Ultrasensitive proteome analysis using paramagnetic bead technology

In order to obtain a systems‐level understanding of a complex biological system, detailed proteome information is essential. Despite great progress in proteomics technologies, thorough interrogation of the proteome from quantity‐limited biological samples is hampered by inefficiencies during processing. To address these challenges, here we introduce a novel protocol using paramagnetic beads, termed Single‐Pot Solid‐Phase‐enhanced Sample Preparation (SP3). SP3 provides a rapid and unbiased means of proteomic sample preparation in a single tube that facilitates ultrasensitive analysis by outperforming existing protocols in terms of efficiency, scalability, speed, throughput, and flexibility. To illustrate these benefits, characterization of 1,000 HeLa cells and single Drosophila embryos is used to establish that SP3 provides an enhanced platform for profiling proteomes derived from sub‐microgram amounts of material. These data present a first view of developmental stage‐specific proteome dynamics in Drosophila at a single‐embryo resolution, permitting characterization of inter‐individual expression variation. Together, the findings of this work position SP3 as a superior protocol that facilitates exciting new directions in multiple areas of proteomics ranging from developmental biology to clinical applications.     

Analysis and isolation of human transferrin receptor using the OKT-9 monoclonal antibody covalently crosslinked to magnetic beads.

A method is described for the use of magnetic beads as a solid phase for the immunoprecipitation of labeled proteins. The anti-human transferrin receptor monoclonal antibody OKT-9 has been coupled to sheep anti-mouse IgG1-coated magnetic beads using the crosslinking agent dimethyl pimelimidate. The transferrin receptor is readily detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography following immunoprecipitation from 35S-labeled cell lysates. When compared with precipitations using OKT-9 coupled to protein G Sepharose the magnetic beads result in fewer nonspecific bands. The protocol described is generally applicable to the identification of labeled proteins. In addition, because magnetic beads are amenable to covalent crosslinking procedures they can be used for the purification of proteins from complex mixtures. Covalently crosslinked OKT-9 sheep anti-mouse IgG1-coated magnetic beads have been used to affinity purify unlabeled transferrin receptor from cell lysates giving comparable purity and yield to transferrin Sepharose isolated transferrin receptor. The major advantages offered by magnetic beads compared to conventional affinity matrices are low nonspecific binding and the rapidity with which the purification can be performed.     

Improving Proteome Coverage for Small Sample Amounts: An Advanced Method for Proteomics Approaches with Low Bacterial Cell Numbers

Proteome analyses are often hampered by the low amount of available starting material like a low bacterial cell number obtained from in vivo settings. Here, the single pot solid‐phase enhanced sample preparation (SP3) protocol is adapted and combined with effective cell disruption using detergents for the proteome analysis of bacteria available in limited numbers only. Using this optimized protocol, identification of peptides and proteins for different Gram‐positive and Gram‐negative species can be dramatically increased and, reliable quantification can also be ensured. This adapted method is compared to already established strain‐specific sample processing protocols for Staphylococcus aureus, Streptococcus suis, and Legionella pneumophila. The highest species‐specific increase in identifications is observed using the adapted method with L. pneumophila samples by increasing protein and peptide identifications up to 300% and 620%, respectively. This increase is accompanied by an improvement in reproducibility of protein quantification and data completeness between replicates. Thus, this protocol is of interest for performing comprehensive proteomics analyses of low bacterial cell numbers from different settings ranging from infection assays to environmental samples.     

Assessment and Refinement of Sample Preparation Methods for Deep and Quantitative Plant Proteome Profiling

A major challenge in the field of proteomics is obtaining high‐quality peptides for comprehensive proteome profiling by LC–MS. Here, evaluation and modification of a range of sample preparation methods using photosynthetically active Arabidopsis leaf tissue are done. It was found that inclusion of filter‐aided sample preparation (FASP) based on filter digestion improves all protein extraction methods tested. Ultimately, a detergent‐free urea‐FASP approach that enables deep and robust quantification of leaf and root proteomes is shown. For example, from 4‐day‐old leaf tissue, up to 11 690 proteins were profiled from a single sample replicate. This method should be broadly applicable to researchers working with difficult to process plant samples.     

