Sensitive Protein Quantification Research Articles

Assessment of Ultra-Deep Mass Spectrometry Based Proteomics Compared to Flow Cytometry and RNA-Based Methods for the Discovery and Validation of Therapeutic Targets in Immune Cells

Immuno-oncology (IO) has substantially improved the survival of cancer patients over the past several years encouraging the discovery of novel IO targets which are typically proteins expressed on the surface of immune cells. Sensitive quantification of proteins in complex biological samples is routinely achieved by immunoassays that use antibodies specific to target proteins. Such approaches can be a limitation in IO drug discovery since the development of de novo antibodies is associated with long lead times, high costs, and high failure rates, often resulting in the substitution of the protein readout by a surrogate transcript readout using RNA sequencing methods. Protein quantification using mass spectrometry (MS) is agnostic to any capture agent and removes the barriers of availability or specificity of antibody-based assays. Furthermore, MS proteomics workflows can support large scale discovery studies with wide proteome coverage, and also represent an attractive antibody-free alternative in targeted quantitative studies for protein measurement. The main purpose of this work was primarily to assess the sensitivity of DIA-based MS-proteomics platform for the deep proteome profiling of human primary immune cells by measuring the maximum protein coverage obtained across cell inputs ranging from 2 million down to single cells ( performance assessment). The secondary purpose of this work was to assess the accuracy of the quantification of targeted MS proteomics workflow in comparison with the gold standard QIFI® flow cytometry approach, and other methods using mRNA readouts in immortalized human hematological cell lines ( accuracy assessment). We found that our MS-based proteomics workflows achieve high sensitivity in detection of immune cell markers down to few immune cells as input. Additionally, we report a strong correlation of the quantitative data derived from our MS-based proteomics workflows compared to the flow cytometry gold standard (r 2= 0.7354). Notably, we observed that this correlation was significantly higher than using transcriptomics approaches, emphasizing the benefit of substituting immunoassays for MS-based proteomics platforms in IO target discovery and validation.

Gold-nanoparticle-embedded hydrogel droplets with enhanced fluorescence for imaging and quantification of proteins in cells.

Conventional cellular protein detection techniques such as immunocytochemistry and flow cytometry require abundant cells, posing multiple challenges, including difficulty and cost for obtaining enough cells and the potential for clogging the instrument when using flow cytometry. Also, it is challenging to conduct cellular protein imaging and quantification simultaneously from a single experiment. We present a novel 3D platform, which integrates highly biocompatible cell-entrapped alginate hydrogel droplet array with gold-nanoparticle (AuNP)-based metal enhanced fluorescence (MEF), to achieve simultaneous imaging and quantification of proteins in intact cells in a sensitive manner. Compared to 2D immunocytochemistry, this 3D system allows for a higher cell loading capacity per unit area; together with the MEF-based signal enhancement from the embedded AuNPs, sensitive protein quantification was realized. Furthermore, compared to flow cytometry, this platform allows for protein imaging from individual cells. Taking the detection of EpCAM protein in ovarian cancer cells as a model, we optimized the AuNP size and concentration for optimal fluorescent signals. The 5nm AuNPs at 6.54 × 1013 particles/mL proved to be the most effective in signal enhancement, providing 2.4-fold higher signals compared to that without AuNPs and 6.4-fold higher signals than that of 2D immunocytochemistry. The number of cells required in our technology is 1-3 orders of magnitude smaller than that of conventional methods. This AuNP-embedded hydrogel platform combines the benefits of immunocytochemistry and flow cytometry, providing increased assay sensitivity while also allowing for qualitative analysis through imaging, suitable for protein determination in a variety of cells.

A Point-of-Care Device for Fully Automated, Fast and Sensitive Protein Quantification via qPCR.

