Protein Mixtures Research Articles

Co-stimulatory pathway competitive assay development using Liquid chromatography–tandem mass spectrometry (LC-MS/MS)

T-cells play a significant role in the development of autoimmune diseases. The CD28-B7 costimulatory pathway is crucial for activating T-cells, and blocking this pathway is essential for treating autoimmune diseases. Therapeutic antibodies and fusion proteins that target costimulatory molecules like CD80, CD86, CTLA-4, and CD28 have been developed to explore the costimulation process and as targeted treatments. To advance our understanding of costimulation in autoimmunity and the inhibition of the costimulatory pathway, it is crucial to have an accurate, precise, and direct method for detecting and quantifying the soluble form of these molecules in body fluids and various biological systems. Herein, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying the four costimulatory proteins depending on the signature peptides derived from the soluble isoform of these proteins in multiple reaction monitoring (MRM) mode. The method was validated using the US FDA guidelines. The LOQ was determined as ∼0.5 nM for the four analytes, with quantification extended to 20 nM with a correlation coefficient of R2>0.998. The developed MRM method was used to analyze on-bead digested protein mixtures to establish a competitive assay for the CD28-B7 costimulatory pathway using CTLA4-Ig (Abatacept ™) as an FDA-approved drug for rheumatoid arthritis. The IC50 was determined to be 2.99 and 159.8 nM for sCD80 and sCD86, respectively. A straightforward MRM-based competitive assay will advance the knowledge about the costimulatory role in autoimmunity and the autoimmune therapeutic drug discovery, with the need for broad application on different in vitro and in vivo models to discover new targeted inhibitors.

Multi-length Scale Approach to Investigate Cleaning of Food-Derived Deposits Adhered to Hard Surfaces: Mixtures of Starch, Whey Protein, and Lard

Fouling, the accumulation of undesirable material on manufacturing equipment surfaces, poses a pervasive challenge in industrial processes. In the food industry, the complex interactions among these compounds can give rise to stubborn deposits that deviate from conventional cleaning protocols. In this work, the forces and removal mechanisms of model fouling agents composed of mixtures of starch, whey protein, and lard deposited on solid surfaces of relevant industrial interest (i.e. stainless steel, aluminium, and PTFE) are investigated using a multi-length scale approach, involving milli-manipulation and a lab-simulated Clean-In-Place (CIP) system. The forces involved in the removal process, the types of failure observed when the deposits are subjected to shear stress (adhesive, mixed, or cohesive), and the performance of the CIP system are systematically analysed as a function of the cleaning treatments applied. For stainless steel surfaces, alkaline treatment seems to facilitate the cleaning of lard and starch deposits, while the whey foulant removal tends to be more effective using hot water under the conditions tested. Hot water is effective for stainless steel and PTFE surfaces, reducing the mechanical shear stress required, while the alkaline treatment demonstrated superior efficacy for aluminium surfaces. These findings emphasise the importance of customising cleaning protocols for CIP optimisation.

Viscometry-based prediction of structural properties of high-moisture meat analogues using gelation properties of soy and pea isolate protein blends

In this study, utilizing the gelation properties of a blend containing soy protein isolate (SPI), pea protein isolate (PPI), and corn starch (CS), we introduced a novel approach to predict the structural attributes of high-moisture meat analogues (HMMA). By leveraging the “Rapid Visco Analyzer 4800″ viscometer, we measured the gelation behavior of SPI and PPI based on temperature profiles, underscoring a method to efficiently forecast fibrous structures and reduce trial errors in complex extrusion processes. As the PPI content increased, we observed a decline in the overall viscosity of the protein mixture, paralleled by a rise in color lightness. Contrary to the sturdy gel network formed by SPI, PPI showcased weaker particle bonds, indicating a structural vulnerability. In line with this, the texture of HMMA diminished as PPI content escalated. The secondary protein structure in both protein gels and HMMA revealed that as PPI content grew, there was a reduction in α-helix and β-sheet structures while random coil structures increased. The molecular strength of the protein gel waned with higher PPI content. Similarly, in HMMA, elevated PPI levels corresponded to fewer protein interactions. The study demonstrates that HMMA's textural properties can be effectively predicted by understanding the gelation characteristics of the SPI-PPI blend, highlighting the need for further research to refine prediction models that incorporate the complexities of molecular interactions in varying processing conditions.

