Intact Protein Research Articles

ProSight Native: Defining Protein Complex Composition from Native Top-Down Mass Spectrometry Data.

Native mass spectrometry has recently moved alongside traditional structural biology techniques in its ability to provide clear insights into the composition of protein complexes. However, to date, limited software tools are available for the comprehensive analysis of native mass spectrometry data on protein complexes, particularly for experiments aimed at elucidating the composition of an intact protein complex. Here, we introduce ProSight Native as a start-to-finish informatics platform for analyzing native protein and protein complex data. Combining mass determination via spectral deconvolution with a top-down database search and stoichiometry calculations, ProSight Native can determine the complete composition of protein complexes. To demonstrate its features, we used ProSight Native to successfully determine the composition of the homotetrameric membrane complex Aquaporin Z. We also revisited previously published spectra and were able to decipher the composition of a heterodimer complex bound with two noncovalently associated ligands. In addition to determining complex composition, we developed new tools in the software for validating native mass spectrometry fragment ions and mapping top-down fragmentation data onto three-dimensional protein structures. Taken together, ProSight Native will reduce the informatics burden on the growing field of native mass spectrometry, enabling the technology to further its reach.

Expedited Evaluation of Conformational Stability-Heterogeneity Associations for Crude Polyclonal Antibodies in Response to Conjugate Vaccines.

Conjugate vaccines have been demonstrated to be a promising strategy for immunotherapeutic intervention in substance use disorder, wherein a hapten structurally similar to the target drug is conjugated to an immunogenic carrier protein. The antibodies generated following immunization with these species can provide long-lasting protection against overdose through sequestration of the abused drug in the periphery, which mitigates its ability to cross the blood-brain barrier. However, these antibodies exhibit a high degree of heterogeneity in structure. The resultant variations in chemical and structural compositions have not yet been clearly linked to the stability that directly affects their in vivo functional performance. In this work, we describe a rapid mass-spectrometry-based analytical workflow capable of simultaneous and comprehensive interrogation of the carrier protein-dependent heterogeneity and stability of crude polyclonal antibodies in response to conjugate vaccines. Quantitative collision-induced unfolding-ion mobility-mass spectrometry with an all-ion mode is adapted to rapidly assess the conformational heterogeneity and stability of crude serum antibodies collected from four different vaccine conditions, in an unprecedented manner. A series of bottom-up glycoproteomic experiments was performed to reveal the driving force underlying these observed heterogeneities. Overall, this study not only presents a generally applicable workflow for fast assessment of crude antibody conformational stability and heterogeneity at the intact protein level but also leverages carrier protein optimization as a simple solution to antibody quality control.

Hydrolyzed rice glutelin as plant-based emulsifier to reduce tween in emulsion facing process stresses and its application in coconut milk

The demand for natural plant–based emulsifier to reduce synthetic emulsifier is attractive to the food industry. The low emulsifying property of native rice glutelin (RG) can be improved by trypsin hydrolysis due to the partial unfolding of intact protein. The solubility and surface hydrophobicity of hydrolyzed RG at degree of hydrolysis (DH) 2.0 (HRG2) (89.48%, 7461.97) were much higher than RG (9.85%, 249.50). HRG2 was substituted for Tween 60 (Tw) in emulsion at ratio 100 : 0, 75 : 25, 50 : 50, 25 : 75, and 0 : 100. The selected 75 : 25 ratio emulsion was subjected to environmental stresses (pH, ionic strength, and temperature). The interfacial pressure of HRG2 and HRG2–Tw was in an increasing trend even after 3 h indicated the presence of HRG2 in the adsorbed interface. After amplitude sweep, the interface formed by HRG2 showed higher elastic modulus (Ed’) indicated a stiffer interface than HRG2–Tw. HRG2–Tw emulsion showed smaller mean droplet size with less concentration dependence than HRG2– and Tw–emulsion. HRG2–Tw–emulsion was precipitated at pI but was stable at pH > 6 and was stabilized under ionic strength similarly to Tw–emulsion. The pasteurized coconut milk with Tw (1%) had the lowest creaming index, followed by HRG2 (1%) and HRG2–Tw (1%) after 7 days which indicated the possibility to replace or reduce the use of synthetic surfactant in coconut milk.

