Quantification Of Protein Biomarkers Research Articles

Nanofluidics Drives Point-of-care Technology for on the Spot Protein Marker Analysis with Rapid Actionable Results

Protein sensing in a nanofluidic environment dramatically shortens immunoassay time-to-result through the combined action of efficient mass transfer and short diffusion distances. Here, we report on a fully automated point-of-care in vitro diagnostic platform based on disposable capsule containing nanofluidic sensors in which fluorescent immunoassays are performed. Performances of the system were established with three model assays for ferritin, immunoglobulin E (IgE) and pancreatic stone protein (PSP). The described system has the typical high capture efficiency of nano-confined spaces combined with forced-flow, which induce a constant maximal concentration gradient on the sensor. Remarkably, analytes are detected in zeptomole quantities from a drop of blood. Dose-response curves show that high precision and accuracy are achieved in the clinically relevant assay ranges. Moreover, accuracy of the system is excellent agreement with laboratory reference method, as illustrated in a method comparison with total IgE as a model. While several academic proof-of-concepts have already described the possibility to exploit the properties of fluids at the nanoscale to develop immunoassay, the transition of these models to a product fulfilling requirements for use at the point-of-care in terms of operability, affordability, reliability and analytic performances remains a challenging endeavor. This study demonstrates that nanofluidic-based immunoassays can efficiently quantify protein biomarkers in the femto- and picomolar range within ultra-short assay time, high precision and accuracy on a closed, small, easyto- operate platform.

Quantitative measurement of selected protein biomarkers of endothelial dysfunction in plasma by micro-liquid chromatography-tandem mass spectrometry based on stable isotope dilution method

The aim of this study was to develop and validate the novel microLC/MS-MRM method for the simultaneous quantification of six proteins: angiopoietin 2 (Angpt-2), soluble form of fms-like tyrosine kinase 1 (sFLT-1), plasminogen activator inhibitor 1 (PAI-1), tissue plasminogen activator (t-PA), endocan (ESM-1), soluble form of E-selectin (sE-sel), and one peptide: adrenomedullin (ADM) in mouse plasma. Two approaches were compared: a stable isotope dilution (SID) method- used as a reference and a modified SID (mSID) procedure. In SID strategy the calibration curves were used, whereas in mSID the ratio between the chromatogram peak area of endogenous tryptic peptides at unknown concentration to chromatogram peak area of exogenous, stable isotope-labelled internal standards (SISs) added to the sample at known concentration was calculated. The microLC/MS-MRM method in the SID approach was linear from 0.250 pmol/mL to 250 pmol/mL for Angpt-2; 5 pmol/mL to 5000 pmol/mL for sFLT-1; 2.5 pmol/mL to 5000 pmol/mL for PAI-1; 0.375 pmol/mL to 250 pmol/mL for t-PA; 0.375 pmol/mL to 187.5 pmol/mL for ESM-1; 2.5 pmol/mL to 5000 pmol/mL for sE-sel and 0.375 pmol/mL to 250 pmol/mL for ADM. LPS-induced changes in plasma assessed based on SID and mSID approaches gave comparable quantitative results and featured LPS-induced dysregulation of endothelial permeability (Angpt-2, sFLT-1), glycocalyx injury (SDC-1) accompanied by a pro-thrombotic response (PAI-1). In addition, we applied microLC/MS-MRM method with mSID strategy to analyze human plasma samples from patients with chronic myeloid leukemia (CML) and obstructive sleep apnoea (OSA) and demonstrated usefulness of the method to characterize endothelial function in humans. In conclusion, the microLC/MS-MRM method with mSID strategy applied for simultaneous quantification of protein biomarkers of endothelial function in plasma represents a novel targeted proteomic platform for the comprehensive evaluation of endothelial function in mice and humans.

Use of a Purpose-Built Impression Cytology Device for Gene Expression Quantification at the Ocular Surface Using Quantitative PCR and Droplet Digital PCR.

