Select cytoplasmic and membrane proteins increase the percentage of immobile integrins but do not affect the average diffusion coefficient of mobile integrins

Integrins are ubiquitous adhesion receptors that are important for signaling and integrating the extracellular matrix and cytoskeleton. The role of cytoplasmic proteins vinculin, focal adhesion kinase (FAK), integrin-linked kinase (ILK), and membrane proteins epidermal growth factor receptor (EGFR) and Notch in altering αPS2CβPS integrin lateral diffusion was measured using single particle tracking (SPT) and RNA interference (RNAi). SPT measures heterogeneous diffusion properties, and RNAi selectively reduces the concentration of a target protein. After systematically reducing the concentration of vinculin, FAK, ILK, EGFR, or Notch, there was a 31 to 80 % increase in the mobile integrin fraction, indicating that these five targeted proteins (or assemblies that contain these proteins) are responsible for immobilizing a fraction of the integrins when all proteins are present at native concentrations. The average diffusion coefficient of all mobile integrins did not change after any of the RNAi treatments, and the percentage of Brownian, directed, or anomalous/constrained trajectories relative to total mobile trajectories did not change after vinculin or EGFR RNAi. However, the fraction of anomalous/constrained trajectories relative to the total mobile trajectories increased 9 to 19 % after FAK, ILK, and Notch RNAi, when the concentration of these proteins was reduced. In the case of FAK, ILK, and Notch, native concentrations of these proteins simultaneously increase the immobile fraction of integrins but decrease the diffusion constraints to those integrins that remain mobile. Comparisons of single receptor and ensemble measurements of diffusion and what is known about the effect of these proteins in altering integrin clustering are discussed.

Nanodisc-solubilized membrane protein library reflects the membrane proteome

The isolation and identification of unknown membrane proteins offers the prospect of discovering new pharmaceutical targets and identifying key biochemical receptors. However, interactions between membrane protein targets and soluble ligands are difficult to study in vitro due to the insolubility of membrane proteins in non-detergent systems. Nanodiscs, nanoscale discoidal lipid bilayers encircled by a membrane scaffold protein belt, have proven to be an effective platform to solubilize membrane proteins and have been used to study a wide variety of purified membrane proteins. This report details the incorporation of an unbiased population of membrane proteins from Escherichia coli membranes into Nanodiscs. This solubilized membrane protein library (SMPL) forms a soluble in vitro model of the membrane proteome. Since Nanodiscs contain isolated proteins or small complexes, the SMPL is an ideal platform for interactomics studies and pull-down assays of membrane proteins. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of the protein population before and after formation of the Nanodisc library indicates that a large percentage of the proteins are incorporated into the library. Proteomic identification of several prominent bands demonstrates the successful incorporation of outer and inner membrane proteins into the Nanodisc library.

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.

Detection and quantification of proteins and cells by use of elemental mass spectrometry: progress and challenges

Much progress has been made in identification of the proteins in proteomes, and quantification of these proteins has attracted much interest. In addition to popular tandem mass spectrometric methods based on soft ionization, inductively coupled plasma mass spectrometry (ICPMS), a typical example of mass spectrometry based on hard ionization, usually used for analysis of elements, has unique advantages in absolute quantification of proteins by determination of an element with a definite stoichiometry in a protein or attached to the protein. In this Trends article, we briefly describe state-of-the-art ICPMS-based methods for quantification of proteins, emphasizing protein-labeling and element-tagging strategies developed on the basis of chemically selective reactions and/or biospecific interactions. Recent progress from protein to cell quantification by use of ICPMS is also discussed, and the possibilities and challenges of ICPMS-based protein quantification for universal, selective, or targeted quantification of proteins and cells in a biological sample are also discussed critically. We believe ICPMS-based protein quantification will become ever more important in targeted quantitative proteomics and bioanalysis in the near future.

