Identification of components of protein complexes using a fluorescent photo-cross-linker and mass spectrometry

This study describes a novel method for improving the specific recognition, detection, and identification of proteins involved in multiprotein complexes. The method is based on a combination of coimmunoprecipitation, chemical cross-linking, and specific fluorescent tagging of protein components in close association with one another. Specific fluorescent tagging of the protein complex components was achieved using the cleavable, fluorescent cross-linker sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido) ethyl-1,3'-dithiopropionate (SAED). Following dissociation and separation by SDS-PAGE, the fluorescently tagged proteins are then visualized by UV illumination, excised, and, following in-gel digestion, identified by mass spectrometry. In this study, a complex of the HIV-envelope protein gp120 and its cellular receptor CD4 was used as a model system. The sensitivity of detection of fluorescent SAED-labeled proteins in SDS gels, and the sensitivity of the mass spectrometric identification of fluorescent proteins after in-gel digestion, is in the range of a few hundred femtomoles of protein. This sensitivity is comparable to that achieved with silver-staining techniques, but fluorescence detection is protein independent and no background interference occurs. Furthermore, fluorescence labeling is significantly more compatible with mass spectrometric identification of proteins than is silver staining. The first application of this strategy was in the investigation of the mechanism of spermiation, the process by which mature spermatids separate from Sertoli cells. For the coimmunoprecipitation experiment, an antibody against paxillin, a protein involved in spermatid-Sertoli cell junctional complexes, was used. More components of the paxillin protein complex were visible by fluorescence detection of SAED-labeled proteins than were visible on comparable silver-stained gels. Mass spectrometric analysis of the fluorescently labeled proteins identified integrin alpha6 precursor as a protein associated in a complex with paxillin. The identification of integrin alpha6 precursor was confirmed by Western blot analysis and verifies the applicability of this novel approach for identifying proteins involved in protein complexes.     

Multiplexed protein analysis using encoded antibody-conjugated microbeads

We describe a method for multiplexed analysis of proteins using fluorescently encoded microbeads. The sensitivity of our method is comparable to the sensitivity obtained by enzyme-linked immunosorbent assay while only 5 µl sample volumes are needed. Streptavidin-coated, 1 µm beads are encoded with a combination of fluorophores at different intensity levels. As a proof of concept, we demonstrate that 27 microbead populations can be readily encoded by affinity conjugation using three intensity levels for each of three different biotinylated fluorescent dyes. Four populations of encoded microbeads are further conjugated with biotinylated capture antibodies and then combined and immobilized in a microfluidic flow cell for multiplexed protein analysis. Using four uniquely encoded microbead populations, we show that a cancer biomarker and three cytokine proteins can be analysed quantitatively in the picogram per millilitre range by fluorescence microscopy in a single assay. Our method will allow for the fabrication of high density, bead-based antibody arrays for multiplexed protein analysis using integrated microfluidic devices and automated sample processing.     

Absolute quantification of specific proteins in complex mixtures using visible isotope-coded affinity tags

The identification of proteins in complex mixtures is most useful when quantitative information is also obtained. We describe a new type of protein tagging reagent called the visible isotope-coded affinity tag (VICAT) which allows the absolute amount of a target protein or proteins to be quantified in a complex biological sample such as a eukaryotic cell lysate. VICAT reagents tag thiol groups of cysteines or thioacetylated amino groups and introduce into the tryptic peptide a biotin affinity handle, a visible moiety for tracking the chromatographic location of the target peptide by a method other than mass spectrometry, a photocleavable linker for removing a portion of the tag, and an isotope tag for distinguishing sample and internal standard peptides. We demonstrate the use of VICAT reagents together with isoelectric focusing of peptides on an immobilized gel strip followed by combined micro-liquid chromatography/electrospray ionization mass spectrometry operating in selected reaction monitoring mode to determine the absolute abundance of a specific protein, human group V phospholipase A(2), in eukaryotic cell lysates. It is found that human lung macrophages contain 66 fmol of this protein per 100 microg of cell protein. Western blot analysis of human group V phospholipase A(2) in macrophages gave inconclusive data. VICAT reagents should be useful for numerous applications including the analysis of candidate disease markers in complex mixtures such as serum.     

Cascaded free-flow isoelectric focusing for improved focusing speed and resolution

This work presents the first implementation of cascaded stages for a microfabricated free-flow isoelectric focusing (FF-IEF) device. Both analytical and computational models for IEF suggest device performance will be improved by utilizing multiple stages to reduce device residence time. These models are shown to be applicable by using focusing of small IEF markers as a demonstration. We also show focusing of fluorescently tagged proteins under different channel geometries, with the most efficient focusing occurring in the cascaded design, as predicted by theory. An additional aim of this work is to demonstrate the compatibility of cascaded FF-IEF with common bioanalytical tools. As an example, outlet fractions from cascaded FF-IEF were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Processing of whole cell lysate followed by immunoblotting for cell signaling markers demonstrates the reduction of albumin from samples, as well as the enrichment of apoptotic markers.     

