Human Blood Proteins Research Articles

Development of a portable and sensitive blood serum test system using LED-based absorption photometry and pump-free microfluidic technology

We reported a novel integrated portable human blood system based on absorption photometry and microfluidic technology. A capillary pump for fluid control was embedded in microfluidic chip instead of heavy syringe pump. The analytes could automatically flow, mix and react in chip. It achieves long-term stability of hydrophilic microfluidic channel surface for rapid and sensitive detection. Herein, we detected three analytes from human blood, including triglyceride (TGL), albumin (ALB) and total protein (TP) which are very important for health examination. All components of this system including light-emitting diode (LED) light source, microfluidic chip, photodiode detector (PD), electronic circuit, Organic LED screen and memory card were fully integrated into a portable instrument with small size (11.2 × 7.2 × 6.1 cm). The pair of LED and PD was used for measuring light absorbance of biochemical assay. The limit of detections (LODs) for TGL, ALB and TP were estimated to be below 0.1 mmol/L, 4 g/L and 10 g/L respectively, which is significantly below the clinical cut-off value. Passing − Boblok regression analyses were used to achieve the evaluation for this system. It showed good accuracy and feasibility of our system and was expected to be useful as a potential clinical tool for human blood serum test.

Interfacial Conformation of Hydrophilic Polyphosphoesters Affects Blood Protein Adsorption.

Synthetic polymers are commonly used as protein repelling materials for a variety of biomedical applications. Despite their widespread use, the fundamental mechanism underlying protein repellence is often elusive. Such insights are essential for improving existing and developing new materials. Here, we investigate how subtle differences in the chemistry of hydrophilic polyphosphoesters influence the adsorption of the human blood proteins serum albumin and fibrinogen. Using thermodynamic measurements, surface-specific vibrational spectroscopy, and Brewster angle microscopy, we investigate protein adsorption, hydration, and steric repulsion properties of the polyphosphoester polymers. Whereas both surface hydration and polymer conformation of the polymers vary substantially as a consequence of the chemical differences in the polymer structure, the protein repellency ability of these hydrophilic materials appears to be dominated by steric repulsion.

Metal affinity-carboxymethyl cellulose functionalized magnetic graphene composite for highly selective isolation of histidine-rich proteins

A metal affinity-carboxymethyl cellulose functionalized magnetic graphene, namely MGCI-Cu composite, was prepared by successive modifications of graphene oxide nanosheets with magnetic nanoparticles, carboxymethyl cellulose (CMC), iminodiacetic acid (IDA) and then chelated with copper ions. The successful modifications of the graphene surface were demonstrated by various characterizations, and a high density of 6.17 μmol m−2 for metal affinity groups was obtained. The composite exhibited high adsorption selectivity toward histidine-rich proteins. The adsorption was governed by strong metal affinity binding force between hisitidine residues of proteins and immobilized Cu2+ ions of MGCI-Cu composite. In particular, highly selective isolation of hemoglobin (Hb) was achieved in 0.2 mol L−1 phosphate buffer at pH 8. The adsorption capacity of Hb significantly increased to 769 mg g−1 in comparison to that of 435 mg g−1 on metal affinity modified magnetic graphene composite (MGI-Cu) without CMC modification. The adsorbed Hb molecules were recovered with a carbonate buffer (0.2 mol L−1 pH 10) containing 0.5 mol L−1 imidazole. MGCI-Cu composite displayed favorable reusability for at least four times after regeneration of the composite by edetic acid (EDTA) and Cu2+ solution. The practical applications demonstrated that MGCI-Cu composite could highly selectively isolate Hb from human whole blood and polyhistidine-tagged recombinant protein from Escherichia coli (E. coli) lysate.

Enhanced Sensitivity in Nanopore Sensing of Cancer Biomarkers in Human Blood via Click Chemistry.

Cancer biomarkers are expected to be indicative of the occurrence of certain cancer diseases before the tumors form and metastasize. However, many biomarkers can only be acquired in extremely low concentrations, which are often beyond the limit of detection (LOD) of current instruments and technologies. A practical strategy for nanopore sensing of cancer biomarkers in raw human blood down to the femtomolar level is developed here. This strategy first converts the detection of cancer biomarkers to the quantification of copper ions by conducting a sandwich assay involving copper oxide nanoparticles. The released Cu2+ is then taken to catalyze the "click" reaction which ligates a host-guest modified DNA probe. Finally, this DNA probe is subjected to single-channel recordings to afford the translocation events that can be used to derive the concentrations of the original biomarkers. Due to the amplification effects of nanoparticle loadings and the "click" reaction, the LOD of this strategy can be as low as the subfemtomolar level. Further, the acid treatment step could effectively eliminate the interferences from plasma proteins in raw human blood and make the strategy highly suitable for the detection of cancer biomarkers in clinical samples.

