Multilevel Proteomic Profiling of Colorectal Adenocarcinoma Caco-2 Cell Differentiation to Characterize an Intestinal Epithelial Model.

Emergent advancements on the role of the intestinal microbiome for human health and disease necessitate well-defined intestinal cellular models to study and rapidly assess host, microbiome, and drug interactions. Differentiated Caco-2 cell line is commonly utilized as an epithelial model for drug permeability studies and has more recently been utilized for investigating host-microbiome interactions. However, its suitability to study such interactions remains to be characterized. Here, we employed multilevel proteomics to demonstrate that both spontaneous and butyrate-induced Caco-2 differentiations displayed similar protein and pathway changes, including the downregulation of proteins related to translation and proliferation and upregulation of functions implicated in host-microbiome interactions, such as cell adhesion, tight junction, extracellular vesicles, and responses to stimuli. Lysine acetylomics revealed that histone protein acetylation levels were decreased along with cell differentiation, while the acetylation in proteins associated with mitochondrial functions was increased. This study also demonstrates that, compared to spontaneous differentiation methods, butyrate-containing medium accelerates Caco-2 differentiation, with earlier upregulation of proteins related to host-microbiome interactions, suggesting its superiority for assay development using this intestinal model. Altogether, this multiomics study emphasizes the controlled progression of Caco-2 differentiation toward a specialized intestinal epithelial-like cell and establishes its suitability for investigating the host-microbiome interactions.

Strategies for the production of isotopically labelled Fab fragments of therapeutic antibodies in Komagataella phaffii (Pichia pastoris) and Escherichia coli for NMR studies.

The importance and fast growth of therapeutic monoclonal antibodies, both innovator and biosimilar products, have triggered the need for the development of characterization methods at high resolution such as nuclear magnetic resonance (NMR) spectroscopy. However, the full power of NMR spectroscopy cannot be unleashed without labelling the mAb of interest with NMR-active isotopes. Here, we present strategies using either Komagataella phaffii (Pichia pastoris) or Escherichia coli that can be widely applied for the production of the antigen-binding fragment (Fab) of therapeutic antibodies of immunoglobulin G1 kappa isotype. The E. coli approach consists of expressing Fab fragments as a single polypeptide chain with a cleavable linker between the heavy and light chain in inclusion bodies, while K. phaffii secretes a properly folded fragment in the culture media. After optimization, the protocol yielded 10-45 mg of single chain adalimumab-Fab, trastuzumab-Fab, rituximab-Fab, and NISTmAb-Fab per liter of culture. Comparison of the 2D-1H-15N-HSQC spectra of each Fab fragment, without their polyhistidine tag and linker, with the corresponding Fab from the innovator product showed that all four fragments have folded into the correct conformation. Production of 2H-13C-15N-adalimumab-scFab and 2H-13C-15N-trastuzumab-scFab (>98% enrichment for all three isotopes) yielded NMR samples where all amide deuterons have completely exchanged back to proton during the refolding procedure.

An economic and robust TMT labeling approach for high throughput proteomic and metaproteomic analysis.

Multiplexed quantitative proteomics using tandem mass tag (TMT) is increasingly used in -omic study of complex samples. While TMT-based proteomics has the advantages of the higher quantitative accuracy, fewer missing values, and reduced instrument analysis time, it is limited by the additional reagent cost. In addition, current TMT labeling workflows involve repeated small volume pipetting of reagents in volatile solvents, which may increase the sample-to-sample variations and is not readily suitable for high throughput applications. In this study, we demonstrated that the TMT labeling procedures could be streamlined by using pre-aliquoted dry TMT reagents in a 96 well plate or 12-tube strip. As little as 50 µg dry TMT per channel was used to label 6-12 µg peptides, yielding high TMT labeling efficiency (∼99%) in both microbiome and mammalian cell line samples. We applied this workflow to analyze 97 samples in a study to evaluate whether ice recrystallization inhibitors improve the cultivability and activity of frozen microbiota. The results demonstrated tight sample clustering corresponding to groups and consistent microbiome responses to prebiotic treatments. This study supports the use of TMT reagents that are pre-aliquoted, dried, and stored for robust quantitative proteomics and metaproteomics in high throughput applications.

Selective reversed-phase high-performance liquid chromatography method for the determination of intact SARS-CoV-2 spike protein.

Protein-based vaccines are playing an increasingly important role in the COVID-19 pandemic. As late-stage clinical data are finalized and released, the number of protein-based vaccines expected to enter the market will increase significantly. Most protein-based COVID-19 vaccines are based on the SARS-CoV-2 spike protein (S-protein), which plays a major role in viral attachment to human cells and infection. As a result, in order to develop and manufacture quality vaccines consistently, it is imperative to have access to selective and efficient methods for the bioanalytical assessment of S-protein. In this study, samples of recombinant S-protein (hexS-protein) and commercial S-protein were used to develop a selective reversed-phase HPLC (RP-HPLC) method that enabled elution of the intact S-protein monomer as a single peak on a wide pore, C8-bonded chromatographic column. The S-protein subunits, S1 and S2 subunits, were clearly separated from intact S-protein and identified. The results of this study set the foundation for reversed-phase HPLC method development and analysis for selective and efficient separation of S-protein monomer from its subunits.

NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods.

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.

A comprehensive proteomics profiling identifies NRP1 as a novel identity marker of human bone marrow mesenchymal stromal cell-derived small extracellular vesicles.

BACKGROUND: Clinical applications have shown extracellular vesicles (EVs) to be a major paracrine effector in therapeutic responses produced by human mesenchymal stromal/stem cells (hMSCs). As the regenerative capacity of EVs is mainly ascribed to the transfer of proteins and RNA composing its cargo, and to the activity attributed by the protein surface markers, we sought to profile the protein composition of small EVs released from hMSCs to identify hMSC-EV biomarkers with potential clinical relevance. METHODS: Small EVs were produced and qualified from five human bone marrow MSC donors at low passage following a 48-h culture in exosome-depleted medium further processed by steps of centrifugation, filtration, and precipitation. Quantitative proteomic analysis comparing the protein profile of the EVs released from hMSCs and their parental cell was conducted using tandem mass tag labeling combined to mass spectrometry (LC-MS/MS) to identify enriched EV protein markers. RESULTS: Nanoparticle tracking analysis showed no differences in the EV concentration and size among the five hMSC donors (1.83 × 1010 ± 3.23 × 109/mL), with the mode particle size measuring at 109.3 ± 5.7 nm. Transmission electron microscopy confirmed the presence of nanovesicles with bilayer membranes. Flow cytometric analysis identified commonly found exosomal (CD63/CD81) and hMSC (CD105/CD44/CD146) markers from released EVs in addition to surface mediators of migration (CD29 and MCSP). Quantitative proteomic identified 270 proteