Proteins In Biofluids Research Articles

Playing pin-the-tail-on-the-protein in extracellular vesicle (EV) proteomics.

Extracellular vesicles (EVs) are anucleate particles enclosed by a lipid bilayer that are released from cells via exocytosis or direct budding from the plasma membrane. They contain an array of important molecular cargo such as proteins, nucleic acids, and lipids, and can transfer these cargoes to recipient cells as a means of intercellular communication. One of the overarching paradigms in the field of EV research is that EV cargo should reflect the biological state of the cell of origin. The true relationship or extent of this correlation is confounded by many factors, including the numerous ways one can isolate or enrich EVs, overlap in the biophysical properties of different classes of EVs, and analytical limitations. This presents a challenge to research aimed at detecting low-abundant EV-encapsulated nucleic acids or proteins in biofluids for biomarker research and underpins technical obstacles in the confident assessment of the proteomic landscape of EVs that may be affected by sample-type specific or disease-associated proteoforms. Improving our understanding of EV biogenesis, cargo loading, and developments in top-down proteomics may guide us towards advanced approaches for selective EV and molecular cargo enrichment, which could aid EV diagnostics and therapeutics research.

A biochemical feedback signal for hypothermia treatment for neonatal hypoxic-ischemic encephalopathy: focusing on central nervous system proteins in biofluids.

Hypothermia has been widely used to treat moderate to severe neonatal hypoxic-ischemic encephalopathy (HIE), yet evaluating the effects of hypothermia relies on clinical neurology, neuroimaging, amplitude-integrated electroencephalography, and follow-up data on patient outcomes. Biomarkers of brain injury have been considered for estimating the effects of hypothermia. Proteins specific to the central nervous system (CNS) are components of nervous tissue, and once the CNS is damaged, these proteins are released into biofluids (cerebrospinal fluid, blood, urine, tears, saliva), and they can be used as markers of brain damage. Clinical reports have shown that CNS-specific marker proteins (CNSPs) were early expressed in biofluids after brain damage and formed unique biochemical profiles. As a result, these markers may serve as an indicator for screening brain injury in infants, monitoring disease progression, identifying damage region of brain, and assessing the efficacy of neuroprotective measures. In clinical work, we have found that there are few reports on using CNSPs as biological signals in hypothermia for neonatal HIE. The aim of this article is to review the classification, origin, biochemical composition, and physiological function of CNSPs with changes in their expression levels after hypothermia for neonatal HIE. Hopefully, this review will improve the awareness of CNSPs among pediatricians, and encourage future studies exploring the mechanisms behind the effects of hypothermia on these CNSPs, in order to reduce the adverse outcome of neonatal HIE.

Proteomics of prostate cancer serum and plasma using low and high throughput approaches

Despite progress, MS-based proteomics in biofluids, especially blood, faces challenges such as dynamic range and throughput limitations in biomarker and disease studies. In this work, we used cutting-edge proteomics technologies to construct label-based and label-free workflows, capable of quantifying approximately 2,000 proteins in biofluids. With 70µL of blood and a single depletion strategy, we conducted an analysis of a homogenous cohort (n = 32), comparing medium-grade prostate cancer patients (Gleason score: 7(3 + 4); TNM stage: T2cN0M0, stage IIB) to healthy donors. The results revealed dozens of differentially expressed proteins in both plasma and serum. We identified the upregulation of Prostate Specific Antigen (PSA), a well-known biomarker for prostate cancer, in the serum of cancer cohort. Further bioinformatics analysis highlighted noteworthy proteins which appear to be differentially secreted into the bloodstream, making them good candidates for further exploration.

Quantitative analysis of fucosylated glycoproteins by immobilized lectin-affinity fluorescent labeling.

