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Förster resonance energy transfer (FRET)-based homogeneous immunoassay obviates tedious washing steps and thus is a promising approach for immunoassays. However, a conventional FRET-based homogeneous immunoassay operating in the visible region is not able to overcome the interference of complex biological samples, thus resulting in insufficient detection sensitivity and poor accuracy. Here, we develop a near-infrared (NIR)-to-NIR FRET platform (Ex = 808 nm, Em = 980 nm) that enables background-free high-throughput homogeneous quantification of various biomarkers in complex biological samples. This NIR-to-NIR FRET platform is portable and easy to operate and is mainly composed of a high-performance NIR-to-NIR FRET pair based on lanthanide-doped nanoparticles (LnNPs) and a custom-made microplate reader for readout of NIR luminescence signals. We demonstrate that this NIR-to-NIR FRET platform is versatile and robust, capable of realizing highly sensitive and accurate detection of various critical biomarkers, including small molecules (morphine and 1,25-dihydroxyvitamin D), proteins (human chorionic gonadotropin), and viral particles (adenovirus) in unprocessed complex biological samples (urine, whole blood, and feces) within 5-10 min. We expect this NIR-to-NIR FRET platform to provide low-cost healthcare for populations living in resource-limited areas and be widely used in many other fields, such as food safety and environmental monitoring.
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Given continuous improvements in industrial production and living standards, the analysis and detection of complex biological sample systems has become increasingly important. Common complex biological samples include blood, serum, saliva, and urine. At present, the main methods used to separate and recognize target analytes in complex biological systems are electrophoresis, spectroscopy, and chromatography. However, because biological samples consist of complex components, they suffer from the matrix effect, which seriously affects the accuracy, sensitivity, and reliability of the selected separation analysis technique. In addition to the matrix effect, the detection of trace components is challenging because the content of the analyte in the sample is usually very low. Moreover, reasonable strategies for sample enrichment and signal amplification for easy analysis are lacking. In response to the various issues described above, researchers have focused their attention on immuno-affinity technology with the aim of achieving efficient sample separation based on the specific recognition effect between antigens and antibodies. Following a long period of development, this technology is now widely used in fields such as disease diagnosis, bioimaging, food testing, and recombinant protein purification. Common immuno-affinity technologies include solid-phase extraction (SPE) magnetic beads, affinity chromatography columns, and enzyme linked immunosorbent assay (ELISA) kits. Immuno-affinity techniques can successfully reduce or eliminate the matrix effect; however, their applications are limited by a number of disadvantages, such as high costs, tedious fabrication procedures, harsh operating conditions, and ligand leakage. Thus, developing an effective and reliable method that can address the matrix effect remains a challenging endeavor. Similar to the interactions between antigens and antibodies as well as enzymes and substrates, biomimetic molecularly imprinted polymers (MIPs) exhibit high specificity and affinity. Furthermore, compared with many other biomacromolecules such as antigens and aptamers, MIPs demonstrate higher stability, lower cost, and easier fabrication strategies, all of which are advantageous to their application. Therefore, molecular imprinting technology (MIT) is frequently used in SPE, chromatographic separation, and many other fields. With the development of MIT, researchers have engineered different types of imprinting strategies that can specifically extract the target analyte in complex biological samples while simultaneously avoiding the matrix effect. Some traditional separation technologies based on MIP technology have also been studied in depth; the most common of these technologies include stationary phases used for chromatography and adsorbents for SPE. Analytical methods that combine MIT with highly sensitive detection technologies have received wide interest in fields such as disease diagnosis and bioimaging. In this review, we highlight the new MIP strategies developed in recent years, and describe the applications of MIT-based separation analysis methods in fields including chromatographic separation, SPE, diagnosis, bioimaging, and proteomics. The drawbacks of these techniques as well as their future development prospects are also discussed.