This paper presents a fully automated point-of-care device for protein quantification using short-DNA aptamers, where no manual sample preparation is needed. The device is based on our novel aptamer-based methodology combined with real-time polymerase chain reaction (qPCR), which we employ for very sensitive protein quantification. DNA amplification through qPCR, sensing and real-time data processing are seamlessly integrated into a point-of-care device equipped with a disposable cartridge for automated sample preparation. The system’s modular nature allows for easy assembly, adjustment and expansion towards a variety of biomarkers for applications in disease diagnostics and personalised medicine. Alongside the device description, we also present a new algorithm, which we named PeakFluo, to perform automated and real-time quantification of proteins. PeakFluo achieves better linearity than proprietary software from a commercially available qPCR machine, and it allows for early detection of the amplification signal. Additionally, we propose an alternative way to use the proposed device beyond the quantitative reading, which can provide clinically relevant advice. We demonstrate how a convolutional neural network algorithm trained on qPCR images can classify samples into high/low concentration classes. This method can help classify obese patients from their leptin values to optimise weight loss therapies in clinical settings.

Entrapping gold nanoparticles in membranes for simple-to-use enhanced fluorescence detection of proteins

Quantifying proteins under different physiological and pathological conditions can give important insights into human health and disease, since proteins are the functional components of cells. In order to be able to use protein expression levels to diagnose diseases, sensitive protein quantification techniques are required, because some proteins can be present in low concentrations in biofluids at early stages of a disease. Here, a novel and simple-to-implement signal enhancement method for fluorescence-based protein detection is presented, in which an immunoassay was conducted on a nitrocellulose membrane, and a solution of gold nanoparticles was then pipetted onto the membrane before signal acquisition with a fluorescence microscope. The gold nanoparticles were entrapped and adsorbed into the 3D membrane matrix upon drying due to the hydrophobic interactions with the membrane. Through optimizing the concentration and size of the nanoparticles and comparison of different membranes, we were able to achieve metal enhanced fluorescence (MEF). This new gold nanoparticle-based MEF method, together with the 3D membrane platform that improves antibody and gold nanoparticle binding capacity, provided higher signal intensity and assay sensitivity for fluorescence-based protein quantification. Fluorescently-labeled IgG protein was spiked in human plasma with different concentrations, and the lower limit of detection was determined to be 29 ng/mL, three orders of magnitude lower than that of conventional 2D assays and one order of magnitude lower than that of 3D membrane assays without applying gold nanoparticles. This method offers a simple way of improving the sensitivity of fluorescence-based immunoassays that can be easily adopted in a biological lab with basic lab set up. Furthermore, it can be potentially extended to the fluorescence detection of other analytes beyond proteins.

Highly Sensitive Protein Quantification by Direct Kinetic Fingerprinting of Single Protein Molecules

The sensitive and accurate quantification of protein biomarkers plays important roles in early stage diagnosis of disease. However, existing methods for protein quantification exhibit significant background signal from the nonspecific binding of detection probes to assay surfaces or matrix components. As a result of this non-specific signal, they generally do not achieve single-molecule sensitivity, and require stringent washing to maximize sensitivity. To filter out this nonspecific signal and to thereby increase the sensitivity and accuracy of protein quantification, here we have applied single-molecule kinetic fingerprinting with dynamically binding probes to the problem of protein detection.

Electrochemical sensing interfaces based on hierarchically architectured zwitterionic peptides for ultralow fouling detection of alpha fetoprotein in serum

Herein, an electrochemical sensing platform based on zwitterionic peptide with a hierarchical structure was constructed for ultralow fouling and highly sensitive protein quantification. Through the combination of CPPPPEKEKEKEK and CPPPPEKEKEK peptides, hierarchical antifouling peptide brushes were formed and exhibited excellent antifouling property, which can be further modified with alpha fetoprotein (AFP) aptamer to achieve highly sensitive detection of AFP. The hierarchical peptide brush-based sensor system achieved an AFP quantification range from 1.0 fg mL−1 to 1.0 ng mL−1, with a very low limit of detection as low as 0.59 fg mL−1. In addition, due to the superior antifouling property of the newly designed hierarchical peptide brushes, the electrochemical biosensor supported the quantification of AFP in solutions with a high concentration of nonspecific proteins without sacrifice in sensitivity. It is worth noting that the constructed antifouling biosensor ensured quantitative recruitment of AFP in clinical serum samples with acceptable accuracy when compared with the commonly used method in the hospital. The strategy of constructing sensing interfaces based on designed hierarchical peptide brushes provided an effective way to develop biosensors with both excellent antifouling capability and high sensitivity.

Developing a Robust Assay to Monitor and Quantify Key Players of Metabolic Pathways during Adipogenesis by Targeted Proteomics.