Human biofluids are often used to discover disease-specific glycosylation, since abnormal changes in protein glycosylation can discern physiopathological states. Highly glycosylated proteins in biofluids make it possible to identify disease signatures. Glycoproteomic studies on saliva glycoproteins showed that fucosylation was significantly increased during tumorigenesis and that glycoproteins became hyperfucosylated in lung metastases, and tumor stage is associated with fucosylation. Quantification of salivary fucosylation can be achieved by mass spectrometric analysis of fucosylated glycoproteins or fucosylated glycans; however, the use of mass spectrometry is non-trivial for clinical practice. Here, we developed a high-throughput quantitative method, lectin-affinity fluorescent labeling quantification (LAFLQ), to quantify fucosylated glycoproteins without relying on mass spectrometry. Lectins with a specific affinity for fucoses are immobilized on the resin and effectively capture fluorescently labeled fucosylated glycoproteins, which are further quantitatively characterized by fluorescence detection in a 96-well plate. Our results demonstrated that serum IgG can be accurately quantified by lectin and fluorescence detection. Quantification in saliva showed significantly higher fucosylation in lung cancer patients compared to healthy controls or other non-cancer diseases, suggesting that this method has the potential to quantify stage-related fucosylation in lung cancer saliva.

Two different protein corona formation modes on Soluplus® nanomicelles

Soluplus® nanomicelles have been widely reported in biomedical field for their excellent drug loading capacity and solubility enhancement ability. However, when administrated in vivo, the protein corona will be formed on Soluplus® nanomicelles, significantly affecting their drug delivery performance. Up to now, few studies examined the protein corona formation process and its impact factors of Soluplus® nanomicelles. The multiple proteins in biofluids may form protein corona in different modes due to their diversified properties. In this study, Bovine serum albumin (BSA), Lysozyme (Lyso) and Bovine hemoglobin (BHb) were chosen as model proteins to investigate the protein corona formation process of Soluplus® nanomicelles. By analyzing the polarity of the protein amino acid residues distributing microenvironments, the results showed that there were two different protein corona formation modes, i.e., surface adsorption and insertion, which were determined by the hydrophilicity of proteins. The hydrophobic BHb followed the insertion mode while hydrophilic BSA and Lyso followed the surface adsorption mode. Ultimately, upon protein corona formation, the size and surface chemistry of nanomicelles was significantly affected. We believe this study will provide a new research paradigm to the design and application of Soluplus® nanomicelles.

Methods for Enhanced Fluorescence Detection of Proteins by using Entrapped Gold Nanoparticles in Membranes.

Measuring protein levels from biofluids can provide important insight into human health and disease during various physiological and pathological conditions. In many situations, sensitive methods are required for protein quantification because at the early stages of many diseases, proteins in biofluids are present at very low concentrations. Here, a new and simple method is presented in the form of Basic and Alternative Protocols for an immunoassay performed on a nitrocellulose membrane, followed by the addition of gold nanoparticles prior to measuring fluorescence with a microscope. The assay protocol was optimized to achieve 3D metal-enhanced fluorescence (MEF) with increased antibody-binding capacity and enhanced fluorescence signals, improving assay sensitivity. Using different concentrations of spiked fluorescently labeled IgGs in pooled normal human plasma, a lower detection limit of 29 ng/ml was achieved. This limit of detection was found to be a thousand-fold lower than the conventional 2D assay and one order of magnitude lower than when the assay was performed on a 3D membrane without MEF. This method provides an easy way to improve immunoassay sensitivity, and it can be simply transferred to other labs. It also can extend to fluorescence detection of other analytes beyond proteins. © 2022 Wiley Periodicals LLC. Basic Protocol: Assay in nitrocellulose membrane with entrapped AuNPs using commercially available AuNPs Alternative Protocol: Assay in nitrocellulose membrane with entrapped AuNPs using lab-made AuNPs.