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Impressão Molecular , Humanos , Cromatografia de Afinidade/métodos , Extração em Fase Sólida/métodos , Ensaio de Imunoadsorção EnzimáticaRESUMO
Efficient separation, enrichment, and detection of bacteria in diverse media are pivotal for identifying bacterial diseases and their transmission pathways. However, conventional bacterial detection methods that split the separation and detection steps are plagued by prolonged processing times. Herein, a multistage annular functionalized carbon nanotube array device designed for the seamless integration of complex biological sample separation and multimarker detection is introduced. This device resorts to the supersmooth fluidity of the liquid sample in the carbon nanotubes interstice through rotation assistance, achieving the ability to quickly separate impurities and capture biomarkers (1 mL sample cost time of 2.5 s). Fluid dynamics simulations show that the reduction of near-surface hydrodynamic resistance drives the capture of bacteria and related biomarkers on the functionalized surface of carbon nanotube in sufficient time. When further assembled as an even detection device, it exhibited fast detection (<30 min), robust linear correlation (101-107 colony-forming units [CFU] mL-1, R2 = 0.997), ultrasensitivity (limit of detection = 1.7 CFU mL-1), and multitarget detection (Staphylococcus aureus, extracellular vesicles, and enterotoxin proteins). Collectively, the material and system offer an expanded platform for real-time diagnostics, enabling integrated rapid separation and detection of various disease biomarkers.
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Nanotubos de Carbono , Staphylococcus aureus , Nanotubos de Carbono/química , Staphylococcus aureus/isolamento & purificação , Limite de Detecção , Biomarcadores/análise , Vesículas Extracelulares/química , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/instrumentação , HidrodinâmicaRESUMO
The development of complex biological sample-compatible fluorescent molecularly imprinted polymers (MIPs) with improved performances is highly important for their real-world bioanalytical and biomedical applications. Herein, we report on the first hydrophilic "turn-on"-type fluorescent hollow MIP microparticles capable of directly, highly selectively, and rapidly optosensing hippuric acid (HA) in the undiluted human urine samples. These fluorescent hollow MIP microparticles were readily obtained through first the synthesis of core-shell-corona-structured nitrobenzoxadiazole (NBD)-labeled hydrophilic fluorescent MIP microspheres by performing one-pot surface-initiated atom transfer radical polymerization on the preformed "living" silica particles and subsequent removal of their silica core via hydrofluoric acid etching. They showed "turn-on" fluorescence and high optosensing selectivity and sensitivity toward HA in the artificial urine (the limit of detection = 0.097 µM) as well as outstanding photostability and reusability. Particularly, they exhibited much more stable aqueous dispersion ability, significantly faster optosensing kinetics, and higher optosensing sensitivity than their solid counterparts. They were also directly used for quantifying HA in the undiluted human urine with good recoveries (96.0%-102.0%) and high accuracy (RSD ≤ 4.0%), even in the presence of several analogues of HA. Such fluorescent hollow MIP microparticles hold much promise for rapid and accurate HA detection in the clinical diagnostic field.
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Impressão Molecular , Polímeros Molecularmente Impressos , Humanos , Polímeros , Corantes , Dióxido de SilícioRESUMO
Quantification of microRNA (miRNA) has attracted intense interest owing to its importance as a biomarker for the early diagnosis of multiple diseases. However, the inefficient capture of microRNAs from complex biological samples due to the passive diffusion of detection probes essentially restricts their accurate quantification. Herein, we report near-infrared (NIR)-powered Janus nanomotors composed of Au nanorods and periodic mesoporous organo-silica microspheres (AuNR/PMO JNMs) as "swimming probes" to assist a lateral flow test strip (LFTS) for direct, amplification-free, and quantitative miRNA-21 detection in serum and cell medium. The AuNR/PMO JNMs were conjugated with designed hDNA as a recognition probe for miRNA-21. Under NIR irradiation, the exposed AuNRs can generate asymmetric thermal gradients around the JNMs to achieve vigorous self-propelled thermophoretic motion. The active movement significantly accelerated the recognition of miRNA-21 targets, which greatly improved the capture efficiency from 59.39 to 86.12% in the reaction buffer. The enhanced miRNA-21 capture enabled direct quantitative miRNA-21 detection on the LFTS assay with both visual and thermal signals. Under the assistance of AuNR/PMO JNMs, a limit-of-detection of 18 fmol/L for miRNA-21 was achieved, which was 12.22-fold compared to that of LFTS assay with static probes. The constructed LFTS assay was further successfully deployed to directly sense the miRNA-21 in spiked serum samples and MDA-MB-231 medium. Overall, the AuNR/PMO JNM-assisted LFTS system unveils a concrete point-of-care testing strategy for precise miRNA detection in real biological samples, which holds great potential for early diagnosis and treatment of miRNA-related diseases.
