Dual Contributions of Analyte Adsorption and Electroosmotic Inhomogeneity to Separation Efficiency in Capillary Electrophoresis of Proteins.

Improving separation efficiency in capillary electrophoresis (CE) requires systematic study of the influence of the electric field (or solute linear velocity) on plate height for a better understanding of the critical parameters controlling peak broadening. Even for poly(diallyldimethylammonium chloride) (PDADMAC)/poly(sodium styrenesulfonate) (PSS) successive multiple ionic-polymer layer (SMIL) coatings, which lead to efficient and reproducible separations of proteins, plate height increases with migration velocity, limiting the use of high electric fields in CE. Solute adsorption onto the capillary wall was generally considered as the main source of peak dispersion, explaining this plate height increase. However, experiments done with Taylor dispersion analysis and CE in the same conditions indicate that other phenomena may come into play. Protein adsorption with slow kinetics and few adsorption sites was established as a source of peak broadening for specific proteins. Surface charge inhomogeneity was also identified as a contribution to plate height due to local electroosmotic fluctuations. A model was proposed and applied to partial PDADMAC/poly(ethylene oxide) capillary coatings as well as PDADMAC/PSS SMIL coatings. Atomic force microscopy with topography and recognition imaging enabled the determination of roughness and charge distribution of the PDADMAC/PSS SMIL surface.

Taylor-Aris Dispersion-Assisted Mass Spectrometry for the Analysis of Native Proteins.

As has recently been shown, Taylor-Aris dispersion-assisted mass spectrometry (TADA-MS) can offer direct injection MS determinations in fields where the targets of the analyses are large molecules present in a matrix that would otherwise cause serious interferences. In the present study, we demonstrated the exceptional utility of TADA-MS in native protein analysis: (i) a dramatic improvement in detection sensitivity was found due to its ability to strongly reduce matrix interferences, (ii) more "native-like" conditions can be used during analyses, (iii) the direct injection of non-MS-compatible matrices is allowed into MS, and (iv) a considerable simplification and economization of the workflow is ensured. We investigated the behavior of different types of proteins and protein complexes present under native conditions, demonstrating the unambiguous benefits and simplicity of the method.

Emerging Designs and Applications for Biomembrane Biosensors.

Nature has inspired the development of biomimetic membrane sensors in which the functionalities of biological molecules, such as proteins and lipids, are harnessed for sensing applications. This review provides an overview of the recent developments for biomembrane sensors compatible with either bulk or planar sensing applications, namely using lipid vesicles or supported lipid bilayers, respectively. We first describe the individual components required for these sensing platforms and the design principles that are considered when constructing them, and we segue into recent applications being implemented across multiple fields. Our goal for this review is to illustrate the versatility of nature's biomembrane toolbox and simultaneously highlight how biosensor platforms can be enhanced by harnessing it.

AI-Assisted Processing Pipeline to Boost Protein Isoform Detection.

Proteomics, the study of proteins within biological systems, has seen remarkable advancements in recent years, with protein isoform detection emerging as one of the next major frontiers. One of the primary challenges is achieving the necessary peptide and protein coverage to confidently differentiate isoforms as a result of the protein inference problem and protein false discovery rate estimation challenge in large data. In this chapter, we describe the application of artificial intelligence-assisted peptide property prediction for database search engine rescoring by Oktoberfest, an approach that has proven effective, particularly for complex samples and extensive search spaces, which can greatly increase peptide coverage. Further, it illustrates a method for increasing isoform coverage by the PickedGroupFDR approach that is designed to excel when applied on large data. Real-world examples are provided to illustrate the utility of the tools in the context of rescoring, protein grouping, and false discovery rate estimation. By implementing these cutting-edge techniques, researchers can achieve a substantial increase in both peptide and isoform coverage, thus unlocking the potential of protein isoform detection in their studies and shedding light on their roles and functions in biological processes.

Data-Independent Acquisition Represents a Promising Alternative for Fast Photochemical Oxidation of Proteins (FPOP) Samples Analysis.

