Elastin-like polypeptide-functionalized nanobody for column-free immunoaffinity purification of aflatoxin B1.

A column-free immunoaffinity purification (CFIP) technique for sample preparation of aflatoxin B1 (AFB1) was developed using an AFB1-specific nanobody (named G8) and an elastin-like polypeptide (ELP). The reversible phase transition between liquid and solid in response to temperature changes was exhibited by the ELP which was derived from human elastin. The G8 was tagged with ELPs of various lengths (20, 40, 60, and 80 repeat units) at the C-terminus using recursive directional ligation (RDL). Coding sequences were then subcloned into pET30a at the multiple cloning sites. Bioactive recombinant proteins were produced by expressing them as inclusion bodies in Escherichia coli BL21 (DE3), then dissolved and refolded. Analysis by indirect competitive enzyme-linked immunosorbent assay (icELISA) and transition temperature (Tt) measurement confirmed that the refolded G8-ELPs preserved the ability to recognize AFB1 as well as phase transition when the temperature rose above Tt. To establish the optimal conditions for cleaning AFB1, the effects of various parameters on recovery were investigated. The recovery in ELISA tests was 95 ± 3.67% under the optimized CFIP workflow. Furthermore, the CFIP-prepared samples were applied for high-performance liquid chromatography (HPLC) detection. The recovery in the CFIP-HPLC test ranged from 54 ± 1.86% to 98 ± 3.58% for maize, rice, soy sauce, and vegetable oil samples. To the best of our knowledge, this is the first report combining the function of both nanobody and ELP to develop a cleanup technique for small molecules in a complex matrix. The CFIP for the sample pretreatment was easy to use and inexpensive. In contrast to conventional immunosensitivity materials, the reagent utilized in the CFIP was entirely biosynthesized without any chemical coupling reactions. This suggests that the nanobody-ELP may serve as a useful dual-functional reagent for the development of sample cleaning or purification methods.

Determination of the occurrence of and exposure to bisphenol A and its analogues in carbonated beverages and canned tuna using liquid chromatography - tandem mass spectrometry.

Bisphenol A (BPA), a known endocrine disruptor, is commonly used in food containers and packaging. Recently, alternatives such as bisphenol AF (BPAF), bisphenol B (BPB), and bisphenol E (BPE) have been introduced to replace BPA. However, these substitutes have been reported to exhibit toxicity levels similar to BPA. In this study, we developed and validated a method for the analysis of trace bisphenols (BPA, BPAF, BPB, and BPE) in food using immunoaffinity column (IAC) clean-up. The method demonstrated satisfactory accuracy and precision. We applied this validated method to analyze 56 carbonated beverage samples and 30 canned tuna samples. In the carbonated beverages, average concentrations of BPA and BPAF were 0.4 and 0.2 µg kg-1, respectively. In canned tuna, BPA and BPAF were found at average concentrations of 22.2 and 0.7 µg kg-1, respectively, while BPB and BPE were not detected in any samples. Estimated exposure levels ranged from 0.13 to 0.18 ng kg bw-1 day-1 in the general population and from 205.2 to 232.0 ng kg bw-1 day-1 among consumers. The commercial IAC-based analytical method used in this study can contribute to the safety management of BPA, BPAF, BPB, and BPE.

Extraction of redox extracellular vesicles using exclusion-based sample preparation.