Human blood proteins and correlations with biochemical parameters after long duration space flights

Human blood proteins and correlations with biochemical parameters after long duration space flights

Human blood proteins after long duration space flights

Human blood proteins after long duration space flights

Nontoxic silver nanocluster-induced folding, fibrillation, and aggregation of blood plasma proteins

In recent years, concerns have been raised considering the potential risks of nanocluster (NC) for the environment and human health. Since the blood circulation system is probably the first entry route of NC into the human body, adsorption of blood proteins on NC may change cellular responses, including cellular uptake efficiency, bio distribution patterns, and nanotoxicity profiles, besides other biological effects. Therefore, the interaction of NCs with proteins and the cellular implications can be therapeutically of great importance. Adsorption of human blood proteins on NCs has been methodically investigated. In the present study, the first analysis of fibrillation was conducted between MBI-AgNCs and human serum (a complex biofluid). AgNCs were prepared by coating with 2-mercaptobenzimidazole. Then, interactions with human blood proteins, such as immunoglobulin, albumin, and insulin were investigated using various experimental approaches. Upon protein association, the fluorescence of proteins significantly decreased, accompanied by a blue shift in the AgNCs-human serum albumin (HSA) system and a red shift in the AgNCs-insulin/γ-globulin. Concomitantly, circular dichroism spectroscopy and atomic force microscopy were employed to investigate the effects of protein binding to NCs. We found that AgNCs induced γ-globulin aggregation. HSA at the AgNC surface was partially unfolded and could promote protein self-assembly into amyloid fibrils, while the surface morphology remained unchanged after insulin incubation. The atomic force microscopy (AFM) data and the ThT and CR analysis of the proteins, as well as circular dichroism (CD) and fluorescence findings, support the use of AgNCs as an indicator for monitoring the progress of HSA fibrillogenesis. Additionally, cytotoxicity assays were used to ensure the biocompatibility of nanoparticles within the applicable limits.

Real-Time Binding Monitoring between Human Blood Proteins and Heavy Metal Ions in Nanoporous Anodic Alumina Photonic Crystals.

This study reports on the real-time binding assessment between heavy metal ions and blood proteins immobilized onto nanoporous anodic alumina photonic crystals (NAA-PCs) by reflectometric interference spectroscopy (RIfS). The surface of NAA-PCs is chemically functionalized with γ-globulin (GG), transferrin (TFN), and serum albumin (HSA), the major proteins present in human blood plasma. Protein-modified NAA-PC platforms are exposed to analytical solutions of mercury ions of different concentrations. Dynamic changes in the effective optical thickness of protein-modified NAA-PCs in response to heavy metal ions are assessed in real time to evaluate the binding kinetics, affinity, and mechanism. Protein molecules undergo conformational changes upon exposure to mercury ions, with HSA exhibiting the strongest affinity. The combination of protein-modified NAA-PCs with RIfS allows real-time monitoring of protein-heavy metal ions interactions under dynamic flow conditions. This system is capable of detecting dynamic conformational changes in these proteins upon exposure to heavy metal ions. Our results provide new insights into these binding events, which could enable new methodologies to study the toxicity of heavy metal ions and other biomolecular interactions.

Interaction of Ochratoxin A and Its Thermal Degradation Product 2'R-Ochratoxin A with Human Serum Albumin.

Ochratoxin A (OTA) is a toxic secondary metabolite produced by several fungal species of the genus Penicillium and Aspergillus. 2′R-Ochratoxin A (2′R-OTA) is a thermal isomerization product of OTA formed during food processing at high temperatures. Both compounds are detectable in human blood in concentrations between 0.02 and 0.41 µg/L with 2′R-OTA being only detectable in the blood of coffee drinkers. Humans have approximately a fifty-fold higher exposure through food consumption to OTA than to 2′R-OTA. In human blood, however, the differences between the concentrations of the two compounds is, on average, only a factor of two. To understand these unexpectedly high 2′R-OTA concentrations found in human blood, the affinity of this compound to the most abundant protein in human blood the human serum albumin (HSA) was studied and compared to that of OTA, which has a well-known high binding affinity. Using fluorescence spectroscopy, equilibrium dialysis, circular dichroism (CD), high performance affinity chromatography (HPAC), and molecular modelling experiments, the affinities of OTA and 2′R-OTA to HSA were determined and compared with each other. For the affinity of HSA towards OTA, a logK of 7.0–7.6 was calculated, while for its thermally produced isomer 2′R-OTA, a lower, but still high, logK of 6.2–6.4 was determined. The data of all experiments showed consistently that OTA has a higher affinity to HSA than 2′R-OTA. Thus, differences in the affinity to HSA cannot explain the relatively high levels of 2′R-OTA found in human blood samples.