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Técnicas Biossensoriais , MicroRNA Circulante , MicroRNAs , MicroRNAs/genética , Sondas de DNA , MicroesferasRESUMO
The efficient preparation of ratiometric fluorescent molecularly imprinted polymer (MIP) microspheres that can directly and selectively optosense a herbicide (i.e., 2,4-dichlorophenoxyacetic acid, 2,4-D) in undiluted pure milk is described. The dual fluorescent MIP microparticles were readily obtained through grafting a green 4-nitrobenzo[c][1,2,5]oxadiazole (NBD)-labeled 2,4-D-MIP layer with hydrophilic polymer brushes onto the preformed uniform "living" red CdTe quantum dot (QD)-labeled SiO2 microspheres via one-pot surface-initiated atom transfer radical polymerization (SI-ATRP) in the presence of a polyethylene glycol macro-ATRP initiator. They proved to be highly promising "turn-on"-type fluorescent chemosensors with red CdTe QD (the maximum emission wavelength λe,max around 710 nm) and green NBD (λe,max around 515 nm) as the reference fluorophore and "turn-on"-type responsive fluorophore, respectively. The sensors showed excellent photostability and reusability, high 2,4-D selectivity and sensitivity (the limit of detection = 0.12 µM), and direct visual detection ability (a fluorescent color change occurs from red to blue-green with the concentration of 2,4-D increasing from 0 to 100 µM) in pure bovine milk. The sensors were used for 2,4-D detection with high recoveries (96.0-104.0%) and accuracy (RSD ≤ 4.0%) in pure goat milk at three spiking levels of both 2,4-D and its mixtures with several analogues. This new strategy lays the foundation for efficiently developing diverse complex biological sample-compatible ratiometric fluorescent MIPs highly useful for real-world bioanalyses and diagnostics.
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Compostos de Cádmio , Herbicidas , Impressão Molecular , Pontos Quânticos , Polímeros Molecularmente Impressos , Microesferas , Telúrio , Dióxido de Silício , Herbicidas/análise , Ácido 2,4-Diclorofenoxiacético/análiseRESUMO
LC-MS/MS is a major analytical platform for metabolomics, which has become a recent hotspot in the research fields of life and environmental sciences. By contrast, structure elucidation of small molecules based on LC-MS/MS data remains a major challenge in the chemical and biological interpretation of untargeted metabolomics datasets. In recent years, several strategies for structure elucidation using LC-MS/MS data from complex biological samples have been proposed, these strategies can be simply categorized into two types, one based on structure annotation of mass spectra and for the other on retention time prediction. These strategies have helped many scientists conduct research in metabolite-related fields and are indispensable for the development of future tools. Here, we summarized the characteristics of the current tools and strategies for structure elucidation of small molecules based on LC-MS/MS data, and further discussed the directions and perspectives to improve the power of the tools or strategies for structure elucidation.
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The slow mass transport of the target molecule essentially limits the biosensing performance. Here, we report a Janus mesoporous microsphere/Pt-based (meso-MS/Pt) nanostructure with greatly enhanced target transport and accelerated recognition process for microRNA (miRNA) amplified detection in complex biological samples. The mesoporous MS was synthesized via double emulsion interfacial polymerization, and Pt nanoparticles (PtNPs) were deposited on the half-MS surface to construct Janus meso-MS/Pt micromotor. The heterogeneous meso-MS/Pt with a large surface available was attached to an entropy-driven DNA recognition system, termed meso-MS/Pt/DNA, and the tremendous pores network was beneficial to enhanced receptor-target interaction. It enabled moving around complex biological samples to greatly enhance target miRNA mass transport and accelerate recognition procedure due to the self-diffusiophoretic propulsion. Coupling with the entropy-driven signal amplification, extremely sensitive miRNA detection in Dulbecco's modified Eagle medium (DMEM), and cell lysate without preparatory and washing steps was realized. Given the free preparatory and washing steps, fast mass transport, and amplified capability, the meso-MS/Pt/DNA micromotor provides a promising method for miRNAs analysis in real biological samples.