To describe an impression cytology (IC) technique using a purpose-built, sterile, EYEPRIM IC device that can be coupled with a TRIzol reagent-based RNA extraction protocol to yield sufficient RNA for gene expression analysis. IC samples using the EYEPRIM device were collected from the bulbar conjunctiva, with and without topical anesthesia, and evaluated for RNA yield, the absence of polymerase chain reaction (PCR) inhibitors, and the ability to detect biomarkers by quantitative real-time PCR and droplet digital PCR. A technique for collecting IC samples in the clinic, while preserving RNA, and a protocol for subsequent laboratory analysis of RNA were developed. The extracted RNA was free of PCR inhibitors and could be synthesized into complementary DNA and used for successful relative quantification of ocular surface biomarkers by quantitative real-time PCR. For gene targets present in low abundance, complementary DNA could also be used for quantification by the relatively new and emerging method of droplet digital PCR. The described method was successfully used to evaluate 3 biomarkers in a clinical trial assessing the tolerability of a proprietary eyelid therapy in 92 IC samples from a study population of 46 participants. IC is a recognized technique for ocular surface cell evaluation and protein biomarker quantification but is infrequently used for quantifying gene expression. The EYEPRIM device allows ease of use and impression-to-impression consistency while accurate gene expression data offers a highly specific and sensitive method of disease characterization for clinician scientists to use in diagnosis.

Impact of biological matrix on inflammatory protein biomarker quantification based on targeted mass spectrometry.

Serum and plasma are widely used matrices in biological and clinical studies. To improve reliability and consistency of markers quantification, the influence of these matrices on proteins was evaluated by targeted mass spectrometry. 65 proteins were quantified in matched blood samples collected in serum, ethylenediaminetetraacetic acid and heparin plasma tubes from 40 healthy and 10 pathological individuals. Only 52% of the proteins were not impacted by any of the biological matrices tested, and the effects on quantification of proteins affected was matrix and protein dependent. Matrix comparisons using mass spectrometry is therefore recommended to assess the relevance of using surrogate matrix, performing biomarker discovery study or evaluating the clinical use of biomarkers in large clinical cohorts.

Analytical convergence in surrogate control matrices.

Analytical convergence in surrogate control matrices.

Optimizing charge state distribution is a prerequisite for accurate protein biomarker quantification with LC-MS/MS, as illustrated by hepcidin measurement.

Targeted quantification of protein biomarkers with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has great potential, but is still in its infancy. Therefore, we elucidated the influence of charge state distribution and matrix effects on accurate quantification, illustrated by the peptide hormone hepcidin. An LC-MS/MS assay for hepcidin, developed based on existing literature, was improved by using 5 mM ammonium formate buffer as mobile phase A and as an elution solution for solid phase extraction (SPE) to optimize the charge state distribution. After extensive analytical validation, focusing on interference and matrix effects, the clinical consequence of this method adjustment was studied by performing receiving operating characteristic (ROC)-curve analysis in patients with iron deficiency anemia (IDA, n=44), anemia of chronic disease (ACD, n=42) and non-anemic patients (n=93). By using a buffered solution during sample preparation and chromatography, the most abundant charge state was shifted from 4+ to 3+ and the charge state distribution was strongly stabilized. The matrix effects which occurred in the 4+ state were therefore avoided, eliminating bias in the low concentration range of hepcidin. Consequently, sensitivity, specificity and positive predictive value (PPV) for detection of IDA patients with the optimized assay (96%, 97%, 91%, respectively) were much better than for the original assay (73%, 70%, 44%, respectively). Fundamental improvements in LC-MS/MS assays greatly impact the accuracy of protein quantification. This is urgently required for improved diagnostic accuracy and clinical value, as illustrated by the validation of our hepcidin assay.

Digital analysis with droplet-based microfluidic for the ultrasensitive detection of β-gal and AFP

In this study, we presented a digital analysis with droplet-based microfluidic, which was applied for the detection of β-galactosidase (β-Gal) and AFP. The β-Gal was quantified according to the Poisson Distribution in digital analysis with droplets containing β-Gal and fluorogenic substrate fluorescein di-β-D-galactopyranoside (FDG). In our method, the lowest concentration of β-Gal we could accurately detect was 5 fM. We found that the digital detection could be applied in quantifying protein biomarkers, take AFP for model sample. The AFP was detected through indirect detection with excess streptavidin-conjugated β-galactosidase (SA-β-gal) as signal tag. To be specific, firstly SA-β-gal was conjugated to biotin-labeled AFP antibody, and then the unbounded signal tag in the reaction solution was taken out for digital detection with droplet-based microfluidic. As a result, the AFP could be also indirectly quantified at an fM level through subtraction. This experimental result demonstrated that the indirect detection with droplet-based digital analysis was able to detect biomarkers at a low level with a few samples on an ordinary flow-focusing microfluidic chip.