Continuous counter-current electrophoretic separation of oleosomes and proteins from oilseeds

Oilseeds, with their high content of oil and protein, will play an important role in the transition to plant based foods. Currently, seed oil extraction involves elevated temperatures and the use of organic solvents, which degrade the quality of both oils and proteins. To take full advantage of the oilseeds, a gentler process is needed. We here propose a mild alkaline extraction followed by a continuous electrophoretic separation process that is based on differences in electrophoretic mobility and recovers intact oleosomes and proteins from the seeds. Rapeseed oleosomes and proteins are both negatively charged at pH ≥ 5, yet exhibit significantly different electrophoretic mobility. Therefore, separation can be achieved by imposing a counter-current hydrodynamic flow rate between their electrophoresis rate. Thus, the compounds with higher mobility, oleosomes, are retained by the electric field, and the compounds with lower mobility, proteins, go along with the flow. The separation was modeled using the Nernst-Planck equation and demonstrated using a PDMS-based microfluidic system. Both the modeling and the experimental studies confirmed that the direction and rate of migration of the compounds can be steered by the electric field strength and the convective flow velocity. The proposed electrophoretic approach is feasible and scaleable, and may be a novel path to separate differently charged components under mild conditions, thereby preserving their original native properties.

The journey of cardosin A in young Arabidopsis seedlings leads to evidence of a Golgi-independent pathway to the protein storage vacuole.

The aspartic proteinase cardosin A is a vacuolar enzyme found to accumulate in protein storage and lytic vacuoles in the flowers and protein bodies in the seeds of the native plant cardoon. Cardosin A was first isolated several decades ago and has since been extensively characterized, both in terms of tissue distribution and enzyme biochemistry. In the native system, several roles have been attributed to cardosin A, such as reproduction, reserve mobilization, and membrane remodeling. To participate in such diverse events, cardosin A must accumulate and travel to different compartments within the cell: protein storage vacuoles, lytic vacuoles, and the cytoplasmic membrane (and eventually outside the cell). Several studies have approached the expression of cardosin A in Arabidopsis thaliana and Nicotiana tabacum with promising results for the use of these systems to study of cardosin A trafficking. A poly-sorting mechanism has been uncovered for this protein, as two different vacuolar sorting determinants, mediating different vacuolar routes, have been described. The first is a conventional C-terminal domain, which delivers the protein to the vacuole via the Golgi, and the second is a more unconventional signal-the plant-specific insert (PSI)-that mediates a Golgi-independent route. The hypothesis that these two signals are activated according to cell needs and in organs with high metabolic activity is investigated here. An Arabidopsis line expressing cardosin A under an inducible promoter was used to understand the dynamics of cardosin A regarding vacuolar accumulation during seed germination events. Using antibodies against different regions of the protein and combining them with immunofluorescence and immunocytochemistry assays in different young seedling tissues, cardosin A was detected along the secretory pathway to the protein storage vacuole, often associated with the endoplasmic reticulum. More interestingly, upon treatment with the drug Brefeldin A, cardosin A was still detected in protein storage vacuoles, indicating that the intact protein can bypass the Golgi in this system, contrary to what was observed in N. tabacum. This study is a good starting point for further research involving the use of fluorescent fusions and exploring in more detail the relationship between cardosin A trafficking and plant development.

Blocking an epitope of misfolded SOD1 ameliorates disease phenotype in a model of amyotrophic lateral sclerosis.