Anti-aquaporin-4 immune complex stimulates complement-dependent Th17 cytokine release in neuromyelitis optica spectrum disorders

Proinflammatory cytokines, such as (IL: interleukin) IL-6 and IL-17A, and complement fixation are critical in the immunopathogenesis of neuromyelitis optica spectrum disorders (NMOSD). Blocking the IL-6 receptor or the C5 complement pathway reduces relapse risk. However, the role of interleukin (IL)-6 and complement in aquaporin-4 (AQP4) autoimmunity remains unclear. To investigate the role of the anti-AQP4 immunoglobulin (AQP4-IgG)/AQP4 immunocomplex on the induction and profile of ex vivo cytokine and surface marker expression in peripheral blood mononuclear cells (PBMC) culture. Isolated PBMCs obtained from 18 patients with AQP4-IgG-seropositive-NMOSD (8 treatment-naive, 10 rituximab-treated) or ten healthy controls were cultured with AQP4-immunocomplex with or without complement. Changes in PBMC surface markers and cytokine expression were profiled using flow cytometry and ELISA. PBMCs derived from treatment-naive NMOSD patients stimulated with a complex mixture of serum complement proteins produced significant elevations of IL-17A and IL-6. Rituximab-treated patients also exhibited higher IL-6 but not IL-17A release. IL-6 and IL-17A elevations are not observed without complement. Co-stimulation of PBMCs with AQP4-IgG/AQP4 immunocomplex and complement prompts a Th17-biased response consistent with the inflammatory paradigm observed in NMOSD. A possible inflammation model is proposed via antigen-specific autoreactive peripheral blood cells, including NK/NKT cells.

Protein interactions and rheological properties of heat-induced gels containing soy and water-soluble mealworm (Tenebrio molitor larvae) protein mixtures

This study investigates the protein interactions, rheological properties, and gel characteristics of complex protein gels prepared using varying ratios of soy protein isolate (SPI) and water-soluble mealworm protein (WS-MP). After heat treatment, the particle size increased for all ratios, and the absolute zeta potential exhibited a decreasing trend with higher WS-MP ratios. A comparison of SDS-PAGE profiles before and after heating indicated that disappeared bands may be associated with gelation. Furthermore, as the WS-MP ratio increased, the dominant molecular forces within the gels shifted from hydrophobic interactions and disulfide (SS) bonds to ionic and SS bonds. The SS content decreased as the WS-MP ratio increased. The surface hydrophobicity displayed an initial decrease followed by increased gels. Rheological analysis revealed that gel elasticity decreased as the WS-MP ratio increased, although no significant difference in gel elasticity was observed when the WS-MP ratio exceeded 50%. Heat-induced gels with different SPI:WS-MP ratios exhibited different color and turbidity characteristics. Heat-induced gels comprising the SPI:WS-MP mixture exhibited lower hardness and gumminess than single SPI and WS-MP heat-induced gels. Furthermore, the water holding capacity and the uniformity of gel microstructure exhibited a decreasing trend with an increasing WS-MP ratio. In conclusion, the results suggest that WS-MP can enhance the texture of SPI gels, with the optimal ratio depending on the desired gel properties.