Multiplex Immuno-MALDI-TOF MS for Targeted Quantification of Protein Biomarkers and Their Proteoforms Related to Inflammation and Renal Dysfunction.

Circulating proteins are widely used as biomarkers in clinical applications for the diagnosis, prediction, and treatment of numerous diseases. Immunoassays are the most common technologies for quantification of protein biomarkers and exist in various formats. Traditional immunoassays offer sensitive and fast analyses but cannot differentiate between proteoforms. Protein microheterogeneity, mainly due to post-translational modification, has been recognized as a fingerprint for different pathologies, and knowledge about proteoforms is an important step toward personalized medicine. Mass spectrometry (MS) has emerged to be a powerful technique for the characterization and quantification of proteoforms. We have established a novel four-plex immunoassay based on Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF) MS for the targeted quantification of the inflammatory markers C-reactive protein (CRP), serum amyloid A (SAA), and calprotectin (S100A8/9) and the kidney function marker cystatin C (CysC). Antibodies were covalently bound to superparamagnetic beads, which delivered robust and fast sample processing. Polyhistidine-tagged recombinant target proteins were used as internal standards for quantification. Our method identified a number of proteoforms for SAA ( n = 11), S100A8/9 ( n = 4) and CysC ( n = 4). The assay was characterized by low limits of detection (0.01-0.06 μg/mL) and low coefficients of variation (3.8-9.4%). Method validation demonstrated good between-assay agreement with immuno-turbidimetry ( R2 = 0.963 for CRP), ELISA ( R2 = 0.958 for SAA; R2 = 0.913 for S100A8/9), and nephelometry ( R2 = 0.963 for CysC). The low sample consumption of 20 μL and the high sample throughput of 384 samples per day make this targeted immuno-MALDI approach suited for assessment of inflammatory and renal status in large cohort studies based on precious biobanks samples.

Biomacromolecules as tools and objects in nanometrology\u2014current challenges and perspectives

Nucleic acids, proteins, and polysaccharides are the most important classes of biopolymers. The inherent properties of biomacromolecules are contrary to those of well-defined small molecules consequently raising a number of specific challenges which become particularly apparent if biomacromolecules are treated as objects in quantitative analysis. At the same time, their specific functional ability of molecular recognition and self-organization (e.g., enzymes, antibodies, DNA) enables us to make biomacromolecules serving as molecular tools in biochemistry and molecular biology, or as precisely controllable dimensional platforms for nanometrological applications. Given the complexity of biomacromolecules, quantitative analysis is not limited to the measurement of their concentration but also involves the determination of numerous descriptors related to structure, interaction, activity, and function. Among the biomacromolecules, glycans set examples that quantitative characterization is not necessarily directed to the measurement of amount-of-substance concentration but instead involves the determination of relative proportions (molar ratios) of structural features for comparison with theoretical models. This article addresses current activities to combine optical techniques such as Raman spectroscopy with isotope dilution approaches to realize reference measurement procedures for the quantification of protein biomarkers as an alternative to mass spectrometry-based techniques. Furthermore, it is explored how established ID-MS protocols are being modified to make them applicable for quantifying virus proteins to measure the HIV viral load in blood samples. As an example from the class of carbohydrates, the challenges in accurate determination of substitution patterns are outlined and discussed. Finally, it is presented that biomacromolecules can also serve as tools in quantitative measurements of dimensions with an example of DNA origami to generate defined dimensional standards to be used for calibration in super-resolution fluorescence microscopy.Graphical abstractQuantitative analysis of biomacromolecules is accompanied with special challenges different from low molecular weight compounds. In addition, they are not only objects but also tools applicable for quantitative measurements