The current strategies to mitigate the toxicity of misfolded SOD1 in familial ALS via blocking SOD1 expression in the CNS are indiscriminative for misfolded and intact proteins, and as such, entail a risk of depriving CNS cells of their essential antioxidant potential. As an alternative approach to neutralize misfolded and spare unaffected SOD1 species, we developed scFv-SE21 antibody that blocks the β6/β7 loop epitope exposed exclusively in misfolded SOD1. The β6/β7 loop epitope has previously been proposed to initiate amyloid-like aggregation of misfolded SOD1 and mediate its prion-like activity. The AAV-mediated expression of scFv-SE21 in the CNS of hSOD1G37R mice rescued spinal motoneurons, reduced the accumulation of misfolded SOD1, decreased gliosis, and thus delayed disease onset and extended survival by 90 days. The results provide evidence for the role of the exposed β6/β7 loop epitope in the mechanism of neurotoxic gain-of-function of misfolded SOD1 and open avenues for the development of mechanism-based anti-SOD1 therapeutics, whose selective targeting of misfolded SOD1 species may entail a reduced risk of collateral oxidative damage to the CNS.

LC-MS detection of amino acids and neurotransmitters by using α-active 2,4,5-triphenylpyrylium salt as an efficient derivatizing material

Herein, a series of pyrylium salts such as 2,4,6-trimethylpyrylium (P1), 2,6-dimethyl,4-phenylpyrylium (P2), 2,4-diphenylpyrylium (P3), 2,6-diphenylpyrylium (P4), 2,4,5-triphenylpyrylium (P5), 2,4,6-triphenylpyrylium (P6), 2-phenyl, 4,5-diphenylmethylprrylium (P7), 2,3,4,5-tetraphenylpyrylium (P8), 2,3,4,5,6-pentaphenylpyrylium (P9) and 3,4-dimethoxybenzyl,6-phenylpyrylium (P10) were used as highly specific derivatizing agent for mass spectrometric detection of neurochemicals (neurotransmitters and amino acids). Neurotransmitters of dopamine (DA), norepinephrine (NE), histamine (His), γ-amino butyric acid (GABA) and 5-hydroxytyramine (5-HT), and the amino acids of alanine (Ala), serine (Ser), proline (Pro), tryptophan (Trp), threonine (Thr), 4-hydroxy proline (4-Hyp), isoleucine (Ile), leucine (Leu), aspargine (Asn), aspartic acid (Asp), glutamine (Gln), lysine (Lys), glutamic acid (Glu), histidine (H), phenylalanine (Phe), arginine (Arg), valine (Val), tyrosine (Tyr) and cysteine (Cys) were successfully derivatized with the pyrylium salts. The hydrophobic and hydrophilic nature of pyrylium salts were modified by changing the side chain groups. The influence of pyrylium concentration, catalyst concentration, temperature and reaction time were thoroughly optimized. The obtained result suggests that the small molecules like neurotransmitters, amino acids and small peptides have better sensitivity than the peptides and intact proteins. We believe that hydrophobic pyrylium salts will be a potential derivatizing agent for the future mass spectrometric analysis using clinical samples.

Paired Capture and FISH Detection of Individual Virions Enable Cell-Free Determination of Infectious Titers.

Early detection of viruses can prevent the uncontrolled spread of viral infections. Determination of viral infectivity is also critical for determining the dosage of gene therapies, including vector-based vaccines, CAR T-cell therapies, and CRISPR therapeutics. In both cases, for viral pathogens and viral vector delivery vehicles, fast and accurate measurement of infectious titers is desirable. The most common methods for virus detection are antigen-based (rapid but not sensitive) and polymerase chain reaction (PCR)-based (sensitive but not rapid). Current viral titration methods heavily rely on cultured cells, which introduces variability within labs and between labs. Thus, it is highly desirable to directly determine the infectious titer without using cells. Here, we report the development of a direct, fast, and sensitive assay for virus detection (dubbed rapid capture fluorescence in situ hybridization (FISH) or rapture FISH) and cell-free determination of infectious titers. Importantly, we demonstrate that the virions captured are "infectious," thus serving as a more consistent proxy of infectious titers. This assay is unique because it first captures viruses bearing an intact coat protein using an aptamer and then detects genomes directly in individual virions using fluorescence in situ hybridization (FISH); thus, it is selective for infectious particles (i.e., positive for coat proteins and positive for genomes).