Targeted data acquisition using nano liquid chromatrography (nano-LC) coupled mass spectrometry is an emerging approach when there is a need to quantify proteins with high accuracy, sensitivity, and reproducibility. Nevertheless, creating assays meeting all those criteria still remains a laborious task, especially when investigating low abundant proteins and small concentration changes. In this work a targeted data acquisition workflow is developed reducing time and effort to target and investigate key players of metabolic pathways during the process of adipocyte differentiation. This leads to accurate and sensitive quantification of proteins involved in the synthesis of fatty acids, glycerolipids, glycerophospholipids, sphingolipids, the production of energy and reduction equivalents. Additionally low abundant signaling molecules part of the peroxisome proliferator-activated receptor gamma (PPARγ) and insulin signaling pathway with ≈400 for the insulin receptor substrate and 1100 copies per cell for PPARγ are determined.

An integrated model for bead-based immunoassays

Bead-based immunoassays have shown great promise for rapid and sensitive protein quantification. However, there still lacks holistic understanding of assay performance that can inform assay design and optimization. In this paper, we present an integrated mathematical model for surface coverage bead-based assays. This model examines the building blocks of surface coverage assays, including heterogeneous binding of analyte molecules on bead or sensor surfaces, attachment of bead labels to sensor surfaces, and generation of electrochemical current by bead labels. To demonstrate and validate this model, we analyze a semi-homogeneous bead-based electronic enzyme-linked immunosorbent assay and find that experimental results agree with various model predictions. We show that the model can provide design guidance for choice of various assay parameters including bead size, bead number, antibody affinity and assay time, and provide a perspective to reconcile the performance of various implementations of surface coverage assays.

HaloTag-based conjugation of proteins to barcoding-oligonucleotides.

Highly sensitive protein quantification enables the detection of a small number of protein molecules that serve as markers/triggers for various biological phenomena, such as cancer. Here, we describe the development of a highly sensitive protein quantification system called HaloTag protein barcoding. The method involves covalent linking of a target protein to a unique molecule counting oligonucleotide at a 1:1 conjugation ratio based on an azido–cycloalkyne click reaction. The sensitivity of the HaloTag-based barcoding was remarkably higher than that of a conventional luciferase assay. The HaloTag system was successfully validated by analyzing a set of protein-protein interactions, with the identification rate of 44% protein interactions between positive reference pairs reported in the literature. Desmoglein 3, the target antigen of pemphigus vulgaris, an IgG-mediated autoimmune blistering disease, was used in a HaloTag protein barcode assay to detect the anti-DSG3 antibody. The dynamic range of the assay was over 104-times wider than that of a conventional enzyme-linked immunosorbent assay (ELISA). The technology was used to detect anti-DSG3 antibody in patient samples with much higher sensitivity compared to conventional ELISA. Our detection system, with its superior sensitivity, enables earlier detection of diseases possibly allowing the initiation of care/treatment at an early disease stage.

Immuno-Matrix-Assisted Laser Desorption/Ionization Assays for Quantifying AKT1 and AKT2 in Breast and Colorectal Cancer Cell Lines and Tumors.

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) pathway is one of the most commonly dysregulated signaling pathways that is linked to cancer development and progression, and its quantitative protein analysis holds the promise to facilitate patient stratification for targeted therapies. Whereas immunohistochemistry (IHC) and immunoassays are routinely used for clinical analysis of signaling pathways, mass spectrometry-based approaches such as liquid chromatography/electrospray ionization multiple reaction monitoring mass spectrometry (LC/ESI-MRM-MS) are more commonly used in clinical research. Both technologies have certain disadvantages, namely, the nonspecificity of IHC and immunoassays, and potentially long analysis times per sample of LC/ESI-MRM-MS. To create a robust, fast, and sensitive protein quantification tool, we developed immuno-matrix-assisted laser desorption/ionization (iMALDI) assays with automated liquid handling. The assays are able to quantify AKT1 and AKT2 from breast cancer and colon cancer cell lines and flash-frozen tumor lysates with a linear range of 0.05-2.0 fmol/μg of total lysate protein and with coefficients of variation < 15%. Compared to other mass spectrometric methods, the developed assays require less sample per analysis-only 25 μg of total protein-and are therefore suitable for analysis of needle biopsies. Furthermore, the presented iMALDI technique is the first MS-based method for absolute quantitation of AKT peptides from cancer tissues. This study demonstrates the suitability of iMALDI for low limit-of-detection and reproducible quantitation of signaling pathway members using a benchtop MALDI mass spectrometer within approximately 6-7 h.