Direct protein quantification in complex sample solutions by surface-engineered nanorod probes

Detecting biomarkers from complex sample solutions is the key objective of molecular diagnostics. Being able to do so in a simple approach that does not require laborious sample preparation, sophisticated equipment and trained staff is vital for point-of-care applications. Here, we report on the specific detection of the breast cancer biomarker sHER2 directly from serum and saliva samples by a nanorod-based homogeneous biosensing approach, which is easy to operate as it only requires mixing of the samples with the nanorod probes. By careful nanorod surface engineering and homogeneous assay design, we demonstrate that the formation of a protein corona around the nanoparticles does not limit the applicability of our detection method, but on the contrary enables us to conduct in-situ reference measurements, thus further strengthening the point-of-care applicability of our method. Making use of sandwich assays on top of the nanorods, we obtain a limit of detection of 110 pM and 470 pM in 10-fold diluted spiked saliva and serum samples, respectively. In conclusion, our results open up numerous applications in direct protein biomarker quantification, specifically in point-of-care settings where resources are limited and ease-of-use is of essence.

Application of volumetric absorptive microsampling for robust, high-throughput mass spectrometric quantification of circulating protein biomarkers

Volumetric absorptive micro sampling (VAMS™) allows accurate sampling of 10µL of blood from a minimally invasive finger prick and could enable remote personalized health monitoring. Moreover, VAMS overcomes effects from hematocrit and sample heterogeneity associated with dried blood spots (DBS). We describe the first application of VAMS with the Mitra® microsampling device for the quantification of protein biomarkers using an automated, high-throughput sample preparation method coupled with mass spectrometric (MS) detection.The analytical performance of the developed workflow was evaluated for 10 peptides from six clinically relevant proteins: apolipoproteins A-I, B, C-I, C-III, E, and human serum albumin (HSA). Extraction recovery from blood with three different levels of hematocrit varied between 100% and 111% for all proteins. Within-day and total assay reproducibility (i.e., 5 replicates on 5days) ranged between 3.2–10.4% and 3.4–12.6%, respectively. In addition, after 22weeks of storage of the Mitra microsampling devices at −80°C, all peptide responses were within ±15% deviation from the initial response. Application to data-independent acquisition (DIA) MS further demonstrated the potential for broad applicability and the general robustness of the automated workflow by reproducible detection of 1661 peptides from 423 proteins (average 15.7%CV (n=3) in peptide abundance), correlating to peptide abundances in corresponding plasma (R=0.8383).In conclusion, we have developed an automated workflow for efficient extraction, digestion, and MS analysis of a variety of proteins in a fixed small volume of dried blood (i.e., 10µL). This robust and high-throughput workflow will create manifold opportunities for the application of remote, personalized disease biomarker monitoring.

Quantification of Protein Biomarkers in Tissues: New Capabilities with Pellet Digestion Peptide Immunoaffinity LC–MS/MS

Quantification of Protein Biomarkers in Tissues: New Capabilities with Pellet Digestion Peptide Immunoaffinity LC–MS/MS

High precision quantification of human plasma proteins using the automated SISCAPA Immuno-MS workflow

Efficient robotic workflows for trypsin digestion of human plasma and subsequent antibody-mediated peptide enrichment (the SISCAPA method) were developed with the goal of improving assay precision and throughput for multiplexed protein biomarker quantification. First, an 'addition only' tryptic digestion protocol was simplified from classical methods, eliminating the need for sample cleanup, while improving reproducibility, scalability and cost. Second, methods were developed to allow multiplexed enrichment and quantification of peptide surrogates of protein biomarkers representing a very broad range of concentrations and widely different molecular masses in human plasma. The total workflow coefficients of variation (including the 3 sequential steps of digestion, SISCAPA peptide enrichment and mass spectrometric analysis) for 5 proteotypic peptides measured in 6 replicates of each of 6 different samples repeated over 6 days averaged 3.4% within-run and 4.3% across all runs. An experiment to identify sources of variation in the workflow demonstrated that MRM measurement and tryptic digestion steps each had average CVs of ∼2.7%. Because of the high purity of the peptide analytes enriched by antibody capture, the liquid chromatography step is minimized and in some cases eliminated altogether, enabling throughput levels consistent with requirements of large biomarker and clinical studies.