Studying specific subpopulations of cancer-derived extracellular vesicles (EVs) could help reveal their role in cancer progression. In cancer, an increase in reactive oxygen species (ROS) happens which results in lipid peroxidation with a major product of 4-hydroxynonenal (HNE). Adduction by HNE causes alteration to the structure of proteins, leading to loss of function. Blebbing of EVs carrying these HNE-adducted proteins as a cargo or carrying HNE-adducted on EV membrane are methods for clearing these molecules by the cells. We have referred to these EVs as Redox EVs. Here, we utilize a surface tension-mediated extraction process, termed exclusion-based sample preparation (ESP), for the rapid and efficient isolation of intact Redox EVs, from a mixed population of EVs derived from human glioblastoma cell line LN18. After optimizing different parameters, two populations of EVs were analyzed, those isolated from the sample (Redox EVs) and those remaining in the original sample (Remaining EVs). Electron microscopic imaging was used to confirm the presence of HNE adducts on the outer leaflet of Redox EVs. Moreover, the population of HNE-adducted Redox EVs shows significantly different characteristics to those of Remaining EVs including smaller size EVs and a more negative zeta potential EVs. We further treated glioblastoma cells (LN18), radiation-resistant glioblastoma cells (RR-LN18), and normal human astrocytes (NHA) with both Remaining and Redox EV populations. Our results indicate that Redox EVs promote the growth of glioblastoma cells, likely through the production of H2O2, and cause injury to normal astrocytes. In contrast, Remaining EVs have minimal impact on the viability of both glioblastoma cells and NHA cells. Thus, isolating a subpopulation of EVs employing ESP-based immunoaffinity could pave the way for a deeper mechanistic understanding of how subtypes of EVs, such as those containing HNE-adducted proteins, induce biological changes in the cells that take up these EVs.

Urinary Extracellular Vesicles as a Readily Available Biomarker Source: A Simplified Stratification Method.

Urine, a common source of biological markers in biomedical research and clinical diagnosis, has recently generated a new wave of interest. It has recently become a focus of study due to the presence of its content of extracellular vesicles (EVs). These uEVs have been found to reflect physiological and pathological conditions in kidney, urothelial, and prostate tissue and can illustrate further molecular processes, leading to a rapid expansion of research in this field In this work, we present the advantages of an immunoaffinity-based method for uEVs' isolation with respect to the gold standard purification approach performed by differential ultracentrifugation [in terms of purity and antigen presence. The immunoaffinity method was made feasible by combining specific antibodies with a functionalized polymethacrylate polymer. Flow cytometry indicated a significant fluorescence shift, validating the presence of the markers (CD9, CD63, CD81) and confirming the effectiveness of the isolation method. Microscopy evaluations have shown that the morphology of the vesicles remained intact and corresponded to the expected shapes and dimensions of uEVs. The described protocol is inexpensive, fast, easy to process, has good reproducibility, and can be applied to further biological samples.

Isolation and Characterization of Exosomes from Cancer Cells Using Antibody-Functionalized Paddle Screw-Type Devices and Detection of Exosomal miRNA Using Piezoelectric Biosensor.

Exosomes are small extracellular vesicles produced by almost all cell types in the human body, and exosomal microRNAs (miRNAs) are small non-coding RNA molecules that are known to serve as important biomarkers for diseases such as cancer. Given that the upregulation of miR-106b is closely associated with several types of malignancies, the sensitive and accurate detection of miR-106b is important but difficult. In this study, a surface acoustic wave (SAW) biosensor was developed to detect miR-106b isolated from cancer cells based on immunoaffinity separation technique using our unique paddle screw device. Our novel SAW biosensor could detect a miR-106b concentration as low as 0.0034 pM in a linear range from 0.1 pM to 1.0 µM with a correlation coefficient of 0.997. Additionally, we were able to successfully detect miR-106b in total RNA extracted from the exosomes isolated from the MCF-7 cancer cell line, a model system for human breast cancer, with performance comparable to commercial RT-qPCR methods. Therefore, the exosome isolation by the paddle screw method and the miRNA detection using the SAW biosensor has the potential to be used in basic biological research and clinical diagnosis as an alternative to RT-qPCR.

Development and validation of extraction and clean-up procedures for UPLC-MS/MS analysis of aflatoxins in spices.

UPLC-MS/MS analytical conditions for the analysis of aflatoxins in spices were optimized and validated in this study. Liquid-liquid partition-based protocols for the cleaning up of extracts using common organic solvents such as acetonitrile, hexane, and ethyl acetate were developed and validated. The developed liquid-liquid partition methods were compared with immuno-affinity column and QuEChERS clean-up methods for the UPLC-MS/MS analysis of aflatoxins in 8 spices. The reduction of lipophilic components using the partition with hexane is particularly useful in spices like red pepper that have higher levels of fatty acids, carotenoids, sterols, triterpenoids, etc. The subsequent partitioning with ethyl acetate considerably reduced the matrix interference from the polar components and increased the sensitivity. The cleaning up of spice extracts using liquid-liquid partition techniques resulted in limits of quantification (LOQ) of 2-5 µgL-1 in UPLC-MS/MS analysis. Trueness, repeatability, and reproducibility of the methods were in acceptable ranges. The accuracy of the developed methods was further verified by analyzing aflatoxins in naturally incurred samples of spices and comparing the results with those obtained from the immuno-affinity column cleanup-HPLC-FD method.