Fast Photochemical Oxidation of Proteins (FPOP) is a protein footprinting method utilizing hydroxyl radicals to provide valuable information on the solvent-accessible surface area. The extensive number of oxidative modifications that are created by FPOP is both advantageous, leading to great spatial resolution, and challenging, increasing the complexity of data processing. The precise localization of the modification together with the appropriate reproducibility is crucial to obtain relevant structural information. In this paper, we propose a novel approach combining validated spectral libraries together with utilizing DIA data. First, the DDA data searched by FragPipe are subsequently validated using Skyline software to form a spectral library. This library is then matched against the DIA data to filter out nonrepresentative IDs. In comparison with FPOP data processing using only a search engine followed by generally applied filtration steps, the manually validated spectral library offers higher confidence in identifications and increased spatial resolution. Furthermore, the reproducibility of quantification was compared for DIA, DDA, and MS-only acquisition modes on timsTOF SCP. Comparison of coefficients of variation (CV) showed that the DIA and MS acquisition modes exhibit significantly better reproducibility in quantification (CV medians 0.1233 and 0.1494, respectively) compared to the DDA mode (CV median 0.2104).

Diagnostic Utility of Selected Matrix Metalloproteinases (MMP-2, MMP-3, MMP-11, MMP-26), HE4, CA125 and ROMA Algorithm in Diagnosis of Ovarian Cancer.

Ovarian cancer (OC) has an unfavorable prognosis. Due to the lack of effective screening tests, new diagnostic methods are being sought to detect OC earlier. The aim of this study was to evaluate the concentration and diagnostic utility of selected matrix metalloproteinases (MMPs) as OC markers in comparison with HE4, CA125 and the ROMA algorithm. The study group consisted of 120 patients with OC; the comparison group consisted of 70 patients with benign lesions and 50 healthy women. MMPs were determined via the ELISA method, HE4 and CA125 by CMIA. Patients with OC had elevated levels of MMP-3 and MMP-11, similar to HE4, CA125 and ROMA values. The highest SE, SP, NPV and PPV values were found for MMP-26, CA125 and ROMA in OC patients. Performing combined analyses of ROMA with selected MMPs increased the values of diagnostic parameters. The topmost diagnostic power of the test was obtained for MMP-26, CA125, HE4 and ROMA and performing combined analyses of MMPs and ROMA enhanced the diagnostic power of the test. The obtained results indicate that the tested MMPs do not show potential as stand-alone OC biomarkers, but can be considered as additional tests to raise the diagnostic utility of the ROMA algorithm.

Extraction of Proteins from Municipal Wastewater and Activated Sludge.

Wastewater treatment plants (WWTPs) are the main barrier to cope with the increased pressure of municipal and industrial wastewater on natural water resources in terms of both polluting load and produced volumes. For this reason, WWTP's efficiency should be the highest; thus, their monitoring becomes critical. In conventional WWTPs, biodegradation of pollutants mainly occurs in the biological reactors, and an increasing interest in a deeper characterization of the biomasses involved in these processes (made of biofilms, granules, and suspended activated sludge) rose up in recent years. In this sense, the meta-omics approaches were recently developed to investigate the entire set of biomolecules of a given class in a microbial community with the same general objective: the identification of the biomolecules through the sequence similarity of high degree in the already available databases. Particularly, metaproteomics concerns the identification of all proteins in a microbial community in a given moment or condition. In this chapter, a protocol for the extraction and separation of proteins from activate sludge sampled at WWTPs is proposed.

Extraction of Proteins from Fermented Food.

The metaproteomic approach allows a deep microbiome characterization in different complex systems. Based on metaproteome data, microbial communities' composition, succession, and functional role in different environmental conditions can be established.The main challenge in metaproteomic studies is protein extraction, and although many protocols have been developed, a few are focused on the protein extraction of fermented foods. In this chapter, a reproducible and efficient method for the extraction of proteins from a traditionally fermented starchy food is described. The method can be applied to any fermented food and aims to enrich the extraction of proteins from microorganisms for their subsequent characterization.

Extraction of Proteins from Soil.

Soil metaproteomics could explore the proteins involved in life activities and their abundance in the soils to overcome the difficulty in pure cultures of soil microorganisms and the limitations of proteomics of pure cultures. However, the complexity and heterogeneity of soil composition, the low abundance of soil proteins, and the presence of massive interfering substances (including humic compounds) generally lead to an extremely low extraction efficiency of soil proteins. Therefore, the efficient extraction of soil proteins is a prerequisite and bottleneck problem in soil metaproteomics. In this chapter, a soil protein extraction method suitable for most types of soils with low cost and enabling simple operation (about 150 µg protein can be extracted from 5.0 g soil) is described. The quantity and purity of the extracted soil proteins could meet the requirements for further analysis using routine mass spectrometry-based proteomics.

