Exploring the fragmentation efficiency of proteins analyzed by MALDI-TOF-TOF tandem mass spectrometry using computational and statistical analyses.

Matrix-assisted laser desorption/ionization time-of-flight-time-of-flight (MALDI-TOF-TOF) tandem mass spectrometry (MS/MS) is a rapid technique for identifying intact proteins from unfractionated mixtures by top-down proteomic analysis. MS/MS allows isolation of specific intact protein ions prior to fragmentation, allowing fragment ion attribution to a specific precursor ion. However, the fragmentation efficiency of mature, intact protein ions by MS/MS post-source decay (PSD) varies widely, and the biochemical and structural factors of the protein that contribute to it are poorly understood. With the advent of protein structure prediction algorithms such as Alphafold2, we have wider access to protein structures for which no crystal structure exists. In this work, we use a statistical approach to explore the properties of bacterial proteins that can affect their gas phase dissociation via PSD. We extract various protein properties from Alphafold2 predictions and analyze their effect on fragmentation efficiency. Our results show that the fragmentation efficiency from cleavage of the polypeptide backbone on the C-terminal side of glutamic acid (E) and asparagine (N) residues were nearly equal. In addition, we found that the rearrangement and cleavage on the C-terminal side of aspartic acid (D) residues that result from the aspartic acid effect (AAE) were higher than for E- and N-residues. From residue interaction network analysis, we identified several local centrality measures and discussed their implications regarding the AAE. We also confirmed the selective cleavage of the backbone at D-proline bonds in proteins and further extend it to N-proline bonds. Finally, we note an enhancement of the AAE mechanism when the residue on the C-terminal side of D-, E- and N-residues is glycine. To the best of our knowledge, this is the first report of this phenomenon. Our study demonstrates the value of using statistical analyses of protein sequences and their predicted structures to better understand the fragmentation of the intact protein ions in the gas phase.

Turn-off enzyme activity of histidine-rich peptides for the detection of lysozyme.

An assay that integrates histidine-rich peptides (HisRPs) with high-affinity aptamers was developed enabling the specific and sensitive determination of the target lysozyme. The enzyme-like activity of HisRP is inhibited by its interaction with a target recognized by an aptamer. In the presence of the target, lysozyme molecules progressively assemble on the surface of HisRP in a concentration-dependent manner, resulting in the gradual suppression of enzyme-like activity. This inhibition of HisRP's enzyme-like activity can be visually observed through color changes in the reaction product or quantified using UV-visible absorption spectroscopy. Under optimal conditions, the proposed colorimetric assay for lysozyme had a detection limit as low as 1 nM and exhibited excellent selectivity against other nonspecific interferents. Furthermore, subsequent research validated the practical applicability of the developed colorimetric approach to saliva samples, indicating that the assay holds significant potential for the detection of lysozymes in samples derived from humans.

Advancements in clinical approaches, analytical methods, and smart sampling for LC-MS-based protein determination from dried matrix spots.

Determination of proteins from dried matrix spots using MS is an expanding research area. Mainly, the collected dried matrix sample is whole blood from a finger or heal prick, resulting in dried blood spots. However as other matrices such as plasma, serum, urine, and tear fluid also can be collected in this way, the term dried matrix spot is used as an overarching term. In this review, the focus is on advancements in the field made from 2017 up to 2023. In the first part reviews concerning the subject are discussed. After this, advancements made for clinical purposes are highlighted. Both targeted protein analyses, with and without the use of affinity extractions, as well as untargeted, global proteomic approaches are discussed. In the last part, both methodological advancements are being reviewed as well as the possibility to integrate sample preparation steps during the sample handling. The focus, of this so-called smart sampling, is on the incorporation of cell separation, proteolysis, and antibody-based affinity capture.

Exploring affinity chromatography in proteomics: A comprehensive review.

Over the past decades, the proteomics field has undergone rapid growth. Progress in mass spectrometry and bioinformatics, together with separation methods, has brought many innovative approaches to the study of the molecular biology of the cell. The potential of affinity chromatography was recognized immediately after its first application in proteomics, and since that time, it has become one of the cornerstones of many proteomic protocols. Indeed, this chromatographic technique exploiting the specific binding between two molecules has been employed for numerous purposes, from selective removal of interfering (over)abundant proteins or enrichment of scarce biomarkers in complex biological samples to mapping the post-translational modifications and protein interactions with other proteins, nucleic acids or biologically active small molecules. This review presents a comprehensive survey of this versatile analytical tool in current proteomics. To navigate the reader, the haphazard space of affinity separations is classified according to the experiment's aims and the separated molecule's nature. Different types of available ligands and experimental strategies are discussed in further detail for each of the mentioned procedures.