Assessment of current mass spectrometric workflows for the quantification of low abundant proteins and phosphorylation sites.

The data described here provide a systematic performance evaluation of popular data-dependent (DDA) and independent (DIA) mass spectrometric (MS) workflows currently used in quantitative proteomics. We assessed the limits of identification, quantification and detection for each method by analyzing a dilution series of 20 unmodified and 10 phosphorylated synthetic heavy labeled reference peptides, respectively, covering six orders of magnitude in peptide concentration with and without a complex human cell digest background. We found that all methods performed very similarly in the absence of background proteins, however, when analyzing whole cell lysates, targeted methods were at least 5–10 times more sensitive than directed or DDA methods. In particular, higher stage fragmentation (MS3) of the neutral loss peak using a linear ion trap increased dynamic quantification range of some phosphopeptides up to 100-fold. We illustrate the power of this targeted MS3 approach for phosphopeptide monitoring by successfully quantifying 9 phosphorylation sites of the kinetochore and spindle assembly checkpoint component Mad1 over different cell cycle states from non-enriched pull-down samples. The data are associated to the research article ‘Evaluation of data-dependent and data-independent mass spectrometric workflows for sensitive quantification of proteins and phosphorylation sites׳ (Bauer et al., 2014) [1]. The mass spectrometry and the analysis dataset have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000964.

Surfactant-aided precipitation/on-pellet-digestion (SOD) procedure provides robust and rapid sample preparation for reproducible, accurate and sensitive LC/MS quantification of therapeutic protein in plasma and tissues.

For targeted protein quantification by liquid chromatography mass spectrometry (LC/MS), an optimal approach for efficient, robust and hi-throughput sample preparation is critical, but often remains elusive. Here we describe a straightforward surfactant-aided-precipitation/on-pellet-digestion (SOD) strategy that provides effective sample cleanup and enables high and constant peptide yields in various matrices, allowing reproducible, accurate and sensitive protein quantification. This strategy was developed using quantification of monocolnocal antibody in tissues and plasma as the model system. Surfactant treatment before precipitation substantially increased peptide recovery and reproducibility from plasma/tissue, likely because surfactant permits extensive denaturation/reduction/alkylation of proteins and inactivation of endogenous protease inhibitors, and facilitates removal of matrix components. The subsequent precipitation procedure effectively eliminates the surfactant and nonprotein matrix components, and the thorough denaturation by both surfactant and precipitation enabled very rapid on-pellet-digestion (45 min at 37 °C) with high peptide recovery. The performance of SOD was systematically compared against in-solution-digestion, in-gel-digestion and filter-aided-sample-preparation (FASP) in plasma/tissues, and then examined in a full pharmacokinetic study in rats. SOD achieved the best peptide recovery (∼21.0-700% higher than the other three methods across various matrices), reproducibility (3.75-10.9%) and sensitivity (28-30 ng/g across plasma and tissue matrices), and its performance was independent of matrix types. Finally, in validation and pharmacokinetic studies in rats, SOD outperformed other methods and provided highly accurate and precise quantification in all plasma samples without using stable isotope labeled (SIL)-protein internal standard (I.S.). In summary, the SOD method has proven to be highly robust, efficient and rapid, making it readily adaptable to large-scale clinical and pharmaceutical quantification of biomarkers or biotherapeutics.

Chemical-free lysis and fractionation of cells by use of surface acoustic waves for sensitive protein assays.