Conjugation-free,visual, and quantitative evaluation of inhibitors on protein tyrosine kinases and phosphatases with a luminescent Tb(III) complex

A straightforward and visual method to assess inhibitors on protein tyrosine kinases (PTKs) and phosphatases (PTPs) has been developed. These enzymes play critical roles in a number of diseases and, thus, their inhibitors are important for effective therapy. With the use of the long-life luminescence emitted from a binuclear Tb(III) complex, enzymatic reactions of PTKs and PTPs were monitored in real-time, and the inhibitor activity was quantitatively evaluated in terms of the decrease in the rate of luminescence change. No conjugation of the probe to a substrate peptide was necessary. The IC₅₀ values of four inhibitors on three kinds of PTKs [Src, Fyn, and epidermal growth factor receptor (EGFR)] were determined. For example, gefitinib, which is a selective inhibitor on EGFR, inhibited this PTK with IC₅₀ of 22 nM. Towards Src and Fyn (non-targeted PTK), however, IC₅₀ of this inhibitor was greater than 20 μM as expected. Inhibition of two kinds of PTPs (Shp-1 and PTP1B) by two inhibitors was also assayed, providing completely consistent results on their known selectivity. Furthermore, the system where both PTK and PTP are active was monitored and the reactions were visualized with the present Tb(III) complex-based method. High potential of the present method to a variety of systems has been evidenced.

Effects of the expression level of epidermal growth factor receptor on the ligand-induced restructuring of focal adhesions: a QCM-D study

Epidermal growth factor receptor (EGFR) plays a major role in cell migration and invasion and is considered to be the primary source of activation of various malignant tumors. To gain insight into how elevated levels of EGFR influence cellular function, particularly cell motility, we used a quartz crystal microbalance with dissipation monitoring (QCM-D) to examine restructuring of focal adhesions in MCF-10A cells induced by epidermal growth factor. Engineered cells that overexpress epidermal growth factor receptor (EGFR) exhibited a very different kinetic profile from wildtype MCF-10A cells that have a lower level of EGFR with a higher rate for the initial disassembly of focal adhesion and a much lower rate for the later reassembly of focal adhesions. It is conceivable that these effects exhibited by EGFR-overexpressing cells may promote the initiation and maintenance of a more favorable adhesion state for cell migration. This study has demonstrated the capability of the dissipation monitoring function of the QCM-D to quantitatively assess kinetic aspects of cellular processes with a high temporal resolution and sensitivity.

Metallomic study on plasma samples from Nile tilapia using SR-XRF and GFAAS after separation by 2D PAGE: initial results

An investigation was made on plasma samples obtained after protein separation. The proteome of the plasma of Nile tilapia (Oreochromis niloticus) was separated by 2D PAGE, and manganese and zinc in protein spots was qualitatively and quantitatively determined by synchrotron radiation X-ray fluorescence (SR-XRF) and graphite furnace atomic absorption spectrometry (GFAAS). Manganese and zinc are present in four and six plasma protein spots, respectively. These ions are bound to proteins with molecular weights ranging from 19 to 70?kDa and with isoelectric point (pI) ranging from 4.7 to 6.3. The concentrations of manganese and zinc bound to these proteins as determined by GFAAS following acid digestion of the spots range from 0.8 to 2.6?mg of manganese, and from 1.0 to 6.3?mg of zinc, respectively, per g of protein.

Neutron-Encoded Protein Quantification by Peptide Carbamylation

We describe a chemical tag for duplex proteome quantification using neutron encoding (NeuCode). The method utilizes the straightforward, efficient, and inexpensive carbamylation reaction. We demonstrate the utility of NeuCode carbamylation by accurately measuring quantitative ratios from tagged yeast lysates mixed in known ratios and by applying this method to quantify differential protein expression in mice fed a either control or high-fat diet.

Exploring Salt Bridge Structures of Gas-Phase Protein Ions using Multiple Stages of Electron Transfer and Collision Induced Dissociation

The gas-phase structures of protein ions have been studied by electron transfer dissociation (ETD) and collision-induced dissociation (CID) after electrospraying these proteins from native-like solutions into a quadrupole ion trap mass spectrometer. Because ETD can break covalent bonds while minimally disrupting noncovalent interactions, we have investigated the ability of this dissociation technique together with CID to probe the sites of electrostatic interactions in gas-phase protein ions. By comparing spectra from ETD with spectra from ETD followed by CID, we find that several proteins, including ubiquitin, CRABP I, azurin, and β-2-microglobulin, appear to maintain many of the salt bridge contacts known to exist in solution. To support this conclusion, we also performed calculations to consider all possible salt bridge patterns for each protein, and we find that the native salt bridge pattern explains the experimental ETD data better than nearly all other possible salt bridge patterns. Overall, our data suggest that ETD and ETD/CID of native protein ions can provide some insight into approximate location of salt bridges in the gas phase.