Microwave-assisted and methanol/acetic acid-free method for rapid staining of proteins in SDS-PAGE gels.

We describe a microwave-assisted, methanol and acetic acid-free, inexpensive method for rapid staining of SDS-PAGE proteins. Only citric acid, benzoic acid, and Coomassie brilliant blue G-250 (CBG) were used. Microwave irradiation reduced the detection duration, and proteins in a clear background were visualized within 30 min of destaining, after 2 min of fixing and 12 min of staining. By using this protocol, comparable band intensities were obtained to the conventional methanol/acetic acid method.

Biochemical changes in lipid and protein metabolism caused by mannose-Raman spectroscopy studies.

Biochemical analysis of human normal bronchial cells (BEpiC) and human cancer lung cells (A549) has been performed by using Raman spectroscopy and Raman imaging. Our approach provides a biochemical compositional mapping of the main cell components: nucleus, mitochondria, lipid droplets, endoplasmic reticulum, cytoplasm and cell membrane. We proved that Raman spectroscopy and Raman imaging can distinguish successfully BEpiC and A549 cells. In this study, we have focused on the role of mannose in cancer development. It has been shown that changes in the concentration of mannose can regulate some metabolic processes in cells. Presented results suggest lipids and proteins can be considered as Raman biomarkers during lung cancer progression. Analysis obtained for bands 1444 cm-1, and 2854 cm-1 characteristic for lipids and derivatives proved that the addition of mannose reduced levels of these compounds. Results obtained for protein compounds based on bands 858 cm-1, 1004 cm-1 and 1584 cm-1 proved that the addition of mannose increases the values of protein in BEpiC cells and blocks protein glycolisation in A549 cells. Noticing Raman spectral changes in BEpiC and A549 cells supplemented with mannose can help to understand the mechanism of sugar metabolism during cancer development and could play in the future an important role in clinical treatment.

Human epididymal protein 4 and its combined detection show good diagnostic value in lung cancer: A retrospective study.

OBJECTIVES: This study aimed to assess the diagnostic value of human epididymal protein 4 (HE4), a potential novel biomarker for lung cancer, and its combined detection with five other conventional biomarkers in lung cancer diagnosis and subtyping. METHODS: In this retrospective study, 115 lung cancer patients, 50 patients with benign pulmonary disease, and 50 healthy controls were included. Serum HE4, progastrin-releasing peptide (ProGRP), squamous cell carcinoma (SCC) antigen, cytokeratin-19 fragment (CYFRA21-1), neuron-specific enolase (NSE), and carcinoembryonic antigen (CEA) were analyzed using the electrochemiluminescence immunoassay and chemiluminescence immunoassay. The receiver operating characteristic curve was performed to analyze the diagnostic efficacy of individual biomarkers in identifying both lung cancer and its histologic subtypes. RESULTS: All six biomarkers showed significantly elevated levels in the lung cancer group compared to both benign pulmonary disease and control groups (P < 0.05). Among the biomarkers evaluated, HE4 exhibited the highest diagnostic performance for lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma with area under the curve (AUC) values of 0.921, 0.891, and 0.937, respectively. ProGRP was the optimal biomarker for small cell lung cancer with an AUC of 0.973. The combination of all six biomarkers yielded the largest AUCs in the diagnosis of lung cancer subtypes (0.937 for lung adenocarcinoma, 0.998 for lung squamous cell carcinoma, and 0.985 for small cell lung cancer). Furthermore, specific combinations, such as HE4 + CEA, HE4 + SCC, and ProGRP + HE4 + NSE, showed strong diagnostic performance in lung cancer. CONCLUSIONS: HE4 and its combined detection held substantial clinical significance in the diagnosis of lung cancer and its histologic subtyping, especially for lung adenocarcinoma and lung squamous cell carcinoma.

Size exclusion chromatography of biopharmaceutical products: From current practices for proteins to emerging trends for viral vectors, nucleic acids and lipid nanoparticles.