Method for the Determination of 2'-Fucosyllactose (2'-FL), 3-Fucosyllactose (3-FL), 6'-Sialyllactose (6'-SL), 3'-Sialyllactose (3'-SL), Lacto-N-Tetraose (LNT), and Lacto-N-neoTetraose (LNnT) by High-Performance Anion-Exchange Chromatography With Pulsed Amperometric Detection (HPAEC-PAD): First Action 2022.04.

A method for simultaneous determination of six human milk oligosaccharides (HMOs) is described. The HMOs include 2'-fucosyllactose (2'-FL, CAS number 41263-94-9), 3-fucosyllactose (3-FL, CAS number 41312-47-4), 6'-sialyllactose (6'-SL, CAS number 35890-39-2), 3'-sialyllactose (3'-SL, CAS number 35890-38-1), lacto-N-tetraose (LNT, CAS number 14116-68-8), and lacto-N-neotetraose (LNnT, CAS number 13007-32-4). The method was designed to comply with the respective Standard Method Performance Requirements (SMPR®; seeTable1), respectively. The method is valid for six HMOs in infant formula and adult nutritional matrixes, including samples with intact protein, protein hydrolysates, elemental formulations free of intact protein, and rice flour over the ranges defined in the SMPR (seeTable2). The method is not valid for determination of difucosyllactose (DFL/DiFL). For most samples, reconstitution with water followed by filtration. For products containing interferences (fructans and maltodextrins), hydrolysis with enzymes is used. After preparation, samples are analyzed using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The method provides for separation of six HMOs and other carbohydrates commonly found in infant formula and adult nutritional products (e.g., lactose, sucrose, and GOS). This study includes data from multiple matrixes evaluated by multiple laboratories globally. RSDr ranged from 0.0068 to 4.8% RSDr, and spike recovery results ranged from 89.4 to 109%. Calibration fit optimal with a quadratic curve; alternately linear fit showed no statistically significant impact to data (when correlation passes). This method was reviewed by the AOAC SPIFAN Expert Review Panel (ERP) and determined to meet the SMPRs for the six noted HMOs. The method was granted First Action Official MethodsSM status.

Hybrid liquid chromatography high resolution and accuracy mass spectrometry approach for quantification of antibody-drug conjugates at the intact protein level in biological samples

Preclinical in vivo and in vitro characterization of Antibody-Drug Conjugates (ADCs) involves the development of several bioanalytical methods to address many drug exposure questions. The current pharma industry approach requires at least three different assays that must be run, i.e., total antibody (mAb), conjugated payload or conjugated mAb, and free payload assays. Herein we present analytical performances of a quantitative hybrid Ligand Binding/Liquid Chromatography High Resolution and Accuracy Mass Spectrometry (LB/LCHRAM) method that can condense much of the necessary bioanalytical information in one method. The method includes an immuno-capture step, and it detects whole ADC molecules. It was applied to plasma mouse samples and showed reliable bioanalytical performance according to full method validation standards. Quantitation using extracted ion chromatograms and deconvoluted mass peaks was evaluated. The limit of quantitation resulted in 0.5ng of protein on column with a linear dynamic range spanning from 0.5 to 10μg/mL. Moreover, lower drug-to-antibody ratio (DAR) ADC species can be simultaneously detected, also enabling qualitative characterization of in vivo ADC conjugation.

An Open-Source Framework for Mass Calculation of Antibody-Based Therapeutic Molecules.