Modification of bacterial cellulose using silk fibroin β-sheet crystals induced by ultrasonication

Silk fibroin (SF) has been continuously explored as a biomaterial due to its biocompatibility, tunability, and self-healing properties. In this work, we present a novel approach to the modification of bacterial cellulose (BC) with SF β-sheet dominant structures induced via ultrasonication. Secondary structure analysis through infrared spectroscopy, thioflavin T assay, and circular dichroism spectropolarimetry revealed a conversion of silk I to silk II structures within the protein mixture. Cold field emission scanning electron microscope images revealed the tightly packed fibers coated with the protein. Thermogravimetric curves demonstrated higher resistance to temperature degradation supplemented by broader and flatter DSC curves attributed to the highly bonded and dense composite. Successful conversion of amide I to amide II and amide III allowed for the more stable β-crystals to contribute to a more thermodynamically stable double-network hydrogel. The conversion of silk I to silk II structures offers a viable and highly biocompatible material that is both thermodynamically and biochemically stable for various potential biomedical applications.

Proteotransciptomics of the Most Popular Host Sea Anemone Entacmaea quadricolor Reveals Not All Toxin Genes Expressed by Tentacles Are Recruited into Its Venom Arsenal.

While the unique symbiotic relationship between anemonefishes and sea anemones is iconic, it is still not fully understood how anemonefishes can withstand and thrive within the venomous environment of their host sea anemone. In this study, we used a proteotranscriptomics approach to elucidate the proteinaceous toxin repertoire from the most common host sea anemone, Entacmaea quadricolor. Although 1251 different toxin or toxin-like RNA transcripts were expressed in E. quadricolor tentacles (0.05% of gene clusters, 1.8% of expression) and 5375 proteins were detected in milked venom, only 4% of proteins detected in venom were putative toxins (230), and they only represent on average 14% of the normalised protein expression in the milked venom samples. Thus, most proteins in milked venom do not appear to have a toxin function. This work raises the perils of defining a dominant venom phenotype based on transcriptomics data alone in sea anemones, as we found that the dominant venom phenotype differs between the transcriptome and proteome abundance data. E. quadricolor venom contains a mixture of toxin-like proteins of unknown and known function. A newly identified toxin protein family, Z3, rich in conserved cysteines of unknown function, was the most abundant at the RNA transcript and protein levels. The venom was also rich in toxins from the Protease S1, Kunitz-type and PLA2 toxin protein families and contains toxins from eight venom categories. Exploring the intricate venom toxin components in other host sea anemones will be crucial for improving our understanding of how anemonefish adapt to the venomous environment.

Identification and semi-quantification of protein allergens in complex mixtures using proteomic and AllerCatPro 2.0 bioinformatic analyses: a proof-of-concept investigation

The demand for botanicals and natural substances in consumer products has increased in recent years. These substances usually contain proteins and these, in turn, can pose a risk for immunoglobulin E (IgE)-mediated sensitization and allergy. However, no method has yet been accepted or validated for assessment of potential allergenic hazards in such materials. In the studies here, a dual proteomic-bioinformatic approach is proposed to evaluate holistically allergenic hazards in complex mixtures of plants, insects, or animal proteins. Twelve commercial preparations of source materials (plant products, dust mite extract, and preparations of animal dander) known to contain allergenic proteins were analyzed by label-free proteomic analyses to identify and semi-quantify proteins. These were then evaluated by bioinformatics using AllerCatPro 2.0 (https://allercatpro.bii.a-star.edu.sg/) to predict no, weak, or strong evidence for allergenicity and similarity to source-specific allergens. In total, 4,586 protein sequences were identified in the 12 source materials combined. Of these, 1,665 sequences were predicted with weak or strong evidence for allergenic potential. This first-tier approach provided top-level information about the occurrence and abundance of proteins and potential allergens. With regards to source-specific allergens, 129 allergens were identified. The sum of the relative abundance of these allergens ranged from 0.8% (lamb’s quarters) to 63% (olive pollen). It is proposed here that this dual proteomic-bioinformatic approach has the potential to provide detailed information on the presence and relative abundance of allergens, and can play an important role in identifying potential allergenic hazards in complex protein mixtures for the purposes of safety assessments.