The 21st century has been particularly productive for the biopharmaceutical industry, with the introduction of several classes of innovative therapeutics, such as monoclonal antibodies and related compounds, gene therapy products, and RNA-based modalities. All these new molecules are susceptible to aggregation and fragmentation, which necessitates a size variant analysis for their comprehensive characterization. Size exclusion chromatography (SEC) is one of the reference techniques that can be applied. The analytical techniques for mAbs are now well established and some of them are now emerging for the newer modalities. In this context, the objective of this review article is: i) to provide a short historical background on SEC, ii) to suggest some clear guidelines on the selection of packing material and mobile phase for successful method development in modern SEC; and iii) to highlight recent advances in SEC, such as the use of narrow-bore and micro-bore columns, ultra-wide pore columns, and low-adsorption column hardware. Some important innovations, such as recycling SEC, the coupling of SEC with mass spectrometry, and the use of alternative detectors such as charge detection mass spectrometry and mass photometry are also described. In addition, this review discusses the use of SEC in multidimensional setups and shows some of the most recent advances at the preparative scale. In the third part of the article, the possibility of SEC for the characterization of new modalities is also reviewed. The final objective of this review is to provide a clear summary of opportunities and limitations of SEC for the analysis of different biopharmaceutical products.

Improved assay development of pharmaceutical modalities using feedback-controlled liquid chromatography optimization.

Development of meaningful and reliable analytical assays in the (bio)pharmaceutical industry can often be challenging, involving tedious trial and error experimentation. In this work, an automated analytical workflow using an AI-based algorithm for streamlined method development and optimization is presented. Chromatographic methods are developed and optimized from start to finish by a feedback-controlled modeling approach using readily available LC instrumentation and software technologies, bypassing manual user intervention. With the use of such tools, the time requirement of the analyst is drastically minimized in the development of a method. Herein key insights on chromatography system control, automatic optimization of mobile phase conditions, and final separation landscape for challenging multicomponent mixtures are presented (e.g., small molecules drug, peptides, proteins, and vaccine products) showcased by a detailed comparison of a chiral method development process. The work presented here illustrates the power of modern chromatography instrumentation and AI-based software to accelerate the development and deployment of new separation assays across (bio)pharmaceutical modalities while yielding substantial cost-savings, method robustness, and fast analytical turnaround.

Insulin-like peptide 3 (INSL3) as an indicator of leydig cell insufficiency (LCI) in Middle-aged and older men with hypogonadism: reference range and threshold.

Insulin-like peptide 3 (INSL3) is a circulating biomarker for Leydig cell functional capacity in men, also indicating Leydig Cell Insufficiency (LCI) and potential primary hypogonadism. Using results from large cohort studies we explore sources of biological and technical variance, and establish a reference range for adult men. It is constitutively secreted with little within-individual variation and reflects testicular capacity to produce testosterone. The main INSL3 assays available indicate good concordance with low technical variance; there is no effect of ethnicity. INSL3 declines with age from 35 years at about 15% per decade. Like low calculated free testosterone, and to a lesser extent low total testosterone, reduced INSL3 is significantly associated with increasing age-related morbidity, including lower overall sexual function, reflecting LCI. Consequently, low INSL3 (≤0.4 ng/ml; ca. <2 SD from the population mean) might serve as an additional biochemical marker in the assessment of functional hypogonadism (late-onset hypogonadism, LOH) where testosterone is in the borderline low range. Excluding individuals with low LCI (INSL3 ≤ 0.4 ng/ml) leads to an age-independent (> 35 years) reference range (serum) for INSL3 in the eugonadal population of 0.4 - 2.3 ng/ml, with low INSL3 prospectively identifying individuals at risk of increased future morbidity.

Deciphering biomolecular complexities: the indispensable role of surface-enhanced Raman spectroscopy in modern bioanalytical research.

Surface-enhanced Raman spectroscopy (SERS) has emerged as an indispensable analytical tool in biomolecular research, providing unmatched sensitivity critical for the elucidation of biomolecular structures. This review presents a thorough examination of SERS, outlining its fundamental principles, cataloging its varied applications within the biomolecular sphere, and contemplating its future developmental trajectories. We begin with a detailed analysis of SERS's mechanistic principles, emphasizing both the phenomena of surface enhancement and the complexities inherent in Raman scattering spectroscopy. Subsequently, we delve into the pivotal role of SERS in the structural analysis of diverse biomolecules, including proteins, nucleic acids, lipids, carbohydrates, and biochromes. The remarkable capabilities of SERS extend beyond mere detection, offering profound insights into biomolecular configurations and interactions, thereby enriching our comprehension of intricate biological processes. This review also sheds light on the application of SERS in real-time monitoring of various bio-relevant compounds, from enzymes and coenzymes to metal ion-chelate complexes and cellular organelles, thereby providing a holistic view and empowering researchers to unravel the complexities of biological systems. We also address the current challenges faced by SERS, such as enhancing sensitivity and resolution, developing stable and reproducible substrates, and conducting thorough analyses in complex biological matrices. Nonetheless, the continual advancements in nanotechnology and spectroscopy solidify the standing of SERS as a formidable force in biomolecular research. In conclusion, the versatility and robustness of SERS not only deepen our understanding of biomolecular intricacies but also pave the way for significant developments in medical research, therapeutic innovation, and diagnostic approaches.