Cellular uptake of protein-bound magnetic nanoparticles in pulsed magnetic field

A method for fast delivery of proteins conjugated to superparamagnetic iron oxide nanoparticles (SPION) into mammalian cells by applying a strong magnetic field in pulses was proposed. Firstly, SPION were prepared from an alkaline solution of divalent and trivalent iron ions and covalently bound with protein through the activation of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC). After fluorescently labelling, the protein-nanoparticle conjugate was mixed with mammalian cell line and exposed to a pulsed magnetic field for short durations of few milliseconds. Results suggested that superparamagnetic nanoparticles were able to carry proteins into living cells immediately. Cellular internalization of the fluorescently labelled protein-nanoparticle conjugate was proved by the observation of cell fluorescence in a fluorescent microscopy, as well as cell analysis by a flow cytometer. We found that the cellular uptake was accomplished dominantly by the process of bombardment of magnetic nanoparticles.     

One-dimensional protein analysis of an HT29 human colon adenocarcinoma cell

A single HT29 human colon adenocarcinoma cell was introduced into a fused-silica capillary and lysed, and the protein content was fluorescently labeled with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde. The labeled proteins were separated by capillary electrophoresis in a submicellar buffer and detected by laser-induced fluorescence in a postcolumn sheath-flow cuvette. Several dozen components were resolved. A number of experiments were done to verify that these components were proteins. Most components of the single-cell electropherogram had the same mobility as components present in the 30-100 kDa fraction of a protein extract prepared from the cell culture. One component was identified as a approximately 100 kDa protein by co-injecting the sample with purified protein obtained from an SDS-PAGE gel. Protein expression varied significantly between cells, but the average expression was consistent with that observed from a protein extract prepared from 10(6) cells.     

Affinity analysis of a protein-aptamer complex using nonequilibrium capillary electrophoresis of equilibrium mixtures

We propose a new method that allows the use of low-affinity aptamers as affinity probes in quantitative analyses of proteins. The method is based on nonequilibrium capillary electrophoresis of the equilibrium mixture (NECEEM) of a protein with its fluorescently labeled aptamer. In general, NECEEM of a protein with a fluorescently labeled aptamer generates an electropherogram with three characteristic features: two peaks and an exponential curve. Two peaks correspond to (i) the equilibrium amount of free aptamer in the equilibrium mixture and (ii) the amount of the protein-aptamer complex that remains intact at the time of detection. The exponential part is ascribed to the complex decaying during separation under nonequilibrium conditions. Simple analysis of the three features in experiments with known concentrations of the protein can be used for the determination of the equilibrium dissociation constant, Kd, of the aptamer-protein complex. Similar analysis of the three features in the experiment with unknown concentration of the protein and known Kd value allows the determination of the protein concentration. In this proof-of-principle work, the NECEEM method was applied to the analysis of thrombin using a fluorescein-labeled aptamer under the conditions at which the protein-aptamer complex completely decayed during the separation. We demonstrated that, despite the decay, as few as 4 x 10(6) molecules of the protein could be detected with NECEEM without sacrificing the accuracy. This sensitivity is comparable with that reported by others for the aptamer-based equilibrium method. Thus, the proposed NECEEM-based method allows the use of aptamers for highly sensitive affinity analysis of proteins even when protein-aptamer complexes are unstable.     

SILACtor: software to enable dynamic SILAC studies

Stable isotope labeling by amino acids in cell culture (SILAC) is a versatile tool in proteomics that has been used to explore protein turnover on a large scale. However, these studies pose a significant undertaking that can be greatly simplified through the use of computational tools that automate the data analysis. While SILAC technology has enjoyed rapid adoption through the availability of several software tools, algorithms do not exist for the automated analysis of protein turnover data generated using SILAC technology. Presented here is a software tool, SILACtor, designed to trace and compare SILAC-labeled peptides across multiple time points. SILACtor is used to profile protein turnover rates for more than 500 HeLa cell proteins using a SILAC label-chase approach. Additionally, SILACtor contains a method for the automated generation of accurate mass and retention time inclusion lists that target peptides of interest showing fast or slow turnover rates relative to the other peptides observed in the samples. SILACtor enables improved protein turnover studies using SILAC technology and also provides a framework for features extensible to comparative SILAC analyses and targeted methods.     