Finding flies in the mushroom soup: Host specificity of fungus‐associated communities revisited with a novel molecular method

Fruiting bodies of fungi constitute an important resource for thousands of other taxa. The structure of these diverse assemblages has traditionally been studied with labour‐intensive methods involving cultivation and morphology‐based species identification, to which molecular information might offer convenient complements. To overcome challenges in DNA extraction and PCR associated with the complex chemical properties of fruiting bodies, we developed a pipeline applicable for extracting amplifiable total DNA from soft fungal samples of any size. Our protocol purifies DNA in two sequential steps: (a) initial salt–isopropanol extraction of all nucleic acids in the sample is followed by (b) an extra clean‐up step using solid‐phase reversible immobilization (SPRI) magnetic beads. The protocol proved highly efficient, with practically all of our samples—regardless of biomass or other properties—being successfully PCR‐amplified using metabarcoding primers and subsequently sequenced. As a proof of concept, we apply our methods to address a topical ecological question: is host specificity a major characteristic of fungus‐associated communities, that is, do different fungus species harbour different communities of associated organisms? Based on an analysis of 312 fungal fruiting bodies representing 10 species in five genera from three orders, we show that molecular methods are suitable for studying this rich natural microcosm. Comparing to previous knowledge based on rearing and morphology‐based identifications, we find a species‐rich assemblage characterized by a low degree of host specialization. Our method opens up new horizons for molecular analyses of fungus‐associated interaction webs and communities. Fruiting bodies of fungi constitute an important resource for thousands of other taxa. The structure of these diverse assemblages has traditionally been studied with labour‐intensive methods involving cultivation and morphology‐based species identification, to which molecular information might offer convenient complements. To overcome challenges in DNA extraction and PCR associated with the complex chemical properties of fruiting bodies, we developed a pipeline applicable for extracting amplifiable total DNA from soft fungal samples of any size. Our protocol purifies DNA in two sequential steps: (a) initial salt–isopropanol extraction of all nucleic acids in the sample is followed by (b) an extra clean‐up step using solid‐phase reversible immobilization (SPRI) magnetic beads. The protocol proved highly efficient, with practically all of our samples—regardless of biomass or other properties—being successfully PCR‐amplified using metabarcoding primers and subsequently sequenced. As a proof of concept, we apply our methods to address a topical ecological question: is host specificity a major characteristic of fungus‐associated communities, that is, do different fungus species harbour different communities of associated organisms? Based on an analysis of 312 fungal fruiting bodies representing 10 species in five genera from three orders, we show that molecular methods are suitable for studying this rich natural microcosm. Comparing to previous knowledge based on rearing and morphology‐based identifications, we find a species‐rich assemblage characterized by a low degree of host specialization. Our method opens up new horizons for molecular analyses of fungus‐associated interaction webs and communities.     

Enrichment of the basic/cationic urinary proteome using ion exchange chromatography and batch adsorption

Anionic or acidic proteins are the main compositions of normal urinary proteome. Efforts to characterize human urinary proteome, thus, have focused mainly on the anionic compartment. The information of cationic or basic proteins present in the normal urine is virtually unknown. In the present study, we applied different methods to enrich cationic urinary proteome. Efficacies of these methods were compared using equal volume (1 L) of urine samples from the same pool obtained from 8 normal healthy individuals. Cation exchange chromatography using RESOURCE-S column provided the least amount of the recovered proteins, whereas batch adsorption using SP Sepharose 4 Fast Flow beads equilibrated with acetic acid (pH 4.8) provided the greatest yield of protein recovery. The recovered proteins were then resolved with 2-DE (pI 7-11) and identified by peptide mass fingerprinting using MALDI-TOF MS. There were several isoforms of immunoglobulin heavy and light chains enriched by these methods. In addition, three isoforms of interferon alpha-3 (IFNalpha3) and six isoforms of eosinophil-derived neurotoxin (EDN), were also enriched. The enrichment of IFNalpha3 and EDN was particularly effective by batch adsorption using SP Sepharose 4 Fast Flow beads equilibrated with acetic acid (pH 6.0). Initial depletion of anionic components using DEAE batch adsorption reduced the recovery yield of these two proteins and did not improve recovery of any other cationic urinary proteins. We conclude that batch adsorption using SP Sepharose Fast Flow beads equilibrated with acetic acid (pH 6.0) is the method of choice to examine the basic/cationic urinary proteome, as this protocol provided the satisfactory yield of protein recovery and provided the greatest amount as well as maximal number of IFNalpha3 and EDN isoforms. Our data will be useful for further highly focused study targeting on cationic/basic urinary proteins. Moreover, the techniques described herein may be applicable for enrichment of cationic proteomes in other body fluids, cells, and tissues.     