We exploit the mechanical action of surface acoustic waves (SAW) to differentially lyse human cancer cells in a chemical-free manner. The extent to which cells were disrupted is reported for a range of SAW parameters, and we show that the presence of 10 μm polystyrene beads is required to fully rupture cells and their nuclei. We show that SAW is capable of subcellular fractionation through the chemical-free isolation of nuclei from whole cells. The concentration of protein was assessed in lysates with a sensitive microfluidic antibody capture (MAC) chip. An antibody-based sandwich assay in a microfluidic microarray format was used to detect unlabeled human tumor suppressor protein p53 in crude lysates, without any purification step, with single-molecule resolution. The results are digital, enabling sensitive quantification of proteins with a dynamic range >4 orders of magnitude. For the conditions used, the efficiency of SAW-induced mechanical lysis was determined to be 12.9% ± 0.7% of that for conventional detergent-based lysis in yielding detectable protein. A range of possible loss mechanisms that could lead to the drop in protein yield are discussed. Our results show that the methods described here are amenable to an integrated point-of-care device for the assessment of tumor protein expression in fine needle aspirate biopsies.

Nonspecific Particle-Based Method with Two-Photon Excitation Detection for Sensitive Protein Quantification and Cell Counting

A novel easy-to-use homogeneous method utilizing two-photon excitation (TPX) for quantification of proteins or counting of eukaryotic cells in solution has been developed. This highly sensitive technique is based on the adsorption competition between the sample and fluorescently labeled protein to micrometer-sized carboxylate modified polystyrene particles and detection of two-photon excited fluorescence. The adsorption of the labeled protein to the particles was detected as a distinct fluorescence on individual microparticles. Analyte protein or eukaryotic cells interacted with particle surface and reduced the adsorption of labeled protein to the particles resulting in a decrease of the fluorescence. The optimizations of assay conditions were performed separately for protein quantification and cell counting, and the principle of the method was confirmed with the fluorescence microscopy imaging. The protein quantification assay allowed the determination of picogram quantities (1.2 μg/L) of protein, and the cell counting assay allowed three cells in the sample with an average variation of approximately 10% in the signal. The protein assay sensitivity was more than 500-fold improved from the common most sensitive commercial methods. Moreover, the dynamic range of the assay was broad, approximately 4 orders of magnitude. The cell assay has sensitivity comparable to the most sensitive commercial method. The developed method tolerates interfering agents such as neutral detergents found in cell lysate samples even at high concentrations. The method is experimentally fairly simple and allows the expansion for the use of the TPX technology.

Large-Scale Quantitative Assessment of Different In-Solution Protein Digestion Protocols Reveals Superior Cleavage Efficiency of Tandem Lys-C/Trypsin Proteolysis over Trypsin Digestion

The complete and specific proteolytic cleavage of protein samples into peptides is crucial for the success of every shotgun LC-MS/MS experiment. In particular, popular peptide-based label-free and targeted mass spectrometry approaches rely on efficient generation of fully cleaved peptides to ensure accurate and sensitive protein quantification. In contrast to previous studies, we globally and quantitatively assessed the efficiency of different digestion strategies using a yeast cell lysate, label-free quantification, and statistical analysis. Digestion conditions include double tryptic, surfactant-assisted, and tandem-combinatorial Lys-C/trypsin digestion. In comparison to tryptic digests, Lys-C/trypsin digests were found most efficient to yield fully cleaved peptides while reducing the abundance of miscleaved peptides. Subsequent sequence context analysis revealed improved digestion performances of Lys-C/trypsin for miscleaved sequence stretches flanked by charged basic and particulary acidic residues. Furthermore, targeted MS analysis demonstrated a more comprehensive protein cleavage only after Lys-C/trypsin digestion, resulting in a more accurrate absolute protein quantification and extending the number of peptides suitable for SRM assay development. Therefore, we conclude that a serial Lys-C/trypsin digestion is highly attractive for most applications in quantitative MS-based proteomics building on in-solution digestion schemes.

Magnetic and electrokinetic manipulations on a microchip device for bead‐based immunosensing applications

The combination of electrophoretic and magnetic manipulations with electrochemical detection for a versatile microfluidic and bead-based biosensing application is demonstrated. Amperometric detection is performed in an off-channel setup by means of a voltammetric cell built at the microchannel outlet and using a gold working electrode. Superparamagnetic particles are introduced and handled inside the channel by means of an external permanent magnet in combination with the electrogenerated flow which allows reproducible loading. The specific detection of phenol as electroactive alkaline phosphatase product is used in this study as proof of concept for a sensitive protein quantification. Characterizations and optimization of different parameters have been carried out in order to achieve the best detection signal. The applicability of the device has been finally demonstrated by the detection of rabbit IgG as model protein after an immunoassay performed on magnetic particles as immobilization platform. A comparison between the electrochemical detection using the developed device and the optical standard detection revealed similar performances with, however, extremely lower amount of reagent used and shorter analysis time. The developed electrophoretic- and magnetic-based chip may open the way to several other biosensing applications with interest not only for other proteins but also for DNA analysis, cell counting, and environmental control.