Aqueous nickel-nitrilotriacetate modified Fe(3)O(4)-NH(3)(+) nanoparticles for protein purification and cell targeting

A comprehensive totally aqueous phase synthesis of nickel-nitrilotriacetate (Ni-NTA) modified superparamagnetic Fe(3)O(4) nanoparticles is presented. The Fe(3)O(4)-NTA-Ni nanoparticles are able to perform efficient and specific purification of 6-His tagged proteins from crude cell lysates, as evidenced by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The average binding capacity, as demonstrated by streptopain (M(W) 42?kDa), is 0.23?mg/mg (protein/Fe(3)O(4)-NTA-Ni). Considering the high affinity and specificity of the binding between hexahistidine motif and Ni-NTA, Ni-NTA modified nanoparticles could act as a module to carry 6-His tagged proteins on the particle surface with molecular orientation control, since only the 6-His domain could be attached. These modularly designed functional nanoparticles enhance cancer cell targeting, as supported by the in vitro receptor mediated targeting assay using RGD-4C-6-His fusion peptide. The nanoparticles show no significant hemolysis for human blood and could be investigated further for their in vivo functional imaging applications.     

Multiphoton microscopy guides neurotrophin modification with poly(ethylene glycol) to enhance interstitial diffusion

Brain-derived neurotrophic factor (BDNF) is a promising therapeutic agent for the treatment of neurodegenerative diseases. However, the limited distribution of this molecule after administration into the brain tissue considerably hampers its efficacy. Here, we show how multiphoton microscopy of fluorescently tagged BDNF in brain-tissue slices provides a useful and rapid screening method for examining the diffusion of large molecules in tissues, and for studying the effects of chemical modifications-for example, conjugating with polyethylene glycol (PEG)-on the diffusion constant. This single variable, obtained by monitoring short-term diffusion in real time, can be effectively used for rational drug design. In this study on fluorescently tagged BDNF and BDNF-PEG, we identify slow diffusion as a major contributing factor to the limited penetration of BDNF, and demonstrate how chemical modification can be used to overcome this barrier.     

Versatile online-offline engine for automated acquisition of high-resolution tandem mass spectra

For automated production of tandem mass spectrometric data for proteins and peptides >3 kDa at >50 000 resolution, a dual online-offline approach is presented here that improves upon standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategies. An integrated hardware and software infrastructure analyzes online LC-MS data and intelligently determines which targets to interrogate offline using a posteriori knowledge such as prior observation, identification, and degree of characterization. This platform represents a way to implement accurate mass inclusion and exclusion lists in the context of a proteome project, automating collection of high-resolution MS/MS data that cannot currently be acquired on a chromatographic time scale at equivalent spectral quality. For intact proteins from an acid extract of human nuclei fractionated by reversed-phase liquid chromatography (RPLC), the automated offline system generated 57 successful identifications of protein forms arising from 30 distinct genes, a substantial improvement over online LC-MS/MS using the same 12 T LTQ FT Ultra instrument. Analysis of human nuclei subjected to a shotgun Lys-C digest using the same RPLC/automated offline sampling identified 147 unique peptides containing 29 co- and post-translational modifications. Expectation values ranged from 10 (-5) to 10 (-99), allowing routine multiplexed identifications.     

Protein capture in silica nanotube membrane 3-D microwell arrays

The microarray format has allowed for rapid and sensitive detection of thousands of analyte DNAs in a single sample, and there is considerable interest in extending this technology to protein biosensing. While glass is the most common substrate for microarrays, its binding capacity is limited because the glass surface is flat. One way to overcome this limitation is to develop arrays based on porous materials. Such "3-D" arrays can provide greater sensitivity because both the capture molecules and the analyte species they bind are immobilized throughout the thickness of the porous material. We describe here 3-D protein microarrays based on nanopore alumina membranes that contain silica nanotubes within the pores. These microarrays are prepared via a plasma-etch method using a TEM grid as the etch mask and consist of individual nanotube-containing microwells imbedded in a Ag film that coats the alumina membrane surface. We show that the microwells can be functionalized with antibodies and that these antibodies can capture their antigen proteins, which serve as prototype analytes. The analyte proteins are fluorescently tagged, which allows for fluorescence microscopy-based imaging of the array. The Ag surrounding the microwells shows very low background fluorescence, thus improving the signal-background ratio obtained from these arrays.     

Evaluation of direct infusion-multiple reaction monitoring mass spectrometry for quantification of heat shock proteins

Protein quantification with liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) has emerged as a powerful platform for assessing panels of biomarkers. In this study, direct infusion, using automated, chip-based nanoelectrospray ionization, coupled with MRM (DI-MRM) is used for protein quantification. Removal of the LC separation step increases the importance of evaluating the ratios between the transitions. Therefore, the effects of solvent composition, analyte concentration, spray voltage, and quadrupole resolution settings on fragmentation patterns have been studied using peptide and protein standards. After DI-MRM quantification was evaluated for standards, quantitative assays for the expression of heat shock proteins (HSPs) were translated from LC-MRM to DI-MRM for implementation in cell line models of multiple myeloma. Requirements for DI-MRM assay development are described. Then, the two methods are compared; criteria for effective DI-MRM analysis are reported on the basis of the analysis of HSP expression in digests of whole cell lysates. The increased throughput of DI-MRM analysis is useful for rapid analysis of large batches of similar samples, such as time course measurements of cellular responses to therapy.     