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Técnicas Biossensoriais , MicroRNAs , Nanopartículas , MicroRNAs/genética , Nanopartículas/química , DNA , Microesferas , Técnicas Biossensoriais/métodosRESUMO
Tracking trace amounts of analytes directly from low volumes of complex biological samples remains an ongoing challenge in precision diagnostics, as the commonly used immunosorbent assays have limited sensitivity. Herein, a CRISPR/Cas12a assisted on-fibre immunosensor (CAFI) was developed based on an antibody-analyte-aptamer sandwich structure, in which a single strand DNA aptamer was applied to detect the analyte while triggering the CRISPR/Cas12a fluorescent detection system to amplify the analyte signal. This novel CAFI biosensing system was fabricated on a glass fibre surface with an antifouling PEG polymer brush modified for the detection of a spectrum of small molecules from complex media. In comparison with a conventional ELISA system, CAFI has a 1,000-fold higher sensitivity with the limit of detection for IFN-γ down to 1 fg mL-1 (58.8 aM). It also has a tuneable linear detection range that can be easily adjusted within the range 1 fg mL-1 to 100 pg mL-1 (5 orders of magnitude), meeting the requirements of the demanding diagnostic scenarios. CAFI has successfully been demonstrated by detecting IFN-γ from a diverse complex biological sample type, including human serum, whole blood, perspiration, and saliva. Moreover, CAFI is applicable for the detection of other analytes by simply modifying the capture antibody and detection aptamer, demonstrated here with insulin. All these superior capabilities of CAFI make it a suitable technology to measure proteins in low (100 µL) volume complex biological samples.
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Técnicas Biossensoriais , Bioensaio , Sistemas CRISPR-Cas , Humanos , ImunoensaioRESUMO
Preparation of molecularly imprinted polymers (MIPs) capable of directly and selectively recognizing small organic analytes in aqueous samples (particularly in the undiluted complex biological samples) is described. Such water-compatible MIPs can be readily obtained by the controlled grafting of appropriate hydrophilic polymer brushes onto the MIP particle surfaces. Two types of synthetic approaches (i.e., "two-step approach" and "one-step approach") for preparing complex biological sample-compatible hydrophilic fluorescent MIP nanoparticles and their applications for direct, selective, sensitive, and accurate optosensing of an antibiotic (i.e., tetracycline (Tc)) in the undiluted pure bovine/porcine serums are presented.
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Impressão Molecular , Animais , Bovinos , Corantes , Interações Hidrofóbicas e Hidrofílicas , Polímeros Molecularmente Impressos , Suínos , ÁguaRESUMO
The health-promoting attributes of bifidobacteria have piqued interest of researchers worldwide. However, scant published studies are available pertinent to bifidobacteria in microbiota/metagenomics datasets due to its intrinsic low abundance and limitations of detection methods. In this work, we designed a procedure to optimize the detection of the bifidobacterial population in complex biological samples with single-molecule real-time sequencing (SMRT) technology, including one primer pair designated as Bif-6 and a Bifidobacterium-specific database. The optimized procedure detected 14 bifidobacterial species/subspecies in ten human stool samples (2024 sequences per sample) and eight breast milk samples (3473 sequences per sample), respectively. Furthermore, by using the optimized procedure of SMRT, we investigated the effect of a 4-week-intervention of probiotic fermented milk (PFM; 200 g/day) on the gut bifidobacteria population of adults. The results showed that consuming PFM changed the structure and enterotype-like clusters of Bifidobacterium. After the consumption of PFM, the level of gut Bifidobacterium animalis increased significantly, replacing several originally dominating taxa in some subjects, including B. catenulatum, B. breve, and B. bifidum. On the other hand, B. adolescentis was, unaffectedly, the representative species in subjects having an original enterotype-like cluster of B. adolescentis. In conclusion, our work designed a procedure for detecting the bifidobacterial population in complex samples. By applying the currently designed procedure, we found that the PFM intervention changed the bifidobacterial enterotype-like cluster of some subjects, and such change was dependent on the basal bifidobacterial population.