Direct label-free protein detection in high ionic strength solution and human plasma using dual-gate nanoribbon-based ion-sensitive field-effect transistor biosensor

We report on direct label-free protein detection in high ionic strength solution and human plasma by a dual-gate nanoribbon-based ion-sensitive field-effect transistor (NR-ISFET) biosensor system with excellent sensitivity and specificity in both solution-gate (SG) and dual-gate (DG) modes. Compared with previously reported results, the NR-ISFET biosensor enables selective prostate specific antigen (PSA) detection based on antibody-antigen binding in broader detection range with lower LOD. For the first time, real-time specific detection of PSA of 10 pM to 1 μM in 100 mM phosphate buffer (PB) was demonstrated by conductance measurements using the polyethylene glycol (PEG)-modified NR-ISFET biosensors in DG mode with the back-gate bias (VBG) of 20 V. Due to larger maximum transconductance value resulting from the modulation of NR-ISFET channel by the back gate in DG mode, the detection range can be broadened with larger linear detection region (100 pM to 100 nM) and lower limit of detection (LOD, 10 pM) as compared to those in SG mode. Moreover, the influence of different back-gate bias from VBG = 5 V to VBG = 25 V on the biosensor performance has been investigated. Furthermore, direct PSA detection of 100 pM to 1 μM in human plasma was demonstrated by using the PEG-modified NR-ISFET in DG mode, enabling direct detection of protein in human blood for clinical applications since the LOD of 100 pM PSA can meet the clinical requirements.

A validated, sensitive electrophoretic method for the detection of activin receptor type II-Fc fusion proteins in human blood.

New therapeutic proteins that trap circulating members of the transforming growth factor (TGF) beta superfamily (activins and growth differentiation factors) show promising effects on erythropoiesis and muscular growth. They are dimeric recombinant fusion proteins composed of the extracellular domain of a human activin receptor (ActRIIA or IIB) linked to the Fc part of human IgG1. Sotatercept (ActRIIA-Fc) and Luspatercept (a modified ActRIIB-Fc) in particular are now in phase 2/3 of clinical trials against anemia and included in the prohibited list established by the World Anti-Doping Agency. To prevent a potential misuse by athletes in the near future, a robust and sensitive method of detection is needed. We validated an approach adapted from an electrophoretic method used for the detection of recombinant erythropoietins that allowed detection of various ActRIIA-Fc and ActRIIB-Fc proteins, including variants produced in different cell types, after a single immuno-extraction step. After separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), an initial testing procedure performed by single-blotting can indicate the presence of an ActRII-Fc (indifferently type IIA or IIB). A confirmation performed by double-blotting using different antibodies for detection allows a more precise identification of the type of ActRII-Fc (IIA, IIB). Starting from a few hundred microliters of serum or plasma, this method is specific, sensitive, and easy to perform. It could easily be adopted by anti-doping laboratories.

Fragmentation patterns involving ammonium adduct fragment ions: A comparison of the determination of metaldehyde in human blood by HPLC-QqQ-MS/MS and UHPLC-Q-TOF-MS

This paper presents a rapid, sensitive and precise method for the determination of metaldehyde in human blood, using ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry and high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry. Separation was performed with a Poroshell 120 EC-C18 column; 2.7 μm atrazine‑d5 (IS) and 200 mg NaCl were added to the blood sample. Proteins in human blood were precipitated using acetonitrile; the supernatant was then analyzed with the UHPLC-Q-TOF-MS or HPLC-QqQ-MS/MS system. The results of selectivity, linearity, accuracy, precision, limits of quantification, recovery, and matrix effects were sufficient to enable the measurement of metaldehyde in human blood samples. In addition, we proposed a fragmentation pathway involving ammonium adduct fragment ions for metaldehyde.