Biotherapeutic masses are a means of verifying identity and structural integrity. Mass spectrometry (MS) of intact proteins or protein subunits provides an easy analytical tool for different stages of biopharmaceutical development. The protein's identity is confirmed when the experimental mass from MS is within a pre-defined mass error range of the theoretical mass. While several computational tools exist for the calculation of protein and peptide molecular weights, they either were not designed for direct application to biotherapeutic entities, have access limitations due to paid licenses, or require uploading protein sequences to host servers. We have developed a modular mass calculation routine that enables the easy determination of the average or monoisotopic masses and elemental compositions of therapeutic glycoproteins, including monoclonal antibodies (mAb), bispecific antibodies (bsAb), and antibody-drug conjugates (ADC). The modular nature of this Python-based calculation framework will allow the extension of this platform to other modalities such as vaccines, fusion proteins, and oligonucleotides in the future, and this framework could also be useful for the interrogation of top-down mass spectrometry data. By creating an open-source standalone desktop application with a graphical user interface (GUI), we hope to overcome the restrictions around use in environments where proprietary information cannot be uploaded to web-based tools. This article describes the algorithms and application of this tool, mAbScale, to different antibody-based therapeutic modalities.

Effects of hydrolysis degree on the functional properties of hydrolysates from sour cherry kernel protein concentrate

During the processing of sour cherries into different foodstuffs, a large amount of kernels is produced as waste material, which creates a significant disposal problem for the food industry. Sour cherry kernels containing 25.3–35.5% of protein can be used as a functional protein source in food production. Therefore, we aimed to study the effects of hydrolysis degree on the sour cherry kernel protein hydrolysates.
 Proteins were extracted from the defatted flour by isoelectric precipitation. The resulting protein concentrate was hydrolyzed (5, 10, and 15% hydrolysis) using Alcalase to yield hydrolysates. We determined their oil and water holding, emulsifying, gelation, and foaming properties, as well as apparent molecular weight distribution and proximate compositions.
 No protein fractions greater than an apparent molecular weight of about 22 kDa were present in the hydrolysates. The hydrolysis of the protein concentrate mostly led to an increase in protein solubility. As the degree of hydrolysis increased from 5 to 15%, the water holding capacity of the hydrolysates decreased from 2.50 ± 0.03 to 2.03 ± 0.02 g water/g, indicating its deterioration. 
 The hydrolysates obtained at different degrees of hydrolysis had a better solubility than the intact protein concentrate. The oil holding capacity, the foaming stability, and the least gelation concentration of the protein concentrate could not be considerably improved by hydrolysis. In contrast, its emulsifying activity index and foaming capacity could be increased with a limited degree of hydrolysis (up to 10%).

Identification of potent antioxidant bioactive peptides from the soluble proteins of chicken egg yolk

Background: Eggs are an excellent nutrient-dense food containing proteins, fats, carbohydrates, minerals, and vitamins. While many proteins are present in egg yolk, there arefew studies on their health benefits. Objective: The aim of this study was to explore the antioxidant peptides derived from the soluble protein fractions of egg yolk. Lipids and lipoproteins were removed with activated carbon and centrifugation after water dilution at acidic pH. Materials and Methods: Compared with 3.6 grams of protein in egg white, egg yolk contains 2.7 grams of protein in a single large egg. The egg yolk soluble protein (EYsP) was digested with pepsin for 2h at pH 3.0 (P-EYsP), followed by digestion with trypsin (P-EYsP-T), α-chymotrypsin (P-EYsP-α), or both enzymes (P-EYsP-T/α). The most active digest was fractionated using sephacryl S-100 gel filtration, followed by reversed phase-HPLC of the most active fraction. The antioxidant activities were evaluated using a superoxide anion-generating system of xanthine oxidase, DPPH-scavenging assay, and yeast cells as an oxidative-stress tolerance cellular model. Results: The intact proteins (EYsP) showed antioxidant activity, but pepsin hydrolysate (P-EYsP) exhibited greater superoxide-scavenging activities than EYsP, while P-EYsP-T, P-EYsP-α and P-EYsP-T/α lacked activities. The P-EYsP and its subsequent proteases (P-EYsP-T, P-EYsP-α and P-EYsP-T/α) exhibited significant DPPH reduction, but P-EYsP-T, P-EYsP-α and P-EYsP-T/α exhibited the strongest DPPH reduction activities. MALDI-TOF-MS analysis revealed five major antioxidant peptides, two derived from yolk glycoprotein 40 (859 Da, 883Da), two from lipovitelline (901Da, 945 Da), and one from livetin (1089 Da). The peptides exhibited potent superoxide anion as well as DPPH scavenging activities and markedly enhanced the tolerance of yeast cells against peroxide-induced oxidative stress.Conclusion: The results show that these bioactive peptides hold a fascinating opportunity for their potential as nutraceuticals in prevention and combating oxidative stress-associated diseases.Keywords: Egg yolk proteins; bioactive peptides; antioxidant; superoxide-scavenging; DPPH-reduction; yeast tolerance