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Bifidobacterium animalis , Probióticos , Adulto , Animais , Bifidobacterium , Feminino , Humanos , LeiteRESUMO
The preparation of well-defined new hydrophilic molecularly imprinted polymer (MIP) microspheres and their use as the dispersive solid-phase microextraction (dSPME) sorbents for direct and selective drug (i.e., propranolol) capture from the undiluted bovine serum are described. These MIPs have surface-grafted dense poly(2-hydroxyethyl methacrylate) (PHEMA) brushes with different molecular weights and grafting densities. They were readily prepared via the facile reversible addition-fragmentation chain transfer (RAFT) coupling chemistry. Both the molecular weights and grafting densities of PHEMA brushes showed significant influence on their complex biological sample-compatibility, and only those MIPs bearing PHEMA brushes with high enough molecular weights and grafting densities could selectively recognize propranolol in the undiluted pure milk and bovine serum. In particular, they have proven to be highly versatile dSPME sorbents for directly and selectively capturing propranolol from the undiluted bovine serum with satisfactory recoveries (85.2-97.4%) and high accuracy (RSD = 2.3-3.7%), even in the presence of one analogue of propranolol. The limit of detection was 0.002 µM with a linear correlation coefficient of 0.9994 in the range of 0.01-100 µM. Excellent precision was verified by both the intraday and interday analytical results. Their good reusability was also confirmed. This work demonstrates the high potential of such hydrophilic MIP-based dSPME sorbents for rapid, accurate, and reliable drug determination in complex biological samples.
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Impressão Molecular , Preparações Farmacêuticas , Animais , Bovinos , Interações Hidrofóbicas e Hidrofílicas , Microesferas , Microextração em Fase SólidaRESUMO
Fluorescence anisotropy (FA) has been widely applied for detecting and monitoring special targets in life sciences. However, matrix autofluorescence restricted its further application in complex biological samples. Herein, we report a near-infrared-II (NIR-II) FA strategy for detecting adenosine triphosphate (ATP) in human serum samples and breast cancer cell lysate, which employed NIR-II fluorescent Ag2Se quantum dots (QDs) as tags to reduce matrix autofluorescence effect and applied graphene oxide (GO) to enhance fluorescence anisotropy signals. In the presence of ATP, the recognition between NIR-II Ag2Se QDs labeled aptamer (QD-pDNA) and ATP led to the release of QD-pDNA from GO, resulting in the obvious decrease of FA values. ATP could be quantitatively detected in concentrations ranged from 3 nM to 2500 nM, with a detection limit down to 1.01 nM. This study showed that the developed NIR-II FA strategy could be applied for detecting targets in complex biological samples and had great potential for monitoring interactions between biomolecules in biomedical research.
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Trifosfato de Adenosina , Pontos Quânticos , Polarização de Fluorescência , Corantes Fluorescentes , HumanosRESUMO
Protein glycosylation is ubiquitous in biological systems and plays essential roles in many cellular events. Global and site-specific analysis of glycoproteins in complex biological samples can advance our understanding of glycoprotein functions and cellular activities. However, it is extraordinarily challenging because of the low abundance of many glycoproteins and the heterogeneity of glycan structures. The emergence of mass spectrometry (MS)-based proteomics has provided us an excellent opportunity to comprehensively study proteins and their modifications, including glycosylation. In this review, we first summarize major methods for glycopeptide/glycoprotein enrichment, followed by the chemical and enzymatic methods to generate a mass tag for glycosylation site identification. We next discuss the systematic and quantitative analysis of glycoprotein dynamics. Reversible protein glycosylation is dynamic, and systematic study of glycoprotein dynamics helps us gain insight into glycoprotein functions. The last part of this review focuses on the applications of MS-based proteomics to study glycoproteins in different biological systems, including yeasts, plants, mice, human cells, and clinical samples. Intact glycopeptide analysis is also included in this section. Because of the importance of glycoproteins in complex biological systems, the field of glycoproteomics will continue to grow in the next decade. Innovative and effective MS-based methods will exponentially advance glycoscience, and enable us to identify glycoproteins as effective biomarkers for disease detection and drug targets for disease treatment. © 2019 Wiley Periodicals, Inc. Mass Spec Rev 9999: XX-XX, 2019.