High-throughput surface epitope immunoaffinity isolation of extracellular vesicles and downstream analysis.

Extracellular vesicles (EVs), including exosomes, have significant potential for diagnostic and therapeutic applications. The lack of standardized methods for efficient and high-throughput isolation and analysis of EVs, however, has limited their widespread use in clinical practice. Surface epitope immunoaffinity (SEI) isolation utilizes affinity ligands, including antibodies, aptamers, or lectins, that target specific surface proteins present on EVs. Paramagnetic bead-SEI isolation represents a fit-for-purpose solution for the reproducible, high-throughput isolation of EVs from biofluids and downstream analysis of RNA, protein, and lipid biomarkers that is compatible with clinical laboratory workflows. This study evaluates a new SEI isolation method for enriching subpopulations of EVs. EVs were isolated from human plasma using a bead-based SEI method designed for on-bead and downstream analysis of EV-associated RNA and protein biomarkers. Western blot analysis confirmed the presence of EV markers in the captured nanoparticles. Mass spectrometry analysis of the SEI lysate identified over 1500 proteins, with the top 100 including known EV-associated proteins. microRNA (miRNA) sequencing followed by RT-qPCR analysis identified EV-associated miRNA transcripts. Using SEI, EVs were isolated using automated high-throughput particle moving instruments, demonstrating equal or higher protein and miRNA yield and recovery compared to manual processing. SEI is a rapid, efficient, and high-throughput method for isolating enriched populations of EVs; effectively reducing contamination and enabling the isolation of a specific subpopulation of EVs. In this study, high-throughput EV isolation and RNA extraction have been successfully implemented. This technology holds great promise for advancing the field of EV research and facilitating their application for biomarker discovery and clinical research.

Investigation of ochratoxin A in air-dry-cured hams.

The European Union legislation regarding ochratoxin A (OTA) in various foodstuffs has changed relatively recently. Nevertheless, the legislation does not regulate OTA in any meat and meat-derived products. In this legislation update, the European Commission requested new studies, including, besides others, the presence of OTA in hams, which raises the concern that its consumption may pose a potential risk of exposure to OTA. This study aims to investigate OTA in a total of 195 samples of air-dry-cured hams acquired at the Czech market from January to June 2023. The analytical technique of high-performance liquid chromatography in combination with a fluorescence detector with pre-treatment employing immunoaffinity columns was used to determine OTA. OTA was found in 93 (48%) samples of air-dry-cured ham, with the OTA concentration reaching up to 14.58 ng/g. Due to the current absence of regulation limits, the results of this study were compared with the Italian maximum limit of 1 ng/g regulating OTA in porcine meat and byproducts. The Italian OTA maximum limit was exceeded in 22 (11%) samples. This study shows that the population of the Czech Republic is exposed to OTA from this pork byproduct. It is essential to set an OTA regulatory limit for meat and food produced from it to protect human health.

Quantifying LAGA mutated mouse IgG2a monoclonal antibody with a rapid pepsin digestion enabled immunoaffinity LC/MS/MS assay.