Detecting nucleic acid fragments in serum using a magnetically modulated sandwich assay

We present a novel assay for rapid and highly sensitive detection of specific nucleic acid fragments in human serum. In a magnetic modulation biosensing (MMB) system, magnetic beads and fluorescently labeled probes are attached to the target analyte and form a “sandwich” complex. An alternating external magnetic field gradient condenses the magnetic beads (and hence the target molecules with the fluorescently labeled probes) to the detection volume and sets them in a periodic motion, in and out of a laser beam. A synchronous detection enables the removal of background signal from the oscillating target signal without complicated sample preparation. The high sensitivity of the MMB system, combined with the specificity of a sandwich hybridization assay, enables detection of DNA fragments without enzymatic signal amplification. Here, we demonstrate the sensitivity of the assay by directly detecting the EML4‐ALK oncogenic translocation sequence spiked in human serum. The calculated limit of detection is 1.4 pM, which is approximately 150 times better than a conventional plate reader. In general, the MMB‐assisted SHA can be implemented in many other applications for which enzymatic amplification, such as PCR, is not applicable and where rapid detection of specific nucleic acid targets is required.     

Surface imprinted beads for the recognition of human serum albumin

The synthesis of poly-aminophenylboronic acid (ABPA) imprinted beads for the recognition of the protein human serum albumin (HSA) is reported. In order to create homogeneous recognition sites, covalent immobilisation of the template HSA was exploited. The resulting imprinted beads were selective for HSA. The indirect imprinting factor (IF) calculated from supernatant was 1.6 and the direct IF, evaluated from the protein recovered from the beads, was 1.9. The binding capacity was 1.4 mg/g, which is comparable to commercially available affinity materials. The specificity of the HSA recognition was evaluated with competitive experiments, indicating a molar ratio 4.5/1 of competitor was necessary to displace half of the bound HSA. The recognition and binding of the imprinted beads was also tested with a complex sample, human serum and targeted removal of HSA without a loss of the other protein components was demonstrated. The easy preparation protocol of derivatised beads and a good protein recognition properties make the approach an attractive solution to analytical and bio-analytical problems in the field of biotechnology.     

Systematic Evaluation of Immobilized Trypsin‐Based Fast Protein Digestion for Deep and High‐Throughput Bottom‐Up Proteomics

Immobilized trypsin (IM) has been recognized as an alternative to free trypsin (FT) for accelerating protein digestion 30 years ago. However, some questions of IM still need to be answered. How does the solid matrix of IM influence its preference for protein cleavage and how well can IM perform for deep bottom‐up proteomics compared to FT? By analyzing Escherichia coli proteome samples digested with amine or carboxyl functionalized magnetic bead–based IM (IM‐N or IM‐C) or FT, it is observed that IM‐N with the nearly neutral solid matrix, IM‐C with the negatively charged solid matrix, and FT have similar cleavage preference considering the microenvironment surrounding the cleavage sites. IM‐N (15 min) and FT (12 h) both approach 9000 protein identifications (IDs) from a mouse brain proteome. Compared to FT, IM‐N has no bias in the digestion of proteins that are involved in various biological processes, are located in different components of cells, have diverse functions, and are expressed in varying abundance. A high‐throughput bottom‐up proteomics workflow comprising IM‐N‐based rapid protein cleavage and fast CZE‐MS/MS enables the completion of protein sample preparation, CZE‐MS/MS analysis, and data analysis in only 3 h, resulting in 1000 protein IDs from the mouse brain proteome.     