Controlling Physical and Biochemical Parameters of Actin Nucleation Using a Patterned Model Lipid Membrane.

Self-assembly of nanoscale actin cytoskeletal proteins into filamentous networks requires organizing actin nucleation areas on the plasma membrane through recruiting actin nucleators and nucleation-promoting factors (NPFs) to the areas. To investigate impacts of the nucleation geometry on actin network assembly, we localized NPF or nucleator on defined micropatterns of laterally mobile lipid bilayers confined in a framework of a polymerized lipid bilayer. We demonstrated that actin network assembly in purified protein mixtures was confined on NPF- or nucleator-localized fluid bilayers. By controlling the shape and size of nucleation areas as well as the density and types of localized NPFs and nucleators, we showed that these parameters regulate actin network architectures. Actin network assembly in Xenopus egg extracts was also spatially controlled by patterning bilayers containing phosphatidylinositol 4,5-bisphoshate (PI(4,5)P2), an essential lipid signaling mediator. Therefore, the system provides a promising platform to investigate the physical and biochemical principles for actin network assembly.

Spatial distribution proteoglycans articular cartilage of femoral condyles and tibial plateau in experimental osteoarthritis in rats

Currently, worldwide research is being conducted on the development of new drugs for viscosupplementation therapy. However, there are a very few studies dedicated to the spatial distribution of proteoglycans in articular cartilage after the use of these drugs. Pulmonary surfactant proteins, which have lubricative functions similar to those of articular cartilage proteoglycans, are possible promising substances for viscosupplementation therapy. The aim of this study is to investigate the effect of a mixture of allogenic surfactant proteins (MASP) on the spatial distribution of proteoglycans and the expression of IL-1 in articular cartilage after their intra-articular injection. Morphological and immunohistochemical studies were conducted using antibodies to aggrecan and lubricin to assess the spatial distribution of proteoglycans, and antibodies to IL-1 were used to identify potential pro-inflammatory processes. Three weeks after intra-articular injection of MASP, the thickness of the articular cartilage and the relief of the joint surface did not change compared to intact rats. However, a decrease in the number of immunopositive chondrocytes to aggrecan in the intermediate zone of the articular cartilage and an increase in IL-1 were observed after MASP injection. The obtained data during the study can be used for further research on MASP in the conditions of experimental osteoarthritis.

Changes in saliva protein profile throughout Rhipicephalus microplus blood feeding

BackgroundWhen feeding on a vertebrate host, ticks secrete saliva, which is a complex mixture of proteins, lipids, and other molecules. Tick saliva assists the vector in modulating host hemostasis, immunity, and tissue repair mechanisms. While helping the vector to feed, its saliva modifies the site where pathogens are inoculated and often facilitates the infection process. The objective of this study is to uncover the variation in protein composition of Rhipicephalus microplus saliva during blood feeding.MethodsTicks were fed on calves, and adult females were collected, weighed, and divided in nine weight groups, representing the slow and rapid feeding phases of blood feeding. Tick saliva was collected, and mass spectrometry analyses were used to identify differentially secreted proteins. Bioinformatic tools were employed to predict the structural and functional features of the salivary proteins. Reciprocal best hit analyses were used to identify conserved families of salivary proteins secreted by other tick species.ResultsChanges in the protein secretion profiles of R. microplus adult female saliva during the blood feeding were observed, characterizing the phenomenon known as “sialome switching.” This observation validates the idea that the switch in protein expression may serve as a mechanism for evading host responses against tick feeding. Cattle tick saliva is predominantly rich in heme-binding proteins, secreted conserved proteins, lipocalins, and protease inhibitors, many of which are conserved and present in the saliva of other tick species. Additionally, another remarkable observation was the identification of host-derived proteins as a component of tick saliva.ConclusionsOverall, this study brings new insights to understanding the dynamics of the proteomic profile of tick saliva, which is an important component of tick feeding biology. The results presented here, along with the disclosed sequences, contribute to our understanding of tick feeding biology and might aid in the identification of new targets for the development of novel anti-tick methods.Graphical