Coagulation and proteolysis of high-protein milks in the gastric environment

Gastric digestion of 2 commercial ultrafiltered milks and milk enriched with skim milk powder (to simulate concentration by reverse osmosis) was investigated and compared with the digestion of nonconcentrated milk. Curd formation and proteolysis of high-protein milks in simulated gastric conditions were studied using oscillatory rheology, extrusion testing, and gel electrophoresis. The presence of pepsin in the gastric fluid triggered coagulation at pH >6 and the elastic modulus of gels from high-protein milks was ~5 times larger than the gel from reference milk. Despite similar protein concentrations, the coagulum from milk enriched with skim milk powder showed higher resistance to shear deformation than the coagula from ultrafiltered milks. The gel structure was also more heterogeneous. During digestion, the degradation of coagula from high-protein milks was slowed down compared with the coagulum from reference milk, and intact milk proteins were still detected after 120 min. Differences in the digestion patterns of coagula from high-protein milks were observed and were associated with the proportion of minerals bound to caseins and the denaturation rate of whey proteins.

Preferential rabbit antibody responses to C-termini of NOTCH3 peptide immunogens

Antibodies raised in peptide-immunized rabbits have been used in biological research for decades. Although there has been wide implementation of this approach, specific proteins are occasionally difficult to target for multiple reasons. One consideration that was noted in mice is that humoral responses may preferentially target the carboxyl terminus of the peptide sequence which is not present in the intact protein. To shed light on the frequency of preferential rabbit antibody responses to C-termini of peptide immunogens, we present our experience with generation of rabbit antibodies to human NOTCH3. A total of 23 antibodies were raised against 10 peptide sequences of human NOTCH3. Over 70% (16 of 23) of these polyclonal antibodies were determined to be C-terminal preferring: NOTCH3 peptide-reactive antibodies largely targeted the terminating free carboxyl group of the immunizing peptide. The antibodies that preferred C-terminal epitopes reacted weakly or not at all with recombinant target sequences with extension the C-terminus that eliminated the free carboxyl group of the immunogen structure; furthermore, each of these antisera revealed no antibody reactivity to proteins truncated before the C-terminus of the immunogen. In immunocytochemical applications of these anti-peptide antibodies, we similarly found reactivity to recombinant targets that best binding to cells expressing the free C-terminus of the immunizing sequence. In aggregate, our experience demonstrates a strong propensity for rabbits to mount antibody responses to C-terminal epitopes of NOTCH3-derived peptides which is predicted to limit their use against the native protein. We discuss some potential approaches to overcome this bias that could improve the efficiency of generation of antibodies in this commonly utilized experimental paradigm.

Plasma Exosome Analysis for Protein Mutation Identification Using a Combination of Raman Spectroscopy and Deep Learning.