Extraction of Proteins from Marine Microbial Communities Sampled by Lab Filters.

A method for the recovery of whole-cell protein extracts from biomass on membrane filters is provided here. The protein extraction method is ideal for biomass captured by filtration of large water volumes, including seawater from marine environments. The protein extraction method includes both chemical disruption and physical disruption to lyse cells and release protein for subsequent metaproteomic analysis.

Extraction of Proteins from Microbiome of Livestocks.

The development of high throughput methods has enabled the study of hundreds of samples and metaproteomics is not the exception. However, the study of thousands of proteins of different organisms represents different challenges from the protein extraction to the bioinformatic analysis. Here, the sample preparation, protein extraction and protein purification for livestock microbiome research throughout metaproteomics are described. These methods are essential because the quality of the final protein pool depends on them. For that reason, the following workflow is a combination of different chemical and physical methods that intend an initial separation of the microbial organisms from the host cells and other organic materials, as well as the extraction of high concentrate pure samples.

Evaluation of a Commercial TIMS-Q-TOF Platform for Native Mass Spectrometry.

Mass-spectrometry based assays in structural biology studies measure either intact or digested proteins. Typically, different mass spectrometers are dedicated for such measurements: those optimized for rapid analysis of peptides or those designed for high molecular weight analysis. A commercial trapped ion mobility-quadrupole-time-of-flight (TIMS-Q-TOF) platform is widely utilized for proteomics and metabolomics, with ion mobility providing a separation dimension in addition to liquid chromatography. The ability to perform high-quality native mass spectrometry of protein complexes, however, remains largely uninvestigated. Here, we evaluate a commercial TIMS-Q-TOF platform for analyzing noncovalent protein complexes by utilizing the instrument's full range of ion mobility, MS, and MS/MS (both in-source activation and collision cell CID) capabilities. The TIMS analyzer is able to be tuned gently to yield collision cross sections of native-like complexes comparable to those previously reported on various instrument platforms. In-source activation and collision cell CID were robust for both small and large complexes. TIMS-CID was performed on protein complexes streptavidin (53 kDa), avidin (68 kDa), and cholera toxin B (CTB, 58 kDa). Complexes pyruvate kinase (237 kDa) and GroEL (801 kDa) were beyond the trapping capabilities of the commercial TIMS analyzer, but TOF mass spectra could be acquired. The presented results indicate that the commercial TIMS-Q-TOF platform can be used for both omics and native mass spectrometry applications; however, modifications to the commercial RF drivers for both the TIMS analyzer and quadrupole (currently limited to m/z 3000) are necessary to mobility analyze protein complexes greater than about 60 kDa.

Determination of physicochemical parameters of (bio)molecules and (bio)particles by capillary electromigration methods.

The review provides an overview of recent developments and applications of capillary electromigration (CE) methods for the determination of important physicochemical parameters of various (bio)molecules and (bio)particles. These parameters include actual and limiting (absolute) ionic mobilities, effective electrophoretic mobilities, effective charges, isoelectric points, electrokinetic potentials, hydrodynamic radii, diffusion coefficients, relative molecular masses, acidity (ionization) constants, binding constants and stoichiometry of (bio)molecular complexes, changes of Gibbs free energy, enthalpy and entropy and rate constants of chemical reactions and interactions, retention factors and partition and distribution coefficients. For the determination of these parameters, the following CE methods are employed: zone electrophoresis in a free solution or in sieving media, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography. In the individual sections, the procedures for the determination of the above parameters by the particular CE methods are described.

The path forward for protein footprinting, covalent labeling, and mass spectrometry-based protein conformational analyses.