Near‐Infrared Multilayer MoS2 Photoconductivity‐Enabled Ultrasensitive Homogeneous Plasmonic Colorimetric Biosensing (Adv. Mater. Interfaces 24/2021)

Plasmonic Colorimetric Biosensing of Proteins in Biofluids In article number 2101291, Younggeun Park, Xiaogan Liang, Katsuo Kurabayashi, and co-workers demonstrate rapid, “mix-and-read” detection of cancer marker proteins at sub-femtomolar concentrations in unprocessed whole blood using a multilayered molybdenum disulfide photoconductive channel and plasmonic nanoparticles. Minimized carrier scattering within the photoconductive channel allows for detecting subtle near-infrared light transmission shifts accompanying antigen-induced plasmonic nanoparticle aggregation in blood.

Near‐Infrared Multilayer MoS2 Photoconductivity‐Enabled Ultrasensitive Homogeneous Plasmonic Colorimetric Biosensing

AbstractThe ability to detect low‐abundance proteins in human body fluids plays a critical role in proteomic research to achieve a comprehensive understanding of protein functions and early‐stage disease diagnosis to reduce mortality rates. Ultrasensitive (sub‐fM), rapid, simple “mix‐and‐read” plasmonic colorimetric biosensing of large‐size (≈180 kDa) proteins in biofluids using an ultralow‐noise multilayer molybdenum disulfide (MoS2) photoconducting channel is reported here. With its out‐of‐plane structure optimized to minimize carrier scattering, the multilayer MoS2 channel operated under near‐infrared illumination enables the detection of a subtle plasmonic extinction shift caused by antigen‐induced nanoprobe aggregation. The demonstrated biosensing strategy allows quantifying carcinoembryonic antigen in unprocessed whole blood with a dynamic range of 106, a sample‐to‐answer time of 10 min, and a limit of detection of 0.1–3 pg mL−1, which is ≈100‐fold more sensitive than the clinical‐standard enzyme‐linked immunosorbent assays. The biosensing methodology can be broadly used to realize timely personalized diagnostics and physiological monitoring of diseases in point‐of‐care settings.

Pushing the detection limits: strategies towards highly sensitive optical-based protein detection.

Proteins are one of the main constituents of living cells. Studying the quantities of proteins under physiological and pathological conditions can give valuable insights into health status, since proteins are the functional molecules of life. To be able to detect and quantify low-abundance proteins in biofluids for applications such as early disease diagnostics, sensitive analytical techniques are desired. An example of this application is using proteins as biomarkers for detecting cancer or neurological diseases, which can provide early, lifesaving diagnoses. However, conventional methods for protein detection such as ELISA, mass spectrometry, and western blotting cannot offer enough sensitivity for certain applications. Recent advances in optical-based micro- and nano-biosensors have demonstrated promising results to detect proteins at low quantities down to the single-molecule level, shining lights on their capacities for ultrasensitive disease diagnosis and rare protein detection. However, to date, there is a lack of review articles synthesizing and comparing various optical micro- and nano-sensing methods of enhancing the limits of detections of the antibody-based protein assays. The purpose of this article is to critically review different strategies of improving assay sensitivity using miniaturized biosensors, such as assay miniaturization, improving antibody binding capacity, sample purification, and signal amplification. The pros and cons of different methods are compared, and the future perspectives of this research field are discussed.

Aptamer-based immunoaffinity LC-MS using an ultra-short column for rapid attomole level quantitation of intact mAbs

Quantification of proteins in biofluids has largely involved either traditional ligand binding assays or “bottom-up” mass spectrometry. Recently, top-down mass spectrometry using reversed-phase liquid chromatography (RPLC) paired with high-resolution mass spectrometry (HRMS) has emerged as a promising technique, due to the potential of better identification of post-translational modifications (PTMs), lack of downstream interferences, and less time-consuming sample preparation and analysis times. However, it can be difficult with this approach to robustly obtain high-fidelity MS data, especially when pushing for low limits of detection. To address these issues, we developed a chromatographic device with an optimized form factor and stationary phase to improve protein recovery, while reducing run times. We have observed that by using this device, it is possible to achieve attomole quantitation of mAbs without the addition of carrier proteins and with over three-fold higher throughput than columns employed in previous studies. Moreover, we have devised a novel affinity capture method, based on repurposing a unique aptamer ligand that can give 93% recovery of mAb using only a 2 h incubation. When hyphenated together, these two technologies greatly improve the ability to analyze proteins in complex matrices.