Quantification of coral heat shock proteins from individual coral polyps

The induction and regulation of heat shock proteins (hsps) is a significant defense mechanism that can preserve metabolic function and foster recovery from short-term stress events. Present coral sampling methodologies that involve hsp analysis often require the harvesting of large samples of live coral colonies that may already be stressed or in poor health. In the present study, 3 novel protocols were developed to: (1) extract single coral polyps, minimizing colony trauma; (2) purify protein from single coral polyps (approximately 12 mm 3 ); and (3) develop a more sensitive protein quantification method. The preliminary testing of 5 separate protein preparation methods resulted in a range of total protein yields from 47 to 77 µg coral polyp -1 . The optimized methods were able to recover, on average, 44 ± 12 µg protein polyp -1 (n = 20). Subsequent SDS-PAGE and immunoblotting analysis of single coral polyps resolved as little as 87 pg hsp70 coral polyp -1 . This minimally invasive sampling protocol reduces coral damage and, thus, reduces stress and diseases caused by sampling.

Multiple sized europium(III) chelate-dyed polystyrene particles as donors in FRET – an application for sensitive protein quantification utilizing competitive adsorption

A variety of particles have been adopted as solid support in bioaffinity assays, and the ability to modify the particle properties makes them a versatile tool in assay development. In separation-free fluorescence resonance energy transfer (FRET) assays, fluorescent particles have been used as donors and acceptors due to their high binding capacity and high specific activity. The availability of multiple fluorophores within the FRET range renders the orientation of individual donors and acceptors a non-factor enabling efficient sensitization. The high specific activity, being proportional to the particle size, may also inflict background fluorescence due to distance dependency of FRET, if the donor emission and detection wavelengths overlap. This is the first study to evaluate the impact of differently sized (47, 68, 92 and 202 nm in diameter) europium(III) chelate-doped polystyrene/acrylic acid donor particles on the core-related background fluorescence in a separation-free FRET-based assay utilizing competitive adsorption of acceptor-labelled protein. Within the particle size range studied, the particle core size-related background fluorescence showed no significant effect on the assay performance at the detection wavelength, which was selected on account of donor emission minimum. The assay sensitivity and dynamics showed compliancy with the rules of competitive assay. The results can be used in designing a competitive separation-free FRET-based assay utilizing particles as donors. In accordance, we applied the method for protein quantification. Sample protein prevented the adsorption of acceptor-labelled protein on the nanoparticle reducing energy transfer. Using this approach, a 100- to 1000-fold lower protein concentration was measured in comparison to traditional photometric protein assays suggesting that a high sensitivity assay can be constructed using FRET-based nanoparticle assay concepts.

Microwave-Enhanced Ink Staining for Fast and Sensitive Protein Quantification in Proteomic Studies

A novel microwave-enhanced ink staining method was developed for rapid and sensitive estimation of protein content in sample buffers containing chaotropes, dyes, detergents, and reducing agents. Dye-based Blue-Black ink was used to quantitatively visualize proteins spotted on a nitrocellulose membrane. The total staining time was greatly reduced to 3 min by brief exposure to microwave radiation. The stained membrane was washed with distilled water, baked in a microwave oven for complete desiccation, transparentized with mineral oil, and documented by a desktop scanner or densitometer. Only 1 microL of protein sample (protein solubilized in SDS-PAGE sample buffer or IEF rehydration buffer) was used for protein spotting. The novel solid-phase protein assay gives a 500-fold dynamic range from 19.5 to 10000 ng/microL and can be scaled up for high-throughput protein quantification analysis. The fast, sensitive and low-cost microwave-enhanced ink staining procedure is ideal for protein quantification in proteomic analysis.

Fluorescent Hydrophobic Probes Based on Intramolecular Charge Transfer State for Sensitive Protein Detection in Solution.

AbstractFor Abstract see ChemInform Abstract in Full Text.