Mass spectrometry-based approaches to assess protein conformation have become widely utilized due to their sensitivity, low sample requirements, and broad applicability to proteins regardless of size and environment. Their wide applicability and sensitivity also make these techniques suitable for the analysis of complex mixtures of proteins, and thus, they have been applied at the cell and even the simple organism levels. These works are impressive, but they predominately employ "bottom-up" workflows and require proteolytic digestion prior to analysis. Once digested, it is not possible to distinguish the proteoform from which any single peptide is derived and therefore, one cannot associate distal-in primary structure-concurrent post-translational modifications (PTMs) or covalent labels, as they would be found on separate peptides. Thus, analyses via bottom-up proteomics report the average PTM status and higher-order structure (HOS) of all existing proteoforms. Second, these works predominately employ promiscuous reagents to probe protein HOS. While this does lead to improved conformational resolution, the formation of many products can divide the signal associated with low-copy number proteins below signal-to-noise thresholds and complicate the bioinformatic analysis of these already challenging systems. In this perspective, I further detail these limitations and discuss the positives and negatives of top-down proteomics as an alternative.

Development and Validation of Miniaturized Assays to Assess Protein Techno-functional Properties.

Techno-functional properties of protein isolates such as emulsification, foaming, and gelling serve as key indicators to determine their food applications. Conventional macro-volume techniques used to measure these techno-functional properties are usually time consuming, require large amounts of protein samples, and are impractical when diverse protein samples are handled at the early screening stage. To overcome these issues, we have developed scaled-down (miniaturized) assays to test techno-functional properties of protein samples. These assays are simple, efficient, and require <400 µl of protein solution. Specifically, the miniaturized emulsification and gelling assays require 25-fold less protein than conventional macro-volume techniques and the miniaturized foaming assay requires 100-fold less sample. The performance of these assays has been thoroughly validated using conventional techno-functional tests for each parameter. The protocols described herein offer high-throughput screening capabilities, accelerating the testing process for protein techno-functional properties and allowing for quick identification of samples of interest from diverse samples. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Miniaturized emulsification assay Alternate Protocol 1: Conventional macro-volume emulsification assay Basic Protocol 2: Miniaturized foaming assay Alternate Protocol 2: Conventional macro-volume foaming assay Basic Protocol 3: Miniaturized gelling assay Alternate Protocol 3: Conventional macro-volume gelling assay.

Electrophoretic Microplate Protein Identification Based on Gold Staining of Molybdenum Disulfide Hydrogels.

Numerous high-performance nanotechnologies have been developed, but their practical applications are largely restricted by the nanomaterials' low stabilities and high operation complexity in aqueous substrates. Herein, we develop a simple and high-reliability hydrogel-based nanotechnology based on the in situ formation of Au nanoparticles in molybdenum disulfide (MoS2)-doped agarose (MoS2/AG) hydrogels for electrophoresis-integrated microplate protein recognition. After the incubation of MoS2/AG hydrogels in HAuCl4 solutions, MoS2 nanosheets spontaneously reduce Au ions, and the hydrogels are remarkably stained with the color of as-synthetic plasmonic Au hybrid nanomaterials (Au staining). Proteins can precisely mediate the morphologies and optical properties of Au/MoS2 heterostructures in the hydrogels. Consequently, Au staining-based protein recognition is exhibited, and hydrogels ensure the comparable stabilities and sensitivities of protein analysis. In comparison to the fluorescence imaging and dye staining, enhanced sensitivity and recognition performances of proteins are implemented by Au staining. In Au staining, exfoliated MoS2 semiconductors directly guide the oriented growth of plasmonic Au nanostructures in the presence of formaldehyde, showing environment-friendly features. The Au-stained hydrogels merge the synthesis and recognition applications of plasmonic Au nanomaterials. Significantly, the one-step incubation of the electrophoretic hydrogels leads to high simplicity of operation, largely challenging those multiple-step Ag staining routes which were performed with high complexity and formaldehyde toxicity. Due to its toxic-free, simple, and sensitive merits, the Au staining integrated with electrophoresis-based separation and microplate-based high-throughput measurements exhibits highly promising and improved practicality of those developing nanotechnologies and largely facilitates in-depth understanding of biological information.

iSERS: from nanotag design to protein assays and ex vivo imaging.