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Glicoproteínas/química , Espectrometria de Massas/métodos , Animais , Glicômica/métodos , Glicopeptídeos/análise , Glicosilação , Humanos , Polissacarídeos/análise , Proteômica/métodosRESUMO
It is great significance to precisely monitor lead (II) ions (Pb2+) for environment protection and human health monitoring. We designed a sensitive detection strategy for sensitive and selective determination of Pb2+, based on a Pb2+-specific DNAzyme as the catalytic unit, Cy3-labeled DNA modified gold nanorods (AuNRs) as SERS reporter. Firstly, AuNRs surface were employed as a platform for the immobilization of thiolated probe DNA, and then hybridized with DNAzyme catalytic beacons. By taking advantage of DNAzyme digest, a molecular beacon, causes a "turn-off" SERS signal by disrupting the labeled probes. Under the optical conditions, the DNAzyme-AuNRs sensor system exhibited high sensitivity, acceptable stability and reproducibility with a wide linear range from 0.5 to 100â¯nMâ¯(R2â¯=â¯0.9973), and an ultra-low detection limit of 0.01â¯nM. The proposed strategy has additional advantages of being less time-consuming, low-cost and remote query, and avoids the interference of some metals such as Fe3+, Cd2+, Ba2+, Cu2+, Zn2+. The SERS biosensor system has been successfully applied for detecting Pb2+ in real samples with a satisfactory result. The result indicated that the proposed sensing strategy not only enriches SERS platform of monitoring Pb2+ but also exhibits potential for the point-of-care diagnostic application of the clinical screening in complicated biological samples.
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There is an urgent need of sensitive bioanalytical platforms for sensitive and precise quantification of low-abundance microRNA targets in complex biological samples, including liquid biopsies of tumors. Many of current miRNA biosensing methods require laborious sample pretreatment procedures, including extraction of total RNA, which largely limits their biomedical and clinical applications. Herein we developed an integrated Microfluidic Exponential Rolling Circle Amplification (MERCA) platform for sensitive and specific detection of microRNAs directly in minimally processed samples. The MERCA system integrates and streamlines solid-phase miRNA isolation, miRNA-adapter ligation, and a dualphase exponential rolling circle amplification (eRCA) assay in one analytical workflow. By marrying the advantages of microfluidics in leveraging bioassay performance with the high sensitivity of eRCA, our method affords a remarkably low limit of detection at <10 zeptomole levels, with the ability to discriminate single-nucleotide difference. Using the MERCA chip, we demonstrated quantitative detection of miRNAs in total RNA, raw cell lysate, and cellderived exosomes. Comparing with the parallel TaqMan RT-qPCR measurements verified the adaptability of the MERCA system for detection of miRNA biomarkers in complex biological materials. In particular, high sensitivity of our method enables direct detection of low-level exosomal miRNAs in as few as 2 × 106 exosomes. Such analytical capability immediately addresses the unmet challenge in sample consumption, a key setback in clinical development of exosome-based liquid biopsies. Therefore, the MERCA would provide a useful platform to facilitate miRNA analysis in broad biological and clinical applications.
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Proteases and their inhibitors have become molecules of increasing fundamental and applicative value. Here we report an integrated strategy to identify and analyze such inhibitors from Caribbean marine invertebrates extracts by a fast and sensitive functional proteomics-like approach. The strategy works in three steps: i) multiplexed enzymatic inhibition kinetic assays, ii) Intensity Fading MALDI-TOF MS to establish a link between inhibitory molecules and the related MALDI signal(s) detected in the extract(s), and iii) ISD-CID-T3 MS fragmentation on the parent MALDI signals selected in the previous step, enabling the partial or total top-down sequencing of the molecules. The present study has allowed validation of the whole approach, identification of a substantial number of novel protein protease inhibitors, as well as full or partial sequencing of reference molecular species and of many unknown ones, respectively. Such inhibitors correspond to six protease subfamilies (metallocarboxypeptidases-A and -B, pepsin, papain, trypsin and subtilisin), are small (1-10KDa) disulfide-rich proteins, and have been found at diverse frequencies among the invertebrates (13 to 41%). The overall procedure could be tailored to other enzyme-inhibitor and protein interacting systems, analyzing samples at medium-throughput level and leading to the functional and structural characterization of proteinaceous ligands from complex biological extracts. SIGNIFICANCE: Invertebrate animals, and marine ones among, display a remarkable diversity of species and contained biomolecules. Many of their proteins-peptides have high biological, biotechnological and biomedical potential interest but, because of the lack of sequenced genomes behind, their structural and functional characterization constitutes a great challenge. Here, looking at the small, disulfide-rich, proteinaceous inhibitors of proteases found in them, it is shown that such problem can be significatively facilitated by integrative multiplexed enzymatic assays, affinity-based Intensity-Fading (IF-) MALDI-TOF mass spectrometry (MS), and on-line MS fragmentation, in a fast and easy approach.