Protein mutation detection using liquid biopsy can be simply performed periodically, making it easy to detect the occurrence of newly emerging mutations rapidly. However, it has low diagnostic accuracy since there are more normal proteins than mutated proteins in body fluids. To increase the diagnostic accuracy, we analyzed plasma exosomes using nanoplasmonic spectra and deep learning. Exosomes, a promising biomarker, are abundant in plasma and stably carry intact proteins originating from mother cells. However, the mutated exosomal proteins cannot be detected sensitively because of the subtle changes in their structure. Therefore, we obtained Raman spectra that provide molecular information about structural changes in mutated proteins. To extract the unique features of the protein from complex Raman spectra, we developed a deep-learning classification algorithm with two deep-learning models. Consequently, controls with wild-type proteins and patients with mutated proteins were classified with high accuracy. As a proof of concept, we discriminated the lung cancer patients with mutations in the epidermal growth factor receptor (EGFR), L858R, E19del, L858R + T790M, and E19del + T790M, from controls with an accuracy of 0.93. Moreover, the protein mutation status of the patients with primary (E19del, L858R) and secondary (+T790M) mutations was clearly monitored. Overall, our technique is expected to be applied as a novel method for companion diagnostic and treatment monitoring.

Orbitrap-Based Mass and Charge Analysis of Single Molecules.

Native mass spectrometry is nowadays widely used for determining the mass of intact proteins and their noncovalent biomolecular assemblies. While this technology performs well in the mass determination of monodisperse protein assemblies, more real-life heterogeneous protein complexes can pose a significant challenge. Factors such as co-occurring stoichiometries, subcomplexes, and/or post-translational modifications, may especially hamper mass analysis by obfuscating the charge state inferencing that is fundamental to the technique. Moreover, these mass analyses typically require measurement of several million molecules to generate an analyzable mass spectrum, limiting its sensitivity. In 2012, we introduced an Orbitrap-based mass analyzer with extended mass range (EMR) and demonstrated that it could be used to obtain not only high-resolution mass spectra of large protein macromolecular assemblies, but we also showed that single ions generated from these assemblies provided sufficient image current to induce a measurable charge-related signal. Based on these observations, we and others further optimized the experimental conditions necessary for single ion measurements, which led in 2020 to the introduction of single-molecule Orbitrap-based charge detection mass spectrometry (Orbitrap-based CDMS). The introduction of these single molecule approaches has led to the fruition of various innovative lines of research. For example, tracking the behavior of individual macromolecular ions inside the Orbitrap mass analyzer provides unique, fundamental insights into mechanisms of ion dephasing and demonstrated the (astonishingly high) stability of high mass ions. Such fundamental information will help to further optimize the Orbitrap mass analyzer. As another example, the circumvention of traditional charge state inferencing enables Orbitrap-based CDMS to extract mass information from even extremely heterogeneous proteins and protein assemblies (e.g., glycoprotein assemblies, cargo-containing nanoparticles) via single molecule detection, reaching beyond the capabilities of earlier approaches. We so far demonstrated the power of Orbitrap-based CDMS applied to a variety of fascinating systems, assessing for instance the cargo load of recombinant AAV-based gene delivery vectors, the buildup of immune-complexes involved in complement activation, and quite accurate masses of highly glycosylated proteins, such as the SARS-CoV-2 spike trimer proteins. With such widespread applications, the next objective is to make Orbitrap-based CDMS more mainstream, whereby we still will seek to further advance the boundaries in sensitivity and mass resolving power.

The Impact of Infant Feeding Regimen on Cow's Milk Protein Allergy, Atopic Dermatitis and Growth in High-Risk Infants during the First 6 Months of Life: The Allergy Reduction Trial.