Preservation Method and Phosphate Buffered Saline Washing Affect the Acute Myeloid Leukemia Proteome.

Acute myeloid leukemia (AML) primary cells can be isolated from peripheral blood, suspended with media containing bovine serum and cryoprotectant, and stored in liquid nitrogen before being processed for proteomic analysis by mass spectrometry (MS). The presence of bovine serum and human blood proteins in AML samples can hamper the identifications of proteins, and thereby reduce the proteome coverage of the study. Herein, we have established the effect of phosphate buffered saline (PBS) washing on AML patient samples stored in media. Although PBS washes effectively removed serum and blood contaminants, the saline wash resulted in cell burst and remarkable protein material loss. We also compared different methods to preserve the AML proteome from THP-1 and Molm-13 cell lines before MS analysis: (1) stored in media containing bovine serum and dimethyl sulfoxide (DMSO); (2) stored as dried cell pellets; and (3) stored as cell lysates in 4% sodium dodecyl sulfate (SDS). MS analysis of differently preserved AML cell samples shows that preservation with DMSO produce a high number of fragile cells that will burst during freezing and thawing. Our studies encourage the use of alternative preservation methods for future MS analysis of the AML proteome.

New HPLC method for determination of cystatin C biomarker in human blood

A rapid and sensitive method for determination of cystatin C (CC) protein in human blood by means of high performance liquid chromatography (HPLC) was developed and validated. Estimated glomerular filtration rate (eGFR) has been calculated for the patients with chronic kidney disease (CKD). Ion pair liquid chromatography technique was utilized to separate CC along with UV detection. Calibration curve with excellent correlation ( r 2 = 0.99) over the range from 0.75 to 20.50 mg/L of CC was accomplished. Limits of detection and quantify-cation were 0.375 and 0.75 mg/L of CC, respectively. The recoveries were in the range of 93.6-102.3%. Intra-assay and inter-assay variabilities were 8.2 and 6.14%, respectively.

"Bottom-up" in situ proteomic differentiation of human and non-human haemoglobins for forensic purposes by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry.

The detection and identification of human blood on crime-related items are of particular relevance to many investigations because shed blood can provide evidence of violent contact between individuals. However, for any detection and identification technique, specificity is a critical performance characteristic to assess; that is, whether the technique has the capability to differentiate between human blood (which usually is of relevance to a criminal investigation) and non-human blood (which usually would not be associated with a crime but may be detected incidentally). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) approaches using "top-down" (detection of intact proteins) and "bottom-up" (detection of tryptic peptide markers) were used to detect and identify haemoglobin in blood from humans and from a range of Australian native mammals; the technique could be carried out directly on blood stains without the need to extract proteins (i.e., in situ measurement). Imaging of haemoglobin was achieved in bloodied fingermarks, including those that had been enhanced using two "industry standard" fingermark enhancement processes. Differentiation of intact haemoglobin proteins in human and non-human blood using "top-down" MALDI-TOF-MS was difficult. However, in situ "bottom-up" approaches using tandem mass spectrometry (MS/MS) and de novo sequencing of tryptic digest peptides allowed unambiguous differentiation. Imaging mass spectrometry of human haemoglobin, even when it was mixed with animal blood, was achieved in bloodied fingermarks that had been enhanced using two common processes (staining with Amido Black or dusted with magnetic powder) and "lifted" using adhesive tape. The MALDI-TOF-MS-based in situ "bottom-up" proteomic methodology described here shows great promise for the detection of human blood and even imaging of blood in bloodied fingermarks. The approach is sensitive, can differentiate between human blood and that from many animals (including several Australian native animals), and can be implemented after traditional crime scene fingermark enhancement techniques have been carried out.

Analysis of Four Circulating Complexes of Insulin-Like Growth Factor Binding Proteins in Human Blood during Aging.

The primary role of insulin-like growth factor binding proteins (IGFBPs) is to regulate availability of IGFs for interacting with receptors, but IGFBPs perform IGF-independent actions as well. The availability and activity of IGFBPs in the circulation is influenced primarily by their concentration and structural modifications, but possibly also by interaction with major plasma proteins such as transferrin, alpha-2-macroglobulin (α2M), and fibrinogen. Four types of circulating IGFBP complexes were examined in this study by immuno- and ligand-binding assays in adults of different age. The amounts of IGFBP-3/transferrin and IGFBP-1/fibrinogen complexes were similar in middle- and old-aged persons, whereas the amounts of IGFBP-1 (or -2)/α2M monomer complexes were lower in the old-aged group and negatively correlated with total IGFBP-1 (or -2) amounts in blood. In contrast to IGFBP-1, IGFBP-2 was present in significantly greater quantities in complexes with α2M dimer than α2M monomer in older individuals. IGFBP complexes did not bind 125I-labeled IGF-I in amounts detectable by ligand blotting. According to the results of this study, the quantities of IGFBP-1 and IGFBP-2, which interact with α2M, are age-dependent and, in the case of complexes with α2M monomer, they are negatively correlated with the total circulating levels of these two IGFBPs.