Development of Optical Differential Sensing Based on Nanomaterials for Biological Analysis.

The discrimination and recognition of biological targets, such as proteins, cells, and bacteria, are of utmost importance in various fields of biological research and production. These include areas like biological medicine, clinical diagnosis, and microbiology analysis. In order to efficiently and cost-effectively identify a specific target from a wide range of possibilities, researchers have developed a technique called differential sensing. Unlike traditional "lock-and-key" sensors that rely on specific interactions between receptors and analytes, differential sensing makes use of cross-reactive receptors. These sensors offer less specificity but can cross-react with a wide range of analytes to produce a large amount of data. Many pattern recognition strategies have been developed and have shown promising results in identifying complex analytes. To create advanced sensor arrays for higher analysis efficiency and larger recognizing range, various nanomaterials have been utilized as sensing probes. These nanomaterials possess distinct molecular affinities, optical/electrical properties, and biological compatibility, and are conveniently functionalized. In this review, our focus is on recently reported optical sensor arrays that utilize nanomaterials to discriminate bioanalytes, including proteins, cells, and bacteria.

Paper-based sensors: affordable, versatile, and emerging analyte detection platforms.

Paper-based sensors, often referred to as paper-based analytical devices (PADs), stand as a transformative technology in the field of analytical chemistry. They offer an affordable, versatile, and accessible solution for diverse analyte detection. These sensors harness the unique properties of paper substrates to provide a cost-effective and adaptable platform for rapid analyte detection, spanning chemical species, biomolecules, and pathogens. This review highlights the key attributes that make paper-based sensors an attractive choice for analyte detection. PADs demonstrate their versatility by accommodating a wide range of analytes, from ions and gases to proteins, nucleic acids, and more, with customizable designs for specific applications. Their user-friendly operation and minimal infrastructure requirements suit point-of-care diagnostics, environmental monitoring, food safety, and more. This review also explores various fabrication methods such as inkjet printing, wax printing, screen printing, dip coating, and photolithography. Incorporating nanomaterials and biorecognition elements promises even more sophisticated and sensitive applications.

A Review for Artificial Intelligence Based Protein Subcellular Localization.

Proteins need to be located in appropriate spatiotemporal contexts to carry out their diverse biological functions. Mislocalized proteins may lead to a broad range of diseases, such as cancer and Alzheimer's disease. Knowing where a target protein resides within a cell will give insights into tailored drug design for a disease. As the gold validation standard, the conventional wet lab uses fluorescent microscopy imaging, immunoelectron microscopy, and fluorescent biomarker tags for protein subcellular location identification. However, the booming era of proteomics and high-throughput sequencing generates tons of newly discovered proteins, making protein subcellular localization by wet-lab experiments a mission impossible. To tackle this concern, in the past decades, artificial intelligence (AI) and machine learning (ML), especially deep learning methods, have made significant progress in this research area. In this article, we review the latest advances in AI-based method development in three typical types of approaches, including sequence-based, knowledge-based, and image-based methods. We also elaborately discuss existing challenges and future directions in AI-based method development in this research field.

A Water-Soluble Wavy Coordination Polymer of Cu(II) as a Turn-On Luminescent Probe for Histidine and Histidine-Rich Proteins/Peptides.

Histidine plays an essential role in most biological systems. Changes in the homeostasis of histidine and histidine-rich proteins are connected to several diseases. Herein, we report a water-soluble Cu(II) coordination polymer, labeled CuCP, for the fluorimetric detection of histidine and histidine-rich proteins and peptides. Single-crystal structure determination of CuCP revealed a two-dimensional wavy network structure in which a carboxylate group connects the individual Cu(II) dimer unit in a syn-anti conformation. The weakly luminescent and water-soluble CuCP shows turn-on blue emission in the presence of histidine and histidine-rich peptides and proteins. The polymer can also stain histidine-rich proteins via gel electrophoresis. The limits of quantifications for histidine, glycine-histidine, serine-histidine, human serum albumin (HSA), bovine serum albumin, pepsin, trypsin, and lysozyme were found to be 300, 160, 600, 300, 600, 800, 120, and 290 nM, respectively. Utilizing the fluorescence turn-on property of CuCP, we measured HSA quantitatively in the urine samples. We also validated the present urinary HSA measurement assay with existing analytical techniques. Job's plot, 1H NMR, high-resolution mass spectrometry (HRMS), electron paramagnetic resonance (EPR), fluorescence, and UV-vis studies confirmed the ligand displacement from CuCP in the presence of histidine.