Accessory gland size increases with sperm competition intensity in Cataglyphis desert ants

In many species, females have multiple mates, whose sperm compete for paternity. Males may subsequently invest in the increased production of sperm and/or seminal fluid. The latter is a complex mixture of proteins, peptides, and other compounds generated by the accessory glands (AGs) and is transferred to females along with a male’s sperm. Seminal fluid is known to be a key determinant of competitive outcomes among sperm, and its production may trade off with that of sperm. We show that AG size—a proxy for seminal fluid production—has a positive and phylogenetically robust correlation with both sperm competition intensity and sperm production in nine species of Cataglyphis desert ants. These results indicate a lack of trade-off between sperm production and seminal fluid production. They underscore that sperm competition may strongly shape sperm traits and could drive reproductive performance in eusocial hymenopterans.

Advances in Research on Pig Salivary Analytes: A Window to Reveal Pig Health and Physiological Status

Saliva is an important exocrine fluid that is easy to collect and is a complex mixture of proteins and other molecules from multiple sources from which considerable biological information can be mined. Pig saliva, as an easily available biological liquid rich in bioactive ingredients, is rich in nucleic acid analytes, such as eggs, enzymes, amino acids, sugars, etc. The expression levels of these components in different diseases have received extensive attention, and the analysis of specific proteins, metabolites, and biological compositions in pig saliva has become a new direction for disease diagnosis and treatment. The study of the changes in analytes in pig saliva can provide a new strategy for early diagnosis, prognosis assessment, and treatment of diseases. In this paper, the detection methods and research progress of porcine salivary analytes are reviewed, the application and research progress of porcine salivary analytes in diseases are discussed, and the future application prospect is presented.

A systematic review of the role of surfactants in bronchial asthma: Implications for pathogenesis and treatment of the disease

The study aims to examine existing literature and highlight the significance of surfactants as a factor in pathogenesis and a potential therapeutic target in bronchial asthma. A systematic search is conducted in Scopus, Web of Science, and PubMed/Medline, utilizing specific keywords related to surfactants and bronchial asthma. The findings demonstrate that surfactants, a unique mixture of lipids and proteins, play a crucial role in the mechanisms of innate and adaptive immunity in the lungs. They act as a mechanical barrier, modulate inflammatory responses, and interact with pathogens. Changes in surfactant composition and levels have been associated with various lung diseases, including bronchial asthma

The concept of enzymatic disintegration of nutrients in the upper gastrointestinal tract

The aim of the study was to study the effect of salivary amylase on improving gastric protein digestion, as well as the effect of gastric protein hydrolysis on improving fat digestion. The work consisted of two parts, in the first part, in two series, the hydrolysis of proteins under the influence of gastric juice in the presence of starch and the effect of saliva amylase on the change in the total proteolytic activity of gastric juice when using substrates of a mixture of starch and proteins were investigated. In the second part of the work, the lipolytic activity of pancreatic juice with the studied proteins was studied in the presence and absence of bile. From the data obtained, it was found that the use of a mixture of starch with proteins helps to reduce the hydrolysis of proteins by gastric juice. The use of salivary amylase promotes an increase in protein hydrolysis by reducing the formation of starch-protein complexes that prevent the digestion of proteins by gastric juice. All the studied proteins, except gelatin, have an inhibitory effect on lipase in the pancreatic juice; the degree of inhibitory effect of each protein is expressed differently. It depends on their degree of digestion in gastric juice. Thus, the preliminary hydrolysis of proteins by pepsins in the stomach contributes not only to the further improvement of their hydrolysis under the influence of proteolytic enzymes of pancreatic juice, but also to the hydrolysis of fats under the influence of pancreatic lipase. It is concluded that the sequence of nutrient breakdown by enzymes of saliva, stomach and pancreas is primarily aimed at enzymatic disintegration between polysaccharides and proteins, as well as proteins and fats, in order to reduce their interaction and the formation of physical complexes. Thus, secondarily, improve the digestibility of nutrients in the upper gastrointestinal tract. The conceptual assumption of enzymatic disintegration of nutrients in the upper gastrointestinal tract allows us to substantiate the existing sequence of initial digestion of polysaccharides by saliva, as well as the initial digestion of proteins by gastric juice.