Rapid capillary electrophoresis approach for the quantification of ewe milk adulteration with cow milk

The substitution of ewe milk with more economic cow milk is a common fraud. Here we present a capillary electrophoresis method for the quantification of ewe milk in ovine/bovine milk mixtures, which allows for the rapid and inexpensive recognition of ewe milk adulteration with cow milk. We utilized a routine CE method for human blood and urine proteins analysis, which fulfilled the separation of skimmed milk proteins in alkaline buffer. Under this condition, ovine and bovine milk exhibited a recognizable and distinct CE protein profiles, with a specific ewe peak showing a reproducible migration zone in ovine/bovine mixtures. Based on ewe specific CE peak, we developed a method for ewe milk quantification in ovine/bovine skimmed milk mixtures, which showed good linearity, precision and accuracy, and a minimum amount of detectable fraudulent cow milk equal to 5%.

Deep-Dive Targeted Quantification for Ultrasensitive Analysis of Proteins in Nondepleted Human Blood Plasma/Serum and Tissues.

Mass spectrometry-based targeted proteomics (e.g., selected reaction monitoring, SRM) is emerging as an attractive alternative to immunoassays for protein quantification. Recently we have made significant progress in SRM sensitivity for enabling quantification of low nanograms per milliliter to sub-naograms per milliliter level proteins in nondepleted human blood plasma/serum without affinity enrichment. However, precise quantification of extremely low abundance proteins (e.g., ≤ 100 pg/mL in blood plasma/serum) using targeted proteomics approaches still remains challenging, especially for these samples without available antibodies for enrichment. To address this need, we have developed an antibody-independent deep-dive SRM (DD-SRM) approach that capitalizes on multidimensional high-resolution reversed-phase liquid chromatography (LC) separation for target peptide separation and enrichment combined with precise selection of target peptide fractions of interest, significantly improving SRM sensitivity by ∼5 orders of magnitude when compared to conventional LC-SRM. Application of DD-SRM to human serum and tissue provides precise quantification of endogenous proteins at the ∼10 pg/mL level in nondepleted serum and at <10 copies per cell level in tissue. Thus, DD-SRM holds great promise for precisely measuring extremely low abundance proteins or protein modifications, especially when high-quality antibodies are not available.

Direct analysis of site-specific N-glycopeptides of serological proteins in dried blood spot samples.

Dried blood spot (DBS) samples have a number of advantages, especially with respect to ease of collection, transportation, and storage and to reduce biohazard risk. N-glycosylation is a major post-translational modification of proteins in human blood that is related to a variety of biological functions, including metastasis, cell-cell interactions, inflammation, and immunization. Here, we directly analyzed tryptic N-glycopeptides from glycoproteins in DBS samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS) without centrifugation of blood samples, depletion of major proteins, desalting of tryptic peptides, and enrichment of N-glycopeptides. Using this simple method, we identified a total of 41 site-specific N-glycopeptides from 16 glycoproteins in the DBS samples, from immunoglobulin gamma 1 (IgG-1, 10mg/mL) down to complement component C7 (50μg/mL). Of these, 32 N-glycopeptides from 14 glycoproteins were consistently quantified over 180days stored at room temperature. The major abundant glycoproteins in the DBS samples were IgG-1 and IgG-2, which contain nine asialo-fucosylated complex types of 16 different N-glycopeptide isoforms. Sialo-non-fucosylated complex types were primarily detected in the other glycoproteins such as alpha-1-acid glycoprotein 1, 2, alpha-1-antitypsin, alpha-2-macroglobulin, haptoglobin, hemopexin, Ig alpha 1, 2 chain C region, kininogen-1, prothrombin, and serotransferrin. We first report the characterization of site-specific N-glycoproteins in DBS samples by LC-MS/MS with minimal sample preparation.

Determination of total protein in human blood by laser

Determination of total protein in human blood by laser