Macro to microfluidics system for biological environmental monitoring

Biological environmental monitoring (BEM) is a growing field of research which challenges both microfluidics and system automation. The aim is to develop a transportable system with analysis throughput which satisfies the requirements: (i) fully autonomous, (ii) complete protocol integration from sample collection to final analysis, (iii) detection of diluted molecules or biological species in a large real life environmental sample volume, (iv) robustness and (v) flexibility and versatility. This paper discusses all these specifications in order to define an original fluidic architecture based on three connected modules, a sampling module, a sample preparation module and a detection module. The sample preparation module highly concentrates on the pathogens present in a few mL samples of complex and unknown solutions and purifies the pathogens' nucleic acids into a few μL of a controlled buffer. To do so, a two-step concentration protocol based on magnetic beads is automated in a reusable macro-to-micro fluidic system. The detection module is a PCR based miniaturized platform using digital microfluidics, where reactions are performed in 64 nL droplets handled by electrowetting on dielectric (EWOD) actuation. The design and manufacture of the two modules are reported as well as their respective performances. To demonstrate the integration of the complete protocol in the same system, first results of pathogen detection are shown.     

A high‐throughput sample preparation method for cellular proteomics using 96‐well filter plates

A high‐throughput sample preparation protocol based on the use of 96‐well molecular weight cutoff (MWCO) filter plates was developed for shotgun proteomics of cell lysates. All sample preparation steps, including cell lysis, buffer exchange, protein denaturation, reduction, alkylation and proteolytic digestion are performed in a 96‐well plate format, making the platform extremely well suited for processing large numbers of samples and directly compatible with functional assays for cellular proteomics. In addition, the usage of a single plate for all sample preparation steps following cell lysis reduces potential samples losses and allows for automation. The MWCO filter also enables sample concentration, thereby increasing the overall sensitivity, and implementation of washing steps involving organic solvents, for example, to remove cell membranes constituents. The optimized protocol allowed for higher throughput with improved sensitivity in terms of the number of identified cellular proteins when compared to an established protocol employing gel‐filtration columns.     

One-step enzyme extraction and immobilization for biocatalysis applications

An extraction/immobilization method for HIs(6) -tagged enzymes for use in synthesis applications is presented. By modifying silica oxide beads to be able to accommodate metal ions, the enzyme was tethered to the beads after adsorption of Co(II). The beads were successfully used for direct extraction of C. antarctica lipase B (CalB) from a periplasmic preparation with a minimum of 58% activity yield, creating a quick one-step extraction-immobilization protocol. This method, named HisSi Immobilization, was evaluated with five different enzymes [Candida antarctica lipase B (CalB), Bacillus subtilis lipase A (BslA), Bacillus subtilis esterase (BS2), Pseudomonas fluorescence esterase (PFE), and Solanum tuberosum epoxide hydrolase 1 (StEH1)]. Immobilized CalB was effectively employed in organic solvent (cyclohexane and acetonitrile) in a transacylation reaction and in aqueous buffer for ester hydrolysis. For the remaining enzymes some activity in organic solvent could be shown, whereas the non-immobilized enzymes were found inactive. The protocol presented in this work provides a facile immobilization method by utilization of the common His(6) -tag, offering specific and defined means of binding a protein in a specific location, which is applicable for a wide range of enzymes.     

Cell-free synthesis and in situ isolation of recombinant proteins

We present a method for rapid expression and isolation of recombinant proteins. Cell-free protein synthesis in the presence of affinity beads enables in situ isolation of translation products, which simplifies the procedures for the preparation of purified protein samples. In the present study, we have made an attempt to carry out in situ isolation of histidine-tagged proteins by using Ni-NTA magnetic agarose beads. The presence of Ni-NTA beads gave no drastic effects on the efficiency of protein synthesis and successfully captured the synthesized proteins. Purified proteins were obtained after subsequent washing and elution steps. In particular, most of the endogenous bead-binding proteins were removed by pre-treating S30 extract with affinity beads and the purity of the target proteins was enhanced up to 95%. The methods described here will provide a basis for fast and convenient preparation of purified proteins from multiple genetic sequences.     