Microfluidic digital isoelectric fractionation for rapid multidimensional glycoprotein analysis

Here we present an integrated microfluidic device for rapid and automated isolation and quantification of glycoprotein biomarkers directly from biological samples on a multidimensional analysis platform. In the first dimension, digital isoelectric fractionation (dIEF) uses discrete pH-specific membranes to separate proteins and their isoforms into precise bins in a highly flexible spatial arrangement on-chip. dIEF provides high sample preconcentration factors followed by immediate high-fidelity transfer of fractions for downstream analysis. We successfully fractionate isoforms of two potential glycoprotein cancer markers, fetuin and prostate-specific antigen (PSA), with 10 min run time, and results are compared qualitatively and quantitatively to conventional slab gel IEF. In the second dimension, functionalized monolithic columns are used to capture and detect targeted analytes from each fraction. We demonstrate rapid two-dimensional fractionation, immunocapture, and detection of C-reactive protein (CRP) spiked in human serum. This rapid, flexible, and automated approach is well-suited for glycoprotein biomarker research and verification studies and represents a practical avenue for glycoprotein isoform-based diagnostic testing.     

A direct nanoflow liquid chromatography-tandem mass spectrometry system for interaction proteomics

One of the strategies of functional proteomics, research aiming to discover gene function at the protein level, is the comprehensive analysis of protein-protein interactions related to the functional linkage among proteins and analysis of functional cellular machinery to better understand the basis of cell functions. Here, we describe the direct nanoflow LC (DNLC) system, which is equipped with a fritless high-resolution electrospray interface column packed with 1-microm reversed-phase (RP) beads and a novel splitless nanoflow gradient elution system to operate the column. Using RP-DNLC at an extremely slow flow rate, <50 nL/min, combined with data-dependent collision-induced dissociation tandem MS (MS/MS) and computer-assisted retrieval of spectra, we identified approximately 100 protein components in a biological complex such as a premature mammalian ribosome pull-down from cultured cells when we used an epitope-tagged protein as bait. Because this analysis is most sensitive, requires approximately 0.2 microg of total protein, and is a fully automated 1-h process, we anticipated that it should be an excellent tool for analyzing a limited amount of functional multi-protein complexes in cells.     

Engineering Protein Nanoparticles Out from Components of the Human Microbiome

Nanoscale protein materials are highly convenient as vehicles for targeted drug delivery because of their structural and functional versatility. Selective binding to specific cell surface receptors and penetration into target cells require the use of targeting peptides. Such homing stretches should be incorporated to larger proteins that do not interact with body components, to prevent undesired drug release into nontarget organs. Because of their low interactivity with human body components and their tolerated immunogenicity, proteins derived from the human microbiome are appealing and fully biocompatible building blocks for the biofabrication of nonreactive, inert protein materials within the nanoscale. Several phage and phage‐like bacterial proteins with natural structural roles are produced in Escherichia coli as polyhistidine‐tagged recombinant proteins, looking for their organization as discrete, nanoscale particulate materials. While all of them self‐assemble in a variety of sizes, the stability of the resulting constructs at 37 °C is found to be severely compromised. However, the fine adjustment of temperature and Zn²⁺ concentration allows the formation of robust nanomaterials, fully stable in complex media and under physiological conditions. Then, microbiome‐derived proteins show promise for the regulatable construction of scaffold protein nanomaterials, which can be tailored and strengthened by simple physicochemical approaches.     

Maskless projection lithography for the fast and flexible generation of grayscale protein patterns

Protein patterns of different shapes and densities are useful tools for studies of cell behavior and to create biomaterials that induce specific cellular responses. Up to now the dominant techniques for creating protein patterns are mostly based on serial writing processes or require templates such as photomasks or elastomer stamps. Only a few of these techniques permit the creation of grayscale patterns. Herein, the development of a lithography system using a digital mirror device which allows fast patterning of proteins by immobilizing fluorescently labeled molecules via photobleaching is reported. Grayscale patterns of biotin with pixel sizes in the range of 2.5 μm are generated within 10 s of exposure on an area of about 5 mm(2) . This maskless projection lithography method permits the rapid and inexpensive generation of protein patterns definable by any user-defined grayscale digital image on substrate areas in the mm(2) to cm(2) range.