Simultaneous serum desalting and total protein determination by macroporous reversed-phase chromatography

Macroporous reversed-phase (mRP) chromatography was successfully used to develop an accurate and precise method for total protein in serum. The limits of detection (0.83 μg, LOD) and quantification (2.51 μg, LOQ) for the mRP method are comparable with those of the widely used micro BCA protein assay. The mRP method can be used to determine the total protein concentration across a wide dynamic range by detecting chromatographic peaks at 215 nm and 280 nm. The method has the added advantage of desalting and denaturing proteins, leading to more complete digestion by trypsin and to better LC–MS–MS identification in shotgun proteomics experiments.

Absorption-Mode Fourier Transform Mass Spectrometry: The Effects of Apodization and Phasing on Modified Protein Spectra

The method of phasing broadband Fourier transform ion cyclotron resonance (FT-ICR) spectra allows plotting the spectra in the absorption-mode; this new approach significantly improves the quality of the data at no extra cost. Herein, an internal calibration method for calculating the phase function has been developed and successfully applied to the top-down spectra of modified proteins, where the peak intensities vary by 100×. The result shows that the use of absorption-mode spectra allows more peaks to be discerned within the recorded data, and this can reveal much greater information about the protein and modifications under investigation. In addition, noise and harmonic peaks can be assigned immediately in the absorption-mode.

Classification of olive leaves and pulps according to their cultivar by using protein profiles established by capillary gel electrophoresis

A method to classify olive leaves and pulps according to their cultivar using protein profiles obtained by capillary gel electrophoresis (CGE) has been developed. For this purpose, proteins were extracted using an enzyme-assisted method, which provided higher protein recoveries than other previously described methods. Ten and nine common peaks, for leaf and pulp samples, respectively, were identified in the 12 cultivars studied in this work. In addition, and using linear discriminant analysis of the CGE data, olive leaf and pulp samples belonging to 12 cultivars from different Spanish regions were correctly classified with an excellent resolution among all the categories, which demonstrated that protein profiles were characteristic of each cultivar.

Synthesis and characterization of poly(ethyleneimine) dendrimers

Poly(ethyleneimine) (PEI) dendrimers up to the third generation (G3) were prepared by a divergent synthesis method from an ethylenediamine (EDA) core. The amine terminals were bonded with vinylbromide by a Michael addition reaction. Then, the bromide terminals were converted to amine groups using a Gabriel amine synthesis method. PEI dendrimers displayed pH-dependent luminescence, and their emission intensities at pH 6 increased over time. Fluorescence intensities also increased with increasing dendrimer generation from G1 to G3. Air-bubbling in aqueous solutions of dendrimers made to incorporate detectable amount of oxygen in dendrimers. EDA also behaved similarly in luminescence and oxygen incorporation.

Examination of an absolute quantity of less than a hundred nanograms of proteins by amino acid analysis

We developed an ultra-sensitive method of amino acid analysis (AAA) for the absolute quantification of less than 100 ng of proteins, in solution or on polyvinylidene difluoride (PVDF) membranes using an oxygen-free chamber for protein hydrolysis. We used a pre-label method with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate for fluorescence detection, ion-pair chromatography with a reversed-phase column, and an ultra-high-pressure high-performance liquid chromatography. We optimized both handling- and instrument-dependent factors for accurate quantification and showed that the least amount of proteins to quantify was determined by handling accuracy rather than instrumental limit for quantification which was 0.6 fmol/amino acid. As a new evaluation method for the handling accuracy, we adopted the protein identification by the obtained amino acid compositions by AAA and the Swiss-Prot database search without the restriction of species. As a result, the least amount of starting material for AAA was 16 ng (0.24 pmol) for a solution of bovine serum albumin (BSA), 33 ng (0.50 pmol) for BSA on a PVDF membrane, and 44 ng (0.15 pmol) for thyroglobulin on a PVDF membrane. These results demonstrate that the ultra-sensitive AAA developed in this study is feasible for absolute quantification of biological significant protein.

