP-TM was achieved by preparative reversed-phase high performance liquid chromatography (HPLC). The final yields of purified hAPP-TM were around 5 mg/l of M9 minimal media.

Acetone Precipitation of Proteins and the Modification of Peptides

Acetone precipitation is a common method for precipitation and concentration of proteins. We show here that a trace amount of residual acetone in the precipitated protein, can, after proteolysis, lead to selective modification of peptides predominantly those in which a glycine residue is the second amino acid, probably generating a relatively stable derivative that, under gas phase conditions, generates a y(1) ion of the same mass as proline. This modification is detectable by either MALDI-ToF or ESI-ion trap mass spectrometry and under normal sample preparation conditions is incomplete. The derivatization occurs in the condensed phase and is sufficiently stable that the modified peptide can elute on reversed phase chromatography at a different time to the unmodified peptide. Acetone precipitation is such a commonly used procedure in protein sample preparation for proteomics that some caution may be warranted. A significant number of peptides (about 5% of a typical proteome) meet the requirements for this reaction and could, therefore, change the outcome of studies.

A microscale protein NMR sample screening pipeline

As part of efforts to develop improved methods for NMR protein sample preparation and structure determination, the Northeast Structural Genomics Consortium (NESG) has implemented an NMR screening pipeline for protein target selection, construct optimization, and buffer optimization, incorporating efficient microscale NMR screening of proteins using a micro-cryoprobe. The process is feasible because the newest generation probe requires only small amounts of protein, typically 30–200 μg in 8–35 μl volume. Extensive automation has been made possible by the combination of database tools, mechanization of key process steps, and the use of a micro-cryoprobe that gives excellent data while requiring little optimization and manual setup. In this perspective, we describe the overall process used by the NESG for screening NMR samples as part of a sample optimization process, assessing optimal construct design and solution conditions, as well as for determining protein rotational correlation times in order to assess protein oligomerization states. Database infrastructure has been developed to allow for flexible implementation of new screening protocols and harvesting of the resulting output. The NESG micro NMR screening pipeline has also been used for detergent screening of membrane proteins. Descriptions of the individual steps in the NESG NMR sample design, production, and screening pipeline are presented in the format of a standard operating procedure.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-009-9386-z) contains supplementary material, which is available to authorized users.

Development of serological proteome analysis of mastitis by Staphylococcus aureus in ewes

Staphylococcus aureus is a major agent of mastitis in ruminants worldwide. So far, efficient measures for its prophylaxis (including vaccination) have proven to be unsuccessful and there is a need for a better understanding of the host response to udder infection by S. aureus. Serological proteome analysis (SERPA) is a promising technique that can be used to identify S. aureus immuno-dominant determinants providing that bacterial culture conditions used to grow S. aureus strains for protein sample preparation mimic the context of mastitis. A S. aureus strain was used in experimental mastitis to generate sheep serum used to determine the best growth conditions for SERPA. Sera collected in the field from different ewes suffering from mastitis by S. aureus were used to confirm experimental observations. Three different culture media (BHI, whey and iron-depleted RPMI) were tested. The influence of aeration and growth phase on protein production was also evaluated by immuno-detection of protein samples prepared from cultures grown in different conditions and obtained from different culture fractions (supernatant, cell wall, and total lysates). Our results showed that culturing in iron-depleted RPMI with (secreted proteins, prepared from stationary phase) or without aeration (cell wall proteins, prepared from early stationary phase, and total proteins, prepared from exponential phase) is the condition that best mimics growth in vivo during mastitis and this in vitro growth condition is to be used henceforth in experiments involving SERPA.