A sensitive and specific bioanalytical method was required to measure the exposure of a LAGA-mutated surrogate mouse IgG2a monoclonal antibody in mouse plasma, but the lack of highly specific reagents for the LAGA mutant hindered the development of a ligand-binding assay. Equally problematic is that no sensitive unique tryptic peptides suitable for quantitative mass spectrometric analysis could be identified in the mIgG2a complementarity-determining regions. To overcome these challenges, a trypsin alternative pepsin, an aspartic protease, was systematically investigated for its use in digesting the mutated mIgG2a antibody to allow generation of signature peptides for the bioanalytical quantification purpose. After a series of evaluations, a rapid one-hour pepsin digestion protocol was established for the mutated Fc backbone. Consequently, a new pepsin digestion-based liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was successfully developed to support the mouse pharmacokinetic (PK) sample analysis. In brief, robust and reproducible C-terminal cleavage of both leucine and phenylalanine near the double mutation site of the mutated mIgG2a was accomplished at pH ≤2 and 37°C. Combined with a commercially available rat anti-mIgG2a heavy-chain antibody, the established immunoaffinity LC/MS/MS assay achieved a limit of quantitation of 20 ng/mL in the dynamic range of interest with satisfactory assay precision and accuracy. The successful implementation of this novel approach in discovery PK studies eliminates the need for tedious and costly generation of specific immunocapturing reagents for the LAGA mutants. The approach should be widely applicable for developing popular LAGA mutant-based biological therapeutics.

Differential Analysis of Cereblon Neosubstrates in Rabbit Embryos Using Targeted Proteomics.

Targeted protein degradation is the selective removal of a protein of interest through hijacking intracellular protein cleanup machinery. This rapidly growing field currently relies heavily on the use of the E3 ligase cereblon (CRBN) to target proteins for degradation, including the immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide which work through a molecular glue mechanism of action with CRBN. While CRBN recruitment can result in degradation of a specific protein of interest (e.g., efficacy), degradation of other proteins (called CRBN neosubstrates) also occurs. Degradation of one or more of these CRBN neosubstrates is believed to play an important role in thalidomide-related developmental toxicity observed in rabbits and primates. We identified a set of 25 proteins of interest associated with CRBN-related protein homeostasis and/or embryo/fetal development. We developed a targeted assay for these proteins combining peptide immunoaffinity enrichment and high-resolution mass spectrometry and successfully applied this assay to rabbit embryo samples from pregnant rabbits dosed with three IMiDs. We confirmed previously reported in vivo decreases in neosubstrates like SALL4, as well as provided evidence of neosubstrate changes for proteins only examined in vitro previously. While there were many proteins that were similarly decreased by all three IMiDs, no compound had the exact same neosubstrate degradation profile as another. We compared our data to previous literature reports of IMiD-induced degradation and known developmental biology associations. Based on our observations, we recommend monitoring at least a major subset of these neosubstrates in a developmental test system to improve CRBN-binding compound-specific risk assessment. A strength of our assay is that it is configurable, and the target list can be readily adapted to focus on only a subset of proteins of interest or expanded to incorporate new findings as additional information about CRBN biology is discovered.

Identification and Quantification of Human Relaxin Proteins by Immunoaffinity-Mass Spectrometry.

The human relaxins belong to the Insulin/IGF/Relaxin superfamily of peptide hormones, and their physiological function is primarily associated with reproduction. In this study, we focused on a prostate tissue-specific relaxin RLN1 (REL1_HUMAN protein) and a broader tissue specificity RLN2 (REL2_HUMAN protein). Due to their structural similarity, REL1 and REL2 proteins were collectively named a 'human relaxin protein' in previous studies and were exclusively measured by immunoassays. We hypothesized that the highly selective and sensitive immunoaffinity-selected reaction monitoring (IA-SRM) assays would reveal the identity and abundance of the endogenous REL1 and REL2 in biological samples and facilitate the evaluation of these proteins for diagnostic applications. High levels of RLN1 and RLN2 transcripts were found in prostate and breast cancer cell lines by RT-PCR. However, no endogenous prorelaxin-1 or mature REL1 were detected by IA-SRM in cell lines, seminal plasma, or blood serum. The IA-SRM assay of REL2 demonstrated its undetectable levels (<9.4 pg/mL) in healthy control female and male sera and relatively high levels of REL2 in maternal sera across different gestational weeks (median 331 pg/mL; N = 120). IA-SRM assays uncovered potential cross-reactivity and nonspecific binding for relaxin immunoassays. The developed IA-SRM assays will facilitate the investigation of the physiological and pathological roles of REL1 and REL2 proteins.

High-Performance Liquid Chromatographic Quantification of the Plant Hormone Abscisic Acid at ppb Levels in Plant Samples after a Single Immunoaffinity Column Cleanup.