Serum Raman spectroscopy: Evaluation of tumour load variations in experimental carcinogenesis.

Several serum Raman spectroscopy (RS) studies have demonstrated its potential as an oral cancer screening tool. This study investigates influence of low tumour load (LTL) and high tumour load (HTL) on serum RS using hamster buccal pouch model of experimental oral carcinogenesis. Sera of untreated control, LTL, and HTL groups at week intervals during malignant transformation were employed. Serum Raman spectra were subjected to multivariate analyses-principal component analysis, principal component-based linear discriminant analysis (for stratification of study groups), and multivariate curve resolution-alternating least squares (MCR-ALS) (to comprehend biomolecular differences). Multivariate analysis revealed misclassifications between LTL and HTL at all week intervals. MCR-ALS components showed statistically significant abundances between control versus LTL and control versus HTL, but could not discern LTL and HTL. MCR-ALS components exhibited spectral mixtures of proteins, lipids, heme and nucleic acids. Thus, these findings support use of serum RS as a screening tool as varying tumour load is not a confounding factor influencing the technique.

Structure and Function of Canine SP-C Mimic Proteins in Synthetic Surfactant Lipid Dispersions.

Lung surfactant is a mixture of lipids and proteins and is essential for air breathing in mammals. The hydrophobic surfactant proteins B and C (SP-B and SP-C) assist in reducing surface tension in the lung alveoli by organizing the surfactant lipids. SP-B deficiency is life-threatening, and a lack of SP-C can lead to progressive interstitial lung disease. B-YL (41 amino acids) is a highly surface-active, sulfur-free peptide mimic of SP-B (79 amino acids) in which the four cysteine residues are replaced by tyrosine. Mammalian SP-C (35 amino acids) contains two cysteine-linked palmitoyl groups at positions 5 and 6 in the N-terminal region that override the β-sheet propensities of the native sequence. Canine SP-C (34 amino acids) is exceptional because it has only one palmitoylated cysteine residue at position 4 and a phenylalanine at position 5. We developed canine SP-C constructs in which the palmitoylated cysteine residue at position 4 is replaced by phenylalanine (SP-Cff) or serine (SP-Csf) and a glutamic acid-lysine ion-lock was placed at sequence positions 20-24 of the hydrophobic helical domain to enhance its alpha helical propensity. AI modeling, molecular dynamics, circular dichroism spectroscopy, Fourier Transform InfraRed spectroscopy, and electron spin resonance studies showed that the secondary structure of canine SP-Cff ion-lock peptide was like that of native SP-C, suggesting that substitution of phenylalanine for cysteine has no apparent effect on the secondary structure of the peptide. Captive bubble surfactometry demonstrated higher surface activity for canine SP-Cff ion-lock peptide in combination with B-YL in surfactant lipids than with canine SP-Csf ion-lock peptide. These studies demonstrate the potential of canine SP-Cff ion-lock peptide to enhance the functionality of the SP-B peptide mimic B-YL in synthetic surfactant lipids.

Optimizing drug discovery for snakebite envenoming via a high-throughput phospholipase A2 screening platform.