Protocols for metagenomic DNA extraction and Illumina amplicon library preparation for faecal and swab samples

Next‐generation sequencing (NGS) technology has extraordinarily enhanced the scope of research in the life sciences. To broaden the application of NGS to systems that were previously difficult to study, we present protocols for processing faecal and swab samples into amplicon libraries amenable to Illumina sequencing. We developed and tested a novel metagenomic DNA extraction approach using solid phase reversible immobilization (SPRI) beads on Western Bluebird (Sialia mexicana) samples stored in RNAlater. Compared with the MO BIO PowerSoil Kit, the current standard for the Human and Earth Microbiome Projects, the SPRI‐based method produced comparable 16S rRNA gene PCR amplification from faecal extractions but significantly greater DNA quality, quantity and PCR success for both cloacal and oral swab samples. We furthermore modified published protocols for preparing highly multiplexed Illumina libraries with minimal sample loss and without post‐adapter ligation amplification. Our library preparation protocol was successfully validated on three sets of heterogeneous amplicons (16S rRNA gene amplicons from SPRI and PowerSoil extractions as well as control arthropod COI gene amplicons) that were sequenced across three independent, 250‐bp, paired‐end runs on Illumina's MiSeq platform. Sequence analyses revealed largely equivalent results from the SPRI and PowerSoil extractions. Our comprehensive strategies focus on maximizing efficiency and minimizing costs. In addition to increasing the feasibility of using minimally invasive sampling and NGS capabilities in avian research, our methods are notably not avian‐specific and thus applicable to many research programmes that involve DNA extraction and amplicon sequencing.     

Automated nano-electrospray mass spectrometry for protein-ligand screening by noncovalent interaction applied to human H-FABP and A-FABP

A method for ligand screening by automated nano-electrospray ionization mass spectrometry (nano-ESI/MS) is described. The core of the system consisted of a chip-based platform for automated sample delivery from a 96-well plate and subsequent analysis based on noncovalent interactions. Human fatty acid binding protein, H-FABP (heart) and A-FABP (adipose), with small potential ligands was analyzed. The technique has been compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation with the found hits was obtained. In the current MS screening method, the cycle time per sample was 1.1 min, which is approximately 50 times faster than NMR for single compounds and approximately 5 times faster for compound mixtures. High reproducibility was achieved, and the protein consumption was in the range of 88 to 100 picomoles per sample. Futhermore, a novel protocol for preparation of A-FABP without the natural ligand is presented. The described screening approach is suitable for ligand screening very early in the drug discovery process before conventional high-throughput screens (HTS) are developed and/or used as a secondary screening for ligands identified by HTS.     

Gel‐aided sample preparation (GASP)—A simplified method for gel‐assisted proteomic sample generation from protein extracts and intact cells

We describe a “gel‐assisted” proteomic sample preparation method for MS analysis. Solubilized protein extracts or intact cells are copolymerized with acrylamide, facilitating denaturation, reduction, quantitative cysteine alkylation, and matrix formation. Gel‐aided sample preparation has been optimized to be highly flexible, scalable, and to allow reproducible sample generation from 50 cells to milligrams of protein extracts. This methodology is fast, sensitive, easy‐to‐use on a wide range of sample types, and accessible to nonspecialists.     

G‐rich sequence‐functionalized polystyrene microsphere‐based instantaneous derivatization for the chemiluminescent amplified detection of DNA

Herein, we develop a novel chemiluminescence (CL) approach with high sensitivity and excellent selectivity, by taking advantage of magnetic beads as preconcentration carriers and polystyrene microspheres as an amplification platform. Briefly, a ‘sandwich‐type’ detection strategy is employed in our design, which involves capture probe DNA immobilized on the surface of carboxyl‐terminated magnetic beads and multiple biotinylated reporter DNA self‐assembled on the surface of streptavidin‐modified polystyrene microspheres. The reporter DNA includes a guanine nucleobase‐rich (G‐rich) sequence domain for the generation of light and an additional tethered nucleic acid domain complementary with the target DNA. The CL signal is obtained via a novel instantaneous derivatization reaction between a specific CL reagent and the guanine nucleo­bases rich in the target and reporter DNA. As a result, we demonstrate that this DNA assay is reproducible, stable, easy to use, and can sensitively detect femtomolar target DNA related to anthrax lethal factors with excellent differentiation ability for single‐base mismatched sequences. Overall, this new CL protocol couples the high sensitivity of CL analysis with effective magnetic separation for discriminating against unwanted constituents such as mismatched sequences, and hence, offers great promise for DNA hybridization analysis. Copyright © 2009 John Wiley & Sons, Ltd.     