Abstract 2847: Multiplex bead array approach for quantitative profiling of 95 cancer-immunity biomarkers in breast cancer

Abstract Immune regulators, including immune checkpoint proteins and cytokines/chemokines, have an emerging role as both disease/treatment biomarkers and targeted agents in cancer therapies. To contribute to the understanding of the roles of these immune regulators in cancer, we have developed 3 new Luminex-based multiplex immunoassay panels, a 48-plex human cytokine/chemokine panel, a 17-plex human immune checkpoint protein panel, and a 30-plex human immune checkpoint protein panel, to simultaneously quantitate the expression levels of 95 key immune regulator proteins in biofluids or cell/tissue homogenates. Here we report the quantitative profiles of these 95 immune regulators in 4 types of breast cancer samples: cancer versus healthy control serum samples, the lysates from breast cancer tumor biopsies versus the adjacent normal tissues, the conditioned media and lysates from the established breast cancer cell lines, and breast cancer cell-derived exosomes. Analysis of the circulating immune protein signatures generated from this multiplex approach reveals an elevated level of IL-6, IL-27, M-CSF, MDC, MIG, sCD27, sTIM3, sCD40, Galectin-3, Galectin-1, FGL-1, BAFF and low level of EGF and sCD40L in breast cancer serum samples compared to the healthy serum controls. The expression profiling of tumor and adjacent normal tissues from 3 patients with metastatic breast cancer revealed differential expression of multiple protein markers including IL-6, IL-8, MIG, IL-1ra, IL-18, IP-10, MCP-1, MIP-1b, VEGF, BTLA, HVEM, CTLA-4, CD40, TLR-2, Siglec-9, CD25, Granzyme B, APRIL, BAFF, Nectin-2, Nectin-4, E-cadherin, and IDO-1 in the matched lysates. We performed similar analysis using cultured breast tumor cells. In these samples we identified markers with significantly altered expression in conditioned media, cell lysates, and tumor cell-derived exosomes from MCF10CA1d tumor cells versus MCF-10A non-tumorigenic mammary epithelial cells as well as triple negative HMT-3522 T4-2 and MDA-MB-231 breast cancer cells. Altogether, our results suggest Luminex-based profiling allows for sensitive and versatile multiplexed analysis of stimulating and inhibitory immune mediators in circulation, tissues, and cell lines which will assist in the discovery of biomarkers and therapeutic targets for cancer interventions. Citation Format: Wen-Rong Lie, Alexander Davies, Danielle Pepin, Michael Godeny. Multiplex bead array approach for quantitative profiling of 95 cancer-immunity biomarkers in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2847.

Diagnosis of ischemic stroke using circulating levels of brain-specific proteins measured via high-sensitivity digital ELISA

Limited lower detection ranges associated with traditional immunoassay techniques have prevented the use of brain-specific proteins as blood biomarkers of stroke in the acute phase of care, as these proteins are often only present in circulation at low concentrations. Digital ELISA is a newly developed technique with allows for quantification of proteins in biofluids with up to 1000 times greater sensitivity than conventional ELISA techniques. The purpose of this study was to determine whether the extended lower limits of detection associated with digital ELISA could enable the use of brain-specific proteins as blood biomarkers of ischemic stroke during triage. Blood was sampled from ischemic stroke patients (n = 14) at emergency department admission, as well as from neurologically normal controls matched in terms of risk factors for cardiovascular disease (n = 33). Plasma levels of two brain-specific axonal proteins, neurofilament light chain (NfL) and tau, were measured via digital ELISA, and receiver-operating characteristic analysis was used to determine their ability to discriminate between groups. Plasma levels of NfL and tau were both significantly elevated in stroke patients versus controls, and could respectively discriminate between groups with 92.9% sensitivity / 84.9% specificity, and 85.7% sensitivity / 54.6% specificity. Furthermore, adjustment of measured NfL and Tau levels according to the lower-limits of detection associated with commercially-available conventional ELISA assays resulted in a dramatic and statistically significant decrease in diagnostic performance. Collectively, our results suggest that the increased analytical sensitivity of digital ELISA could enable the use of brain-specific proteins as blood biomarkers of ischemic stroke during triage.