The development of early-onset cow's milk protein allergy and atopic dermatitis during the first months of life is multifactorial, including both genetic and nutritional aspects. This study aims to assess the impact of different feeding patterns on the incidence of cow's milk protein allergy, atopic dermatitis, and growth among infants with a family history of allergy. A total of 551 high-risk infants were randomly recruited from 3 European countries in three feeding regimens: exclusive breastfeeding, partially hydrolyzed formula, or standard formula with intact protein either exclusively or supplementary to breastfeeding. During the first 6 months of intervention, amongst infants with a family history of atopic dermatitis, 6.5% of partially hydrolyzed formula-fed infants and 22.7% of exclusively breastfed infants (p = 0.007) presented with atopic dermatitis respectively. Growth as assessed by weight increase did not differ between the aforementioned groups. Although cow's milk protein allergy was not related to the different milk feeding regimens in the whole cohort, when adjusting for high breast milk intake, the respective incident was significantly lower in the infants consuming partially hydrolyzed formula (p < 0.001). This data indicates that a specific partially hydrolyzed formula could serve as a more appropriate complement to breast milk compared to a standard intact protein formula in high-risk infants, to reduce the incidence of atopic dermatitis.

In-depth characterization of myofibrillar muscle proteome changes in lambs fed hazelnut skin by-products

This study investigated the effect of hazelnut skin by-products supplementation on lamb myofibrillar proteome changes during post-mortem storage (0, 4, and 7 days). Gel-based proteomics and bioinformatics approaches were applied to reveal the underlying biochemical pathways and their importance in lamb meat texture development. Twenty-two Valle del Belice male lambs were randomly assigned to two dietary treatments: control (C) for lambs fed with maize-barley diet, and hazelnut skin (H) for lambs fed hazelnut skin by-product as maize partial replacer in the concentrate diet (150 g/kg DM basis). A greater myofibrillar fragmentation index (MFI) was found in meat from the C group at day 0 of storage (91.33 vs 98.38 in H and C groups, respectively). Conversely, starting from 4 days of storage, higher MFI values were observed in meat from lambs fed hazelnut skin (113.74 and 116.1 vs 99.28 and 107.26 in H and C groups at 4 and 7 days, respectively). Myofibrillar proteome changes estimated by SDS-PAGE and immunoblotting revealed a degradation of desmin (P<0.01) and troponin T (P<0.001) intact proteins, and an increase in the abundance of the appearing 30 kDa fragment (P<0.001) after 4 days of storage in meat from H than the C group. In-depth proteomics and bioinformatics revealed 44 proteoforms (26 unique proteins), mainly involved in actin filament-based process/cytoskeleton organization, energy metabolism, and heat shock proteins, as the major interconnected pathways impacted by hazelnut by-product feeding strategy on lamb meat quality. Twelve proteins were proposed in this trial as potential biomarkers of lamb meat texture as a consequence of hazelnut skin by-products supplementation.

Development of a Liquid Chromatography and High-Resolution and -Accuracy Mass Spectrometry Method to Evaluate New Biotherapeutic Entity Processing in Human Liver Lysosomes.

Biotherapeutic immunogenicity remains a great challenge for researchers because multiple factors trigger immune responses. Predicting and assessing the potential human immune response against biological drugs could represent an impressive breakthrough toward generating potentially safer and more efficacious therapeutic proteins. This article describes an invitro assay that can contribute to evaluating the potential immunogenicity of biotherapeutics by focusing on lysosomal proteolysis. We selected human liver lysosomes (hLLs) from four different donors as a surrogate invitro model instead of APC lysosomes because they are a ready-to-use lysosomal source. To assess the biological comparability of this surrogate to APC lysosomal extract, we compared the proteome content of hLLs with literature data of lysosomal fractions extracted from murine bone marrow and human blood-derived dendritic cells. Then we tested infliximab (IFX; Remicade) under different proteolytic conditions using liquid chromatography and high-resolution and -accuracy mass spectrometry to better define the degradation kinetics inside the lysosomes. hLLs revealed similar enzymatic content compared with human and murine dendritic cell lysosomes. Degradation assays demonstrated that our liquid chromatography and high-resolution and -accuracy mass spectrometry method could identify both the intact protein and the peptides resulting from proteolysis with high specificity and resolution. The rapid and easy assay described in this article can be extremely useful for evaluating the immunogenic risk associated with therapeutic proteins. In addition, this method can complement information from MHC class II-associated peptide proteomics assays and other invitro and in silico techniques.