Snakebite envenoming is a neglected tropical disease that causes as many as 1.8 million envenomings and 140,000 deaths annually. To address treatment limitations that exist with current antivenoms, the search for small molecule drug-based inhibitors that can be administered as early interventions has recently gained traction. Snake venoms are complex mixtures of proteins, peptides and small molecules and their composition varies substantially between and within snake species. The phospholipases A2 (PLA2) are one of the main pathogenic toxin classes found in medically important viper and elapid snake venoms, yet varespladib, a drug originally developed for the treatment of acute coronary syndrome, remains the only PLA2 inhibitor shown to effectively neutralise venom toxicity in vitro and in vivo, resulting in an extremely limited drug portfolio. Here, we describe a high-throughput drug screen to identify novel PLA2 inhibitors for repurposing as snakebite treatments. We present method optimisation of a 384-well plate, colorimetric, high-throughput screening assay that allowed for a throughput of ∼2,800 drugs per day, and report on the screening of a ∼3,500 post-phase I repurposed drug library against the venom of the Russell's viper, Daboia russelii. We further explore the broad-spectrum inhibitory potential and efficacy of the resulting top hits against a range of medically important snake venoms and demonstrate the utility of our method in determining drug EC50s. Collectively, our findings support the future application of this method to fully explore the chemical space to discover novel PLA2-inhibiting drugs of value for preventing severe pathology caused by snakebite envenoming.

Lupin protein-stabilized oil droplets contribute to structuring whey protein emulsion-filled gels

This work aimed to understand the role of lupin protein or mixed lupin-whey protein stabilized oil droplets on the texture and microstructure of a heat-induced whey protein gel. Protein-stabilized emulsions were compared to surfactant-stabilized emulsions to investigate the potential of their interfacial interactions to impart unique structures in the filled gels. The structure development was followed in situ using rheology and the final heat-induced gels were characterized by small and large amplitude oscillatory rheology and confocal microscopy. The development of the gel modulus as well as the final gel properties were linked to the type of interactions between the whey protein matrix and the protein adsorbed at the oil interface. The final gels were selectively dissolved in various buffers, and the results showed that replacing interfacial whey protein with lupin protein resulted in a reduced amount of disulfide bridges, explaining the softer gel in the lupin containing gels compared to those with whey protein. Non-covalent interactions were the main forces involved in the formation of actively filled droplets in the gel network. This work demonstrated that by modulating the interfacial composition of the oil droplets, differing gel structures could be achieved due to differences in the protein–protein interactions between the continuous and the interfacial phase. There is therefore potential for the development of innovative products using lupin-whey protein mixtures, by careful control of the processing steps and the matrix composition.

Absolute Quantification of Human Serum Albumin Isoforms by Internal Calibration Based on a Top-Down LC-MS Approach.

Well-characterized biomarkers using reliable quantitative methods are essential for the management of various pathologies such as diabetes, kidney, and liver diseases. Human serum albumin (HSA) isoforms are gaining interest as biomarkers of advanced liver pathologies. In view of the structural alterations observed for HSA, insights into its isoforms are required to establish them as reliable biomarkers. Therefore, a robust absolute quantification method seems necessary. In this study, we developed and validated a far more advanced top-down liquid chromatography-mass spectrometry (LC-MS) method for the absolute quantification of HSA isoforms, using myoglobin (Mb) as an internal standard for quantification and for mass recalibration. Two different quantification approaches were investigated based on peak integration from the deconvoluted spectrum and extracted ion chromatogram (XIC). The protein mixture human serum albumin/myoglobin eluted in well-shaped separated peaks. Mb allowed a systematic mass recalibration for every sample, resulting in extremely low mass deviations compared to conventional deconvolution-based methods. In total, eight HSA isoforms of interest were quantified. Specific-isoform calibration curves showing good linearity were obtained by using the deconvoluted peaks. Noticeably, the HSA ionization behavior appeared to be isoform-dependent, suggesting that the use of an enriched isoform solution as a calibration standard for absolute quantification studies of HSA isoforms is necessary. Good repeatability, reproducibility, and accuracy were observed, with better sensitivity for samples with low albumin concentrations compared to routine biochemical assays. With a relatively simple workflow, the application of this method for absolute quantification shows great potential, especially for HSA isoform studies in a clinical context, where a high-throughput method and sensitivity are needed.