Quantitation of low abundant soluble biomarkers using high sensitivity Single Molecule Counting technology.

Quantitation of biomarkers in biofluids plays a central role in basic research to management of patient care and is routinely used in clinical laboratories and academic institutions. Standard immunoassays, such as an enzyme-linked immunosorbent assay (ELISA), have provided understanding of both normal and pathological processes for many decades. However, in more recent decades, new immunoassay technologies have uncovered numerous analytes in blood that were once undetectable using traditional ELISAs. To meet this new challenge for quantifying low abundant proteins in biofluids, Single Molecule Counting (SMC™) technology was developed. This new technology is a combination of improvements to both the immunoassay procedure as well as the instrument. The aim of this article is to introduce the new SMCxPRO™ instrument, xPRO Acquisition and Analysis software, and the high sensitivity immunoassay kits validated on this instrument for the detection of low abundant proteins in biofluids, such as serum and plasma. Using this new technology platform, biomarkers that were once unquantifiable can now be quantitated in both normal and diseased biofluids.

Expression and secretion of synaptic proteins during stem cell differentiation to cortical neurons

Synaptic function and neurotransmitter release are regulated by specific proteins. Cortical neuronal differentiation of human induced pluripotent stem cells (hiPSC) provides an experimental model to obtain more information about synaptic development and physiology in vitro. In this study, expression and secretion of the synaptic proteins, neurogranin (NRGN), growth-associated protein-43 (GAP-43), synaptosomal-associated protein-25 (SNAP-25) and synaptotagmin-1 (SYT-1) were analyzed during cortical neuronal differentiation. Protein levels were measured in cells, modeling fetal cortical development and in cell-conditioned media which was used as a model of cerebrospinal fluid (CSF), respectively. Human iPSC-derived cortical neurons were maintained over a period of at least 150 days, which encompasses the different stages of neuronal development. The differentiation was divided into the following stages: hiPSC, neuro-progenitors, immature and mature cortical neurons. We show that NRGN was first expressed and secreted by neuro-progenitors while the maximum was reached in mature cortical neurons. GAP-43 was expressed and secreted first by neuro-progenitors and its expression increased markedly in immature cortical neurons. SYT-1 was expressed and secreted already by hiPSC but its expression and secretion peaked in mature neurons. SNAP-25 was first detected in neuro-progenitors and the expression and secretion increased gradually during neuronal stages reaching a maximum in mature neurons. The sensitive analytical techniques used to monitor the secretion of these synaptic proteins during cortical development make these data unique, since the secretion of these synaptic proteins has not been investigated before in such experimental models. The secretory profile of synaptic proteins, together with low release of intracellular content, implies that mature neurons actively secrete these synaptic proteins that previously have been associated with neurodegenerative disorders, including Alzheimer's disease. These data support further studies of human neuronal and synaptic development in vitro, and would potentially shed light on the mechanisms underlying altered concentrations of the proteins in bio-fluids in neurodegenerative diseases.