Direct and multiplex quantification of protein biomarkers in serum samples using an immuno-magnetic platform.

A direct and ultrasensitive multiplex assay using an immuno-magnetic platform has been developed for the quantification of trace amounts of circulating cancer-associated antigens in serum. The detection is based on the specific immuno-interactions among the target antigen, detection antibody and capture antibody that is immobilized on the surface of magnetic nanoparticles. The sandwiched immuno-assembly is then labelled with turn-on fluorophores and detected with a fluorescence imaging system. To afford a high signal-to-noise ratio, three turn-on fluorophores with unique optical properties have been designed and synthesized to label the target antigens. The developed assay has achieved a remarkable LOD down to the femto-molar regime without sample pre-treatment. This versatile assay can efficiently differentiate the target antigen from a protein matrix and simultaneously quantify multiple cancer-associated antigens, for instance, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and prostate specific antigen (PSA) using only 6 μL of serum sample in an hour. This novel system has a high applicability to serve as a universal and useful tool for early disease diagnostics.

Rapid dry-reagent immunomagnetic biosensing platform based on volumetric detection of nanoparticles on 3D structures

A dry-reagent immunomagnetic (DRIM) biosensing platform is developed for rapid high-precision quantitative analyses for in vitro diagnostics. The platform combines the advantages of immunochromatography with highly sensitive quantification of 200-nm magnetic nanoparticles (MP) from the entire volume of lateral flow membranes. The MP are registered by their non-linear magnetization at combinatorial frequencies by a portable reader that offers the detection limit of 60 zeptomoles or 0.4ng of magnetic nanolabels and extremely wide linear dynamic range of 7 orders. The efficient combination of small MP with large reaction surface of the 3D membranes has permitted the detection in human serum of as low as 25pg/ml total prostate specific antigen (PSA) during 30-min assay. The featured 3-fold signal increase per every order of concentration within 3.5 orders of magnitude allows precise analysis of antigen concentrations in a wide range. The system also provides the first tool for quantitative MP mapping along the lateral flow strip for easy development and optimization of assays. The DRIM detection can be used for simple, rapid and sensitive quantification of protein biomarkers for in vitro diagnostics both in laboratory and near-patient conditions, for food analysis, environmental monitoring, security, and safety applications.

Electronic Quantification of Protein Biomarkers Based on Bead Aggregate Sizing

We demonstrate a novel protein sensing method using detection of bead aggregates of different sizes in a micro-fluidic impedance cytometer. We utilize micro-fabricated coplanar electrodes at the base of our micro-channel to measure the impedance. When the single beads or bead aggregates pass through the electrodes, a difference in impedance change is detected. We measure these signals using a lock-in amplifier. Due to the difference in impedance change for unbound beads compared with protein-bound bead aggregates, we can differentiate the two types from each other electronically. We demonstrate proof-of-concept using streptavidin and biotin coated beads.

PicoMolar level detection of protein biomarkers based on electronic sizing of bead aggregates: theoretical and experimental considerations.

We demonstrate a novel method for electronically detecting and quantifying protein biomarkers using microfluidic impedance cytometry. Our biosensor, which consists of gold electrodes micro-fabricated in a microchannel, detects the differences between bead aggregates of varying sizes in a micro-pore sandwiched between two micro channels. We perform a sandwich immunoassay, where the complementary antibody pairs are immobilized on two different bead types, and the presence of antigen results in bead aggregation, the amount of which depends on antigen quantity. When single beads or bead aggregates pass through the impedance sensor, differences in impedance change are detected. In this manuscript, we perform a comprehensive theoretical study on the limits imposed on sensitivity of this technique due to electronic noise and also mass transfer and reaction limits. We also experimentally characterize the performance of this technique by validating the technique on an IgG detection assay. A detection limit at the picoMolar level is demonstrated, thus comparable in sensitivity to a sandwich ELISA.

Proteomic profiling of healthy and diseased hybrid soft corals Sinularia maxima × S. polydactyla.