High-performance liquid chromatography with ultraviolet detection (HPLC-UV) is a common analysis technique due to its high versatility and simple operation. In the present study, HPLC-UV detection was integrated with immunoaffinity cleanup (IAC) of the sample extracts. The matrix effect was greatly reduced, and the limit of detection was as low as 1 ng/g of free abscisic acid (ABA) in fresh plant tissues. A monoclonal antibody 3F1 (mAb 3F1) was developed to specifically recognize free ABA but not ABA analogues. The mAb 3F1-immobilized immunoaffinity column exhibited a capacity of 850 ng/mL and an elution efficiency of 88.8-105% for standards. The extraction recoveries of the column for ABA ranged from 80.4 to 108.9%. ABA content was detected in various plant samples with IAC-HPLC-UV. The results were verified with ultraperformance liquid chromatography-electrospray tandem mass spectrometry. IAC-HPLC-UV can be a sensitive and cost-efficient method for plant hormone analysis.

Improved elution strategy and new monoclonal anti-biotin antibody for LC-MS/MS characterization of protein biotinylation sites.

Biotin labeling in combination with mass spectrometry has been widely applied in large-scale biological studies, such as determination of protein partners, protein subcellular localization, and protein post-translational modifications. Previous studies have shown that immunoaffinity enrichment is a better method than streptavidin/avidin purification for site-specific studies of biotinylated molecules. In this study, we made a crucial improvement to the elution phase of the immunoaffinity enrichment step for biotinylated peptides, which involves the addition of a highly organic solvent, and developed a monoclonal anti-biotin antibody that improved the identification number for biotinylated peptides. We then demonstrated its application in the characterization of protein interaction sites for the ß2 adrenergic receptor (ß2AR) by proximity labeling in living cells. Our research provides an improved and reproducible immunoaffinity enrichment method for site-specific biotin-related research.

Precursor carboxy-silica for functionalization with interactive ligands. IV. Carbodiimide assisted preparation of immobilized antibody stationary phases for high performance immuno-affinity chromatography of human serum.

In this Part IV of the article series dealing with the functionalization of the precursor carboxy silica with various chromatographic ligands, immuno affinity (IA) columns were prepared with immobilized anti-apolipoprotein B (AAP B) and anti-haptoglobin (AHP) antibodies for use in immuno affinity chromatography (IAC) in the aim of selectivily capturing their corresponding antigens from healthy and cancer human sera. Diseased human serum with adenocarcinoma cancer was selected as a typical diseased biological fluid. Besides preferentially capturing their corresponding antigens, the AAP B column captured from disease-free and cancer sera, 34 proteins and 33 proteins, respectively, while the AHP column enriched 38 and 47 proteins, respectively. This nonspecific binding can be attributed to the many proteins human serum have, which could mediate protein-protein interactions thus leading to the so-called "sponge effect". This kind of behavior can be exploited positively in the determination of differentially expressed proteins (DEPs) for diseased serum with respect to healthy serum and in turn allow the identification of an array of potential biomarkers for cancer. In fact, For AHP column, 13 upregulated and 22 downregulated proteins were identified whereas for AAP B column the numbers were 23 and 10, respectively. The DEPs identified with both columns match those reported in the literature for other types of cancers. The different expression of proteins in each IAC column can be related to the variability of protein-protein interactions. In addition, an array of a few biomarkers is more indicative of a certain disease than a single biomarker.

Immunoaffinity proteomics for kidney injury biomarkers in male beagle dogs.