Needle lost in the haystack: multiple reaction monitoring fails to detect Treponema pallidum candidate protein biomarkers in plasma and urine samples from individuals with syphilis

Background: Current syphilis diagnostic strategies are lacking a sensitive manner of directly detecting Treponema pallidum antigens. A diagnostic test that could directly detect T. pallidum antigens in individuals with syphilis would be of considerable clinical utility, especially for the diagnosis of reinfections and for post-treatment serological follow-up. Methods: In this study, 11 candidate T. pallidum biomarker proteins were chosen according to their physiochemical characteristics, T. pallidum specificity and predicted abundance. Thirty isotopically labelled proteotypic surrogate peptides (hPTPs) were synthesized and incorporated into a scheduled multiple reaction monitoring assay. Protein extracts from undepleted/unenriched plasma (N = 18) and urine (N = 4) samples from 18 individuals with syphilis in various clinical stages were tryptically digested, spiked with the hPTP mixture and analysed with a triple quadruple mass spectrometer. Results: No endogenous PTPs corresponding to the eleven candidate biomarkers were detected in any samples analysed. To estimate the Limit of Detection (LOD) of a comparably sensitive mass spectrometer (LTQ-Orbitrap), two dilution series of rabbit cultured purified T. pallidum were prepared in PBS. Polyclonal anti- T. pallidum antibodies coupled to magnetic Dynabeads were used to enrich one sample series; no LOD improvement was found compared to the unenriched series. The estimated LOD of MS instruments is 300 T. pallidum/ml in PBS. Conclusions: Biomarker protein detection likely failed due to the low (femtomoles/liter) predicted concentration of T. pallidum proteins. Alternative sample preparation strategies may improve the detectability of T. pallidum proteins in biofluids.

Conductive Polymer Spray Ionization Mass Spectrometry for Biofluid Analysis.

We present a conductive polymer spray ionization (CPSI) method for the direct mass spectrometric analysis of hydrophilic drugs, saccharides, peptides, and proteins in biofluids. Carbon nanotubes (CNTs) were introduced into poly(methyl methacrylate) (PMMA) to fabricate a conductive composite substrate CNT/PMMA in the shape of a triangle (8 mm wide and 10 mm long) with its apex pointed toward the inlet of a mass spectrometer. In comparison with a traditional paper spray substrate, the conductive polymer absorbs less hydrophilic compounds owing to its hydrophobic nature. When aqueous biofluid samples are loaded, they also exhibit less diffusion on this nonporous surface. Only 1.0-2.0 μL solvent suffices to extract the components in a dried biofluid spot and to form charged microdroplets (4.5 kV high voltage applied). Furthermore, the hydrophobic polymer surface only needs to overcome weak surface tension to emit charged microdroplets, so that the signal has a typical duration of 7.5 min. For sunitinib, acarbose, melamine, and angiotensin II, the ion intensity of the target compound from the conductive polymer support is significantly higher than paper spray, typically by a factor of 20 to 100. These results suggest that the CNT/PMMA conductive polymer spray has great potential in the analysis of hydrophilic drugs, saccharides, peptides, and proteins in biofluids.

Improving Huntington's disease detection

Huntington's Disease Early detection of Huntington's disease (HD) could help the development of therapeutic strategies to block or delay disease progression. Byrne et al. found that blood and cerebrospinal fluid concentrations of mutant huntingtin (mHTT) and neurofilament light (NfL) proteins correlated with disease severity in HD patients. Alterations in circulating mHTT and NfL concentrations were among the earliest detectable changes in HD. Thus, concentrations of these proteins in biofluids might be used in combination with other clinical measures for improving the accuracy and efficiency of early HD detection. Sci. Transl. Med. 10 , eaat7108 (2018).

Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI).