基于功能化磁珠快速提取植物病毒RNA方法的建立与优化

植物病毒病是制约作物生产的主要病害之一。及时明确其病毒病原和发展规律是有效控制其大规模传播的前提。而现有植物病毒病检测技术存在周期长、步骤繁琐、检测环境严苛等缺点。本研究以烟草花叶病毒 (Tobacco mosaic virus,TMV)为模型,基于碱基互补配对原则设计针对TMV的功能化磁珠（CMBs-ACPTMV）进行RNA提取,并对功能化磁珠的制备条件、提取反应条件以及灵敏性和稳定性等性能进行优化分析。结果表明,当添加4 μmol捕获探针（ACPTMV）、0.08 mg羧基磁珠（CMBs）时,所制备的CMBs-ACPTMV吸附RNA的能力最好;当提取时间为3 min时,CMBs-ACPTMV提取RNA的效果最好,而改变CMBs-ACPTMV的提取温度时其提取能力无明显变化;性能评价分析发现,CMBs-ACPTMV的灵敏度可达2.5 ng/μL,且检测稳定性较好。与常规RNA提取技术相比,CMBs-ACPTMV在检测时间和样品消耗量上具有突出优势。本研究所建立的功能化磁珠提取法快速、安全和简便,只需简易设备便可实现植物病毒RNA的快速提取,具有广阔的应用前景。     

Phosphorylation and Desensitization of the Neurokinin-1 Receptor Expressed in Epithelial Cells

A rat kidney epithelial cell line expressing the rat neurokinin-1 receptor (NK-1 R) was used to investigate the relationship between receptor phosphorylation and desensitization. Substance P (SP) maximally stimulated cellular inositol 1,4,5-trisphosphate (IP3) production 14-fold within 3 s, after which cellular IP3 levels rapidly diminished to near basal levels in the continuing presence of SP. SP also caused concentration-dependent phosphorylation of the NK-1R, and this effect was blocked by a receptor antagonist. Stimulation with 100 nM SP for as little as 2 s resulted in 90% desensitization of the receptor to restimulation by SP, and near-maximal receptor phosphorylation was observed at 5 s. Receptor desensitization was not affected by agents that affect protein kinase A. Phorbol 12-myristate 13-acetate (PMA) also caused phosphorylation and desensitization of the receptor but with slower kinetics and to a lesser extent than SP. PMA- but not SP-induced NK-1 R desensitization and phosphorylation were abolished by the protein kinase C inhibitor bisindolylmaleimide 1. The concentration-response curves for SP-stimulated IP3 signaling and desensitization were similar, but the curve for NK-1R phosphorylation was shifted to the right and was steeper, suggesting that the relationship between desensitization and phosphorylation is complex. These results show that both rapid homologous and rapid heterologous NK-1R desensitizations may be mediated by receptor phosphorylation but occur via distinct mechanisms with different kinetics and efficacies.     

A sensitive magnetic bead method for the detection and identification of tyrosine phosphorylation in proteins by MALDI‐TOF/TOF MS

Phosphorylation is one of the most important PTMs and is estimated to occur on 30% of the mammalian proteome. Its perturbed regulation has been implicated in many pathologies. The rarity of phosphotyrosine compared with phosphoserine or phosphothreonine is prompting the development of more sensitive approaches because proteomic technologies that are currently used to assess tyrosine phosphorylation in proteins are inadequate, identifying only a fraction of the predicted tyrosine phosphoproteome. Here we describe the development of a reproducible, high‐sensitivity methodology for the detection and mapping of phosphotyrosine residues by MS. The anti‐phosphotyrosine antibody 4G10 was coupled covalently to super para‐magnetic beads or by affinity to super para‐magnetic beads with protein G covalently attached. Using this approach, we successfully enriched phosphotyrosine peptides mixed with non‐phosphorylated peptides at a ratio of up to 1:200, enabling detection at a level representing the highest sensitivity reported for tyrosine phosphorylation. The beads were subsequently used to enrich tyrosine phosphopeptides from a digest of the in vitro‐phosphorylated recombinant β‐intracellular region of the granulocyte‐macrophage colony‐stimulating factor receptor, which was subsequently analysed by MALDI‐TOF/TOF MS. Our results define this methodology as a sensitive approach for tyrosine phosphoproteome analysis.