Drug-induced kidney injury (DIKI) is a cause of drug development failure. Dogs represent a common non-rodent animal model in pre-clinical safety studies; however, biomarker assays for detecting nephrotoxicity in dogs are limited. To identify novel proteins and gain insight into the molecular mechanisms involved in DIKI, we developed an assay to evaluate proteomic changes associated with DIKI in male beagle dogs that received nephrotoxic doses of tobramycin for 10 consecutive days. Label-free quantitative discovery proteomics analysis on representative kidney cortex tissues collected on Day 11 showed that the tobramycin-induced kidney injury led to a significant differential regulation of 94 proteins mostly associated with mechanisms of nephrotoxicity such as oxidative stress and proteasome degradation. For verification of the proteomic results, we developed a multiplex peptide-centric immunoaffinity liquid chromatography tandem mass spectrometry assay (IA LC-MS/MS) to evaluate the association of eight DIKI protein biomarker candidates using kidney cortices collected on Day 11 and urine samples collected on Days -4, 1, 3, 7 and 10. The results showed that most biomarkers evaluated were detected in the kidney cortices and their expression profile in tissue aligned with the label-free data. Cystatin C was the most consistent marker regardless of the magnitude of the renal injury while fatty acid-binding protein-4 (FABP4) and kidney injury molecule-1 (KIM-1) were the most affected biomarkers in response to moderate proximal tubular injury in absence of changes in serum-based concentrations of blood urea nitrogen or creatinine. In the urine, clusterin is considered the most consistent biomarker regardless of the magnitude and time of the renal injury. To our knowledge, this is the most comprehensive multiplex assay for the quantitative analysis of mechanism-based proximal tubular injury biomarkers in dogs.

Bifunctional immunoaffinity magnetic nanoparticles for high-efficiency separation of exosomes based on host-guest interaction.

The investigation of exosomes holds substantial importance in the field of disease diagnosis and prognosis, where in the rapid and low-loss isolation of exosomes emerges as a crucial step owing to their exceedingly low concentration in bodily fluids. Herein, bifunctional immunoaffinity magnetic nanoparticles (BI-NPs) were successfully constructed by binding Ti4+ and antibody to magnetic nanoparticles through host-guest interaction. Exosomes in the cell culture supernatant could be effectively captured by the BI-NPs and then gently eluted with α-CD and 10% ammonia solution. Following the elution process, the content of captured exosome protein was determined to be up to 97 µg/mL. A total of 2822 protein groups were identified in the exosomes isolated by BI-NPs, 1060 protein groups were derived from exosomes. At the same time, the mass of exosome protein obtained by BI-NPs was more than 2 times that of UC isolation, and the above results indicated that BI-NPs had high purity enrichment performance. This is attributed to the fact that BI-NPs combine the dual affinity of Ti4+ and antibody to achieve efficient enrichment of exosomes, in addition to the mild elution property of BI-NPs due to the presence of the host-guest system. BI-NPs offer a novel approach for the isolation of exosomes, in order to further promote the application of exosomes in the required fields.

Exploration of Nitrotyrosine-Containing Proteins and Peptides by Antibody-Based Enrichment Strategies.

Nitrotyrosine, or 3-nitrotyrosine, is an oxidative post-translational modification induced by reactive nitrogen species. Although nitrotyrosine is considered a marker of oxidative stress and has been associated with inflammation, neurodegeneration, cardiovascular disease, and cancer, identification of nitrotyrosine-modified proteins remains challenging owing to its low stoichiometric levels in biological samples. To facilitate a comprehensive analysis of proteins and peptides containing nitrotyrosine, we optimized an immunoprecipitation-based enrichment workflow using a cell line model. The identification of proteins and peptides containing nitrotyrosine residues was carried out after peroxynitrite treatment of cell lysates, which generated modified nitrotyrosine residues on susceptible sites on proteins. We evaluated the efficacy of enriching nitrotyrosine-modified proteins and peptides by employing four different commercially available monoclonal antibodies directed against nitrotyrosine. LC-MS/MS analysis resulted in the identification of 1377 and 1624 nitrotyrosine-containing peptides from protein- and peptide-based enrichment experiments, respectively. Although the yield of nitrotyrosine-containing peptides was higher in experiments where peptides rather than proteins were enriched, we found a substantial proportion (37-65%) of identified nitrotyrosine-containing peptides contained nitrotyrosine at the N-terminus. However, in protein-based immunoprecipitation <9% of nitrotyrosine-containing peptides had nitrotyrosine modification at the N-terminus of the peptide. Overall, our study resulted in the identification of 2603 nitrotyrosine-containing peptides of which >2000 have not previously been reported. We synthesized 101 novel nitrotyrosine-containing peptides identified in our analysis and analyzed them by LC-MS/MS to validate our findings. We have confirmed the validity of 70% of these peptides, as they demonstrated a similarity score exceeding 0.7 when compared to peptides identified through experimental methods. Finally, we also validated the presence of nitrotyrosine modification on PKM and EF2 proteins in peroxynitrite-treated samples by immunoblot analysis. The large catalog presented in this study along with the workflow should facilitate the investigation of nitrotyrosine as an oxidative modification in a variety of settings in greater detail.