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a result of the direct detection of the mass-to-charge ratio of each target molecule. However, MS-based proteomics, like most other analytical techniques, has a bias towards measuring high-abundance analytes, so it is challenging to achieve detection limits of low ng/mL or pg/mL in complex samples, and this is the concentration range for many disease-relevant proteins in biofluids such as human plasma. To assist in the detection of low-abundance analytes, immuno-enrichment has been integrated into the assay to concentrate and purify the analyte before MS measurement, significantly improving assay sensitivity. In this work, the immuno- Matrix-Assisted Laser Desorption/Ionization (iMALDI) technology is presented for the quantification of proteins and peptides in biofluids, based on immuno-enrichment on beads, followed by MALDI-MS measurement without prior elution. The anti-peptide antibodies are functionalized on magnetic beads, and incubated with samples. After washing, the beads are directly transferred onto a MALDI target plate, and the signals are measured by a MALDI-Time of Flight (MALDI-TOF) instrument after the matrix solution has been applied to the beads. The sample preparation procedure is simplified compared to other immuno-MS assays, and the MALDI measurement is fast. The whole sample preparation is automated with a liquid handling system, with improved assay reproducibility and higher throughput. In this article, the iMALDI assay is used for determining the peptide angiotensin I (Ang I) concentration in plasma, which is used clinically as readout of plasma renin activity for the screening of primary aldosteronism (PA).

Influence of the Surface Functional Group Density on the Carbon-Nanotube-Induced α-Chymotrypsin Structure and Activity Alterations.

Because of the special properties of carbon nanotubes (CNTs), their applications have been introduced to many fields. The biosafety of these emerging materials is of high concern concomitantly. Because CNTs may initially bind with proteins in biofluids before they exert biological effects, it is of great importance to understand how the target proteins interact with these exogenous nanomaterials. Here we investigated the interaction between α-chymotrypsin (α-ChT) and carboxylized multiwalled CNTs in a simulated biophysical environment utilizing the techniques of fluorescence, UV-vis, circular dichroism spectroscopy, ζ potential, atomic force microscopy, and bicinchoninic acid analysis. It was demonstrated that CNTs interacted with α-ChT through electrostatic forces, causing a decrement in the α-helix and an increment in the β-sheet content of the protein. The protein fluorescence was quenched in a static mode. The increase in the surface modification density of CNTs enhanced the protein absorption and decreased the enzymatic activity correspondingly. α-ChT activity inhibition induced by CNTs with low surface modification density exhibited noncompetitive characteristics; however, a competitive feature was observed when CNTs with high surface modification density interacted with the protein. An increase of the ionic strength in the reaction buffer may help to reduce the interaction between CNTs and α-ChT because the high ionic strength may favor the release of the protein from binding on a CNT surface modified with functional groups. Accordingly, the functionalization density on the CNT surface plays an important role in the regulation of their biological effects and is worthy of concern when new modified CNTs are developed.

Comparative Proteomics for Studying Muscular Dystrophy: Intrinsic Biological and Analytical Issues Associated with the Systematic Utilization of Tissue Specimens

Over the past decade, mass spectrometry-based proteomics has been instrumental for the detailed elucidation of pathobiochemical mechanisms involved in major neuromuscular diseases. Although the identification of musclederived proteins in biofluids is the main focus of diagnostic biomarker research, the large-scale proteomic analysis of pathological muscle tissue is of central importance for furthering our general understanding of the dysregulation that underlies complex muscle diseases. Here, we discuss intrinsic biological issues and bioanalytical difficulties that are generally associated with comparative muscle tissue proteomics. The systematic utilization of cellular mixtures or whole tissue specimens as starting material for studying neuromuscular pathology is seriously complicated by the cellular heterogeneity and physiological plasticity of contractile tissues. The comprehensive biochemical analysis of the skeletal muscle proteome is often hampered by the wide dynamic expression range of proteins, the greatly differing physicochemical properties of dissimilar muscle protein species and the potential cross-contamination of samples by highly abundant proteins. Thus, neither gel electrophoretic methodology nor liquid chromatography, is capable of appropriately separating all constituents of the skeletal muscle proteome. However, the application of advanced extraction strategies, the usage of subcellular fractionation protocols to reduce sample complexity and the affinity purification of distinct protein fractions prior to mass spectrometric analysis promises to overcome some of the inherent problems associated with muscle tissue proteomics.