Multivalent chelators for spatially and temporally controlled protein functionalization

Site-specific protein modification—e.g. for immobilization or labelling—is a key prerequisite for numerous bioanalytical applications. Although modification by use of short peptide tags is particularly attractive, efficient and bio-orthogonal systems are still lacking. Here, we review the application of multivalent chelators (MCH) for high-affinity yet reversible recognition of oligohistidine (His)-tagged proteins. MCH are based on multiple nitrilotriacetic acid (NTA) moieties grafted on to molecular scaffolds suitable for conjugation to surfaces, probes or other biomolecules. Reversible interaction with the His-tag is mediated via transition metal ions chelated by the NTA moieties. The small size and biochemical compatibility of these recognition units and the possibility of rapid dissociation of the interaction with His-tagged proteins despite sub-nanomolar binding affinity, enable distinct and versatile handling and modification of recombinant proteins. In this review, we briefly introduce the key principles and features of MCH–His-tag interactions and recapitulate the broad spectrum of bioanalytical applications with a focus on quantitative protein interaction analysis on micro or nano-patterned solid surfaces and specific protein labelling in living cells.

Identification of selenium in the leaf protein of sunflowers by a combination of 2D-PAGE and laser ablation ICP-MS

We report on a method for the identification of selenium-containing proteins in an extract of sunflower leafs. It is based on the separation of the proteins by 2-dimensional gel electrophoresis, followed by detection of selenium via laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The laser system was operated in a raster mode at 100?μm?s^-1 and proved to be an efficient alternative in the search for selenoproteins in the spots of the gels. The instrumental parameters were optimized in terms of plasma energy and application of optimal reaction cell conditions, and the detection of the mass ⁸⁰Se¹⁶O⁺ which enabled the elimination of interfering species. Selenium was identified in 9.6% of the analyzed spots, indicating its random incorporation into the primary structure of the proteins.

Sensitive detection of trace hemoglobin using fluorescence method based on functionalized quantum dots

The fluorescence quenching of quantum dots by hemoglobin has been demonstrated to depend on surface functionalization, and this property has been utilized to construct a novel fluorescent method for rapid, sensitive, and selective detection of trace hemoglobin in urine at microgram level. This method shows low interference and high selectivity for hemoglobin with a limit of detection of 4.3 μg L^?1 in water and 66.1 μg L^?1 in urine, which are lower than those of currently used methods in labs and clinics. Spike and recovery tests in raw, acidified, and alkalized urine samples exhibit good recovery rates for the spiked concentrations close to the limit of detection.

Interpretation and Deconvolution of Nanodisc Native Mass Spectra

Nanodiscs are a promising system for studying gas-phase and solution complexes of membrane proteins and lipids. We previously demonstrated that native electrospray ionization allows mass spectral analysis of intact Nanodisc complexes at single lipid resolution. This report details an improved theoretical framework for interpreting and deconvoluting native mass spectra of Nanodisc lipoprotein complexes. In addition to the intrinsic lipid count and charge distributions, Nanodisc mass spectra are significantly shaped by constructive overlap of adjacent charge states at integer multiples of the lipid mass. We describe the mathematical basis for this effect and develop a probability-based algorithm to deconvolute the underlying mass and charge distributions. The probability-based deconvolution algorithm is applied to a series of dimyristoylphosphatidylcholine Nanodisc native mass spectra and used to provide a quantitative picture of the lipid loss in gas-phase fragmentation.

Jim Morrison, Friend and Colleague

Our long-time association with Jim Morrison and the work that came from it is the result of a series of fortunate coincidences. We are pleased to be able to share recollections here of our interactions with Jim and how his life and work have influenced us and the field of mass spectrometry.

High-throughput,multiparameter analysis of single cells

Heterogeneity of cell populations in various biological systems has been widely recognized, and the highly heterogeneous nature of cancer cells has been emerging with clinical relevance. Single-cell analysis using a combination of high-throughput and multiparameter approaches is capable of reflecting cell-to-cell variability, and at the same time of unraveling the complexity and interdependence of cellular processes in the individual cells of a heterogeneous population. In this review, analytical methods and microfluidic tools commonly used for high-throughput, multiparameter single-cell analysis of DNA, RNA, and proteins are discussed. Applications and limitations of currently available technologies for cancer research and diagnostics are reviewed in the light of the ultimate goal to establish clinically applicable assays.