Proteins are an eminently important class of ubiquitous biomacromolecules with diverse biological functions, and numerous techniques for their detection, quantification, and localisation have been developed. Many of these methods exploit the selectivity arising from molecular recognition of proteins/antigens by immunoglobulins. The combination of surface-enhanced Raman scattering (SERS) with such "immuno"-techniques to immuno-SERS (iSERS) is the central topic of this review, which is focused on colloidal SERS nanotags, i.e., molecularly functionalised noble metal nanoparticles conjugated to antibodies, for their use in protein assays and ex vivo imaging. After contrasting the fundamental differences between label-free SERS and iSERS, including a balanced description of the advantages and drawbacks of the latter, we describe the usual workflow of iSERS experiments. Milestones in the development of the iSERS technology are summarised from a historical perspective. By highlighting selected examples from the literature, we illustrate the conceptual progress that has been achieved in the fields of iSERS-based protein assays and ex vivo imaging. Finally, we attempt to predict what is necessary to fully exploit the transformative potential of the iSERS technology by stimulating the transition from research in academic labs into applications for the benefit of our society.

Suborgan Level Quantitation of Proteins in Tissues Delivered by Polymeric Nanocarriers.

Amidst the rapid growth of protein therapeutics as a drug class, there is an increased focus on designing systems to effectively deliver proteins to target organs. Quantitative monitoring of protein distributions in tissues is essential for optimal development of delivery systems; however, existing strategies can have limited accuracy, making it difficult to assess suborgan dosing. Here, we describe a quantitative imaging approach that utilizes metal-coded mass tags and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to quantify the suborgan distributions of proteins in tissues that have been delivered by polymeric nanocarriers. Using this approach, we measure nanomole per gram levels of proteins as delivered by guanidinium-functionalized poly(oxanorborneneimide) (PONI) polymers to various tissues, including the alveolar region of the lung. Due to the multiplexing capability of the LA-ICP-MS imaging, we are also able to simultaneously quantify protein and polymer distributions, obtaining valuable information about the relative excretion pathways of the protein cargo and carrier. This imaging approach will facilitate quantitative correlations between nanocarrier properties and protein cargo biodistributions.

Comparison of the precursor, amino acid oxidation, and end-product methods for the evaluation of protein turnover in senior dogs.

Stable isotope methods have been used to study protein metabolism in humans; however, there application in dogs has not been frequently explored. The present study compared the methods of precursor (13C-Leucine), end-products (15N-Glycine), and amino acid oxidation (13C-Phenylalanine) to determine the whole-body protein turnover rate in senior dogs. Six dogs (12.7 ± 2.6 years age, 13.6 ± 0.6 kg bodyweight) received a dry food diet for maintenance and were subjected to all the above-mentioned methods in succession. To establish 13C and 15N kinetics, according to different methodologies blood plasma, urine, and expired air were collected using a specifically designed mask. The volume of CO2 was determined using respirometry. The study included four methods viz. 13C-Leucine, 13C-Phenylalanine evaluated with expired air, 13C-Phenylalanine evaluated with urine, and 15N-Glycine, with six dogs (repetitions) per method. Data was subjected to variance analysis and means were compared using the Tukey test (P<0.05). In addition, the agreement between the methods was evaluated using Pearson correlation and Bland-Altman statistics. Protein synthesis (3.39 ± 0.33 g.kg-0,75. d-1), breakdown (3.26 ± 0.18 g.kg-0.75.d-1), and flux estimations were similar among the four methods of study (P>0.05). However, only 13C-Leucine and 13C-Phenylalanine (expired air) presented an elevated Pearson correlation and concordance. This suggested that caution should be applied while comparing the results with the other methodologies.

Comprehensive Micro-SPE-Based Bottom-Up Proteomic Workflow for Sensitive Analysis of Limited Samples.

Clinical and biological samples are often scarce and precious (e.g., rare cell isolates, microneedle tissue biopsies, small-volume liquid biopsies, and even single cells or organelles). Typical large-scale proteomic methods, where significantly higher protein amounts are analyzed, are not directly transferable to the analysis of limited samples due to their incompatibility with pg-, ng-, and low-µg-level protein sample amounts. Here, we report the on-microsolid-phase extraction tip (OmSET)-based sample preparation workflow for sensitive analysis of limited biological samples to address this challenge. The developed platform was successfully tested for the analysis of 100-10,000 typical mammalian cells and is scalable to allow for lower and larger protein amounts and more samples to be analyzed (i.e., higher throughput of analysis).