Protein Tyrosine Amination: Detection, Imaging, and Chemoproteomic Profiling with Synthetic Probes.

Protein tyrosine nitration (PTN) by oxidative and nitrative stress is a well-known post-translational modification that plays a role in the initiation and progression of various diseases. Despite being recognized as a stable modification for decades, recent studies have suggested the existence of a reduction in PTN, leading to the formation of 3-aminotyrosine (3AT) and potential denitration processes. However, the vital functions of 3AT-containing proteins are still unclear due to the lack of selective probes that directly target the protein tyrosine amination. Here, we report a novel approach to label and enrich 3AT-containing proteins with synthetic salicylaldehyde (SAL)-based probes: SALc-FL with a fluorophore and SALc-Yn with an alkyne tag. These probes exhibit high selectivity and efficiency in labeling and can be used in cell lysates and live cells. More importantly, SALc-Yn offers versatility when integrated into multiple platforms by enabling proteome-wide quantitative profiling of cell nitration dynamics. Using SALc-Yn, 355 proteins were labeled, enriched, and identified to carry the 3AT modification in oxidatively stressed RAW264.7 cells. These findings provide compelling evidence supporting the involvement of 3AT as a critical intermediate in nitrated protein turnover. Moreover, our probes serve as powerful tools to investigate protein nitration and denitration processes, and the identification of 3AT-containing proteins contributes to our understanding of PTN dynamics and its implications in cellular redox biology.

Perspective: The complex relationship between charge, mobility, and gas-phase protein structure.

Ion mobility spectrometry coupled to mass spectrometry (IMS/MS) is a widely used tool for biomolecular separations and structural elucidation. The application of IMS/MS has resulted in exciting developments in structural proteomics and genomics. This perspective gives a brief background of the field, addresses some of the important issues in making structural measurements, and introduces complementary techniques.

Ultrasensitive and Multiplexed Protein Imaging with Clickable and Cleavable Fluorophores.

Single-cell spatial proteomic analysis holds great promise to advance our understanding of the composition, organization, interaction, and function of the various cell types in complex biological systems. However, the current multiplexed protein imaging technologies suffer from low detection sensitivity, limited multiplexing capacity, or are technically demanding. To tackle these issues, here, we report the development of a highly sensitive and multiplexed in situ protein profiling method using off-the-shelf antibodies. In this approach, the protein targets are stained with horseradish peroxidase (HRP) conjugated antibodies and cleavable fluorophores via click chemistry. Through repeated cycles of target staining, fluorescence imaging, and fluorophore cleavage, many proteins can be profiled in single cells in situ. Applying this approach, we successfully quantified 28 different proteins in human formalin-fixed paraffin-embedded (FFPE) tonsil tissue, which represents the highest multiplexing capacity among the tyramide signal amplification (TSA) methods. Based on their unique protein expression patterns and their microenvironment, ∼820,000 cells in the tissue are classified into distinct cell clusters. We also explored the cell-cell interactions between these varied cell clusters and observed that different subregions of the tissue are composed of cells from specific clusters.

Two-Dimensional SEC-SEC-UV-MALS-dRI Workflow for Streamlined Analysis and Characterization of Biopharmaceuticals.

The emergence of complex biological modalities in the biopharmaceutical industry entails a significant expansion of the current analytical toolbox to address the need to deploy meaningful and reliable assays at an unprecedented pace. Size exclusion chromatography (SEC) is an industry standard technique for protein separation and analysis. Some constraints of traditional SEC stem from its restricted ability to resolve complex mixtures and notoriously long run times while also requiring multiple offline separation conditions on different pore size columns to cover a wider molecular size distribution. Two-dimensional liquid chromatography (2D-LC) is becoming an important tool not only to increase peak capacity but also to tune selectivity in a single online method. Herein, an online 2D-LC framework in which both dimensions utilize SEC columns with different pore sizes is introduced with a goal to increase throughput for biomolecule separation and characterization. In addition to improving the separation of closely related species, this online 2D SEC-SEC approach also facilitated the rapid analysis of protein-based mixtures of a wide molecular size range in a single online experimental run bypassing time-consuming deployment of different offline SEC methods. By coupling the second dimension with multiangle light scattering (MALS) and differential refractive index (dRI) detectors, absolute molecular weights of the separated species were obtained without the use of calibration curves. As illustrated in this report for protein mixtures and vaccine processes, this workflow can be used in scenarios where rapid development and deployment of SEC assays are warranted, enabling bioprocess monitoring, purity assessment, and characterization.