Emerging diseases of marine invertebrates have been implicated as one of the major causes of the continuing decline in coral reefs worldwide. To date, most of the focus on marine diseases has been aimed at hard (scleractinian) corals, which are the main reef builders worldwide. However, soft (alcyonacean) corals are also essential components of tropical reefs, representing food, habitat and the 'glue' that consolidates reefs, and they are subject to the same stressors as hard corals. Sinularia maxima and S. polydactyla are the dominant soft corals on the shallow reefs of Guam, where they hybridize. In addition to both parent species, the hybrid soft coral population in Guam is particularly affected by Sinularia tissue loss disease. Using label-free shotgun proteomics, we identified differences in protein expression between healthy and diseased colonies of the hybrid S. maxima × S. polydactyla. This study provided qualitative and quantitative data on specific proteins that were differentially expressed under the stress of disease. In particular, metabolic proteins were down-regulated, whereas proteins related to stress and to symbiont photosynthesis were up-regulated in the diseased soft corals. These results indicate that soft corals are responding to pathogenesis at the level of the proteome, and that this label-free approach can be used to identify and quantify protein biomarkers of sub-lethal stress in studies of marine disease.

A Colorimetric Enzyme-Linked Immunosorbent Assay (ELISA) Detection Platform for a Point-of-Care Dengue Detection System on a Lab-on-Compact-Disc.

The enzyme-linked Immunosorbent Assay (ELISA) is the gold standard clinical diagnostic tool for the detection and quantification of protein biomarkers. However, conventional ELISA tests have drawbacks in their requirement of time, expensive equipment and expertise for operation. Hence, for the purpose of rapid, high throughput screening and point-of-care diagnosis, researchers are miniaturizing sandwich ELISA procedures on Lab-on-a-Chip and Lab-on-Compact Disc (LOCD) platforms. This paper presents a novel integrated device to detect and interpret the ELISA test results on a LOCD platform. The system applies absorption spectrophotometry to measure the absorbance (optical density) of the sample using a monochromatic light source and optical sensor. The device performs automated analysis of the results and presents absorbance values and diagnostic test results via a graphical display or via Bluetooth to a smartphone platform which also acts as controller of the device. The efficacy of the device was evaluated by performing dengue antibody IgG ELISA on 64 hospitalized patients suspected of dengue. The results demonstrate high accuracy of the device, with 95% sensitivity and 100% specificity in detection when compared with gold standard commercial ELISA microplate readers. This sensor platform represents a significant step towards establishing ELISA as a rapid, inexpensive and automatic testing method for the purpose of point-of-care-testing (POCT) in resource-limited settings.

Quantifying protein measurands by peptide measurements: where do errors arise?

Clinically actionable quantification of protein biomarkers by mass spectrometry (MS) requires analytical performance in concordance with quality specifications for diagnostic tests. Laboratory-developed tests should, therefore, be validated in accordance with EN ISO 15189:2012 guidelines for medical laboratories to demonstrate competence and traceability along the entire workflow, including the selected standardization strategy and the phases before, during, and after proteolysis. In this study, bias and imprecision of a previously developed MS method for quantification of serum apolipoproteins A-I (Apo A-I) and B (Apo B) were thoroughly validated according to Clinical and Laboratory Standards Institute (CLSI) guidelines EP15-A2 and EP09-A3, using 100 patient sera and either stable-isotope labeled (SIL) peptides or SIL-Apo A-I as internal standard. The systematic overview of error components assigned sample preparation before the first 4 h of proteolysis as major source (∼85%) of within-sample imprecision without external calibration. No improvement in imprecision was observed with the use of SIL-Apo A-I instead of SIL-peptides. On the contrary, when the use of SIL-Apo A-I was combined with external calibration, imprecision improved significantly (from ∼9% to ∼6%) as a result of the normalization for matrix effects on linearity. A between-sample validation of bias in 100 patient sera further supported the presence of matrix effects on digestion completeness and additionally demonstrated specimen-specific biases associated with modified peptide sequences or alterations in protease activity. In conclusion, the presented overview of bias and imprecision components contributes to a better understanding of the sources of errors in MS-based protein quantification and provides valuable recommendations to assess and control analytical quality in concordance with the requirements for clinical use.