Innovative approaches for mycotoxin detection in various food categories.

Mycotoxins (MTs), produced by filamentous fungi, represent a severe hazard to the health of humans and food safety, affecting the quality of various agricultural products. They can contaminate a wide range of foods, during any processing phase before or after harvest. Animals and humans who consume MTs-contaminated food or feed may experience acute or chronic poisoning, which may result in serious pathological consequences. Accordingly, developing rapid, easy, and accurate methods of MTs detection in food becomes highly urgent and critical as a quality control and to guarantee food safety and lower health hazards. In this review, we highlighted and discussed innovative approaches like biosensors, fluorescent polarization, capillary electrophoresis, infrared spectroscopy, and electronic noses for MT identification pointing out current challenges and future directions. The limitations, current challenges, and future directions of conventional detection methods versus innovative methods have also been highlighted and discussed.

Proteomic applications in identifying protein-protein interactions.

There are many things that can be used to characterize a protein. Size, isoelectric point, hydrophobicity, structure (primary to quaternary), and subcellular location are just a few parameters that are used. The most important feature of a protein, however, is its function. While there are many experiments that can indicate a protein's role, identifying the molecules it interacts with is probably the most definitive way of determining its function. Owing to technology limitations, protein interactions have historically been identified on a one molecule per experiment basis. The advent of high throughput multiplexed proteomic technologies in the 1990s, however, made identifying hundreds and thousands of proteins interactions within single experiments feasible. These proteomic technologies have dramatically increased the rate at which protein-protein interactions (PPIs) are discovered. While the improvement in mass spectrometry technology was an early driving force in the rapid pace of identifying PPIs, advances in sample preparation and chromatography have recently been propelling the field. In this chapter, we will discuss the importance of identifying PPIs and describe current state-of-the-art technologies that demonstrate what is currently possible in this important area of biological research.

A high throughput immuno-affinity mass spectrometry method for detection and quantitation of SARS-CoV-2 nucleoprotein in human saliva and its comparison with RT-PCR, RT-LAMP, and lateral flow rapid antigen test.

OBJECTIVES: Many reverse transcription polymerase chain reaction (RT-PCR) methods exist that can detect SARS-CoV-2 RNA in different matrices. RT-PCR is highly sensitive, although viral RNA may be detected long after active infection has taken place. SARS-CoV-2 proteins have shorter detection windows hence their detection might be more meaningful. Given salivary droplets represent a main source of transmission, we explored the detection of viral RNA and protein using four different detection platforms including SISCAPA peptide immunoaffinity liquid chromatography-mass spectrometry (SISCAPA-LC-MS) using polyclonal capture antibodies. METHODS: The SISCAPA-LC MS method was compared to RT-PCR, RT-loop-mediated isothermal amplification (RT-LAMP), and a lateral flow rapid antigen test (RAT) for the detection of virus material in the drool saliva of 102 patients hospitalised after infection with SARS-CoV-2. Cycle thresholds (Ct) of RT-PCR (E gene) were compared to RT-LAMP time-to-positive (TTP) (NE and Orf1a genes), RAT optical densitometry measurements (test line/control line ratio) and to SISCAPA-LC-MS for measurements of viral protein. RESULTS: SISCAPA-LC-MS showed low sensitivity (37.7 %) but high specificity (89.8 %). RAT showed lower sensitivity (24.5 %) and high specificity (100 %). RT-LAMP had high sensitivity (83.0 %) and specificity (100.0 %). At high initial viral RNA loads (<20 Ct), results obtained using SISCAPA-LC-MS correlated with RT-PCR (R2 0.57, p-value 0.002). CONCLUSIONS: Detection of SARS-CoV-2 nucleoprotein in saliva was less frequent than the detection of viral RNA. The SISCAPA-LC-MS method allowed processing of multiple samples in <150 min and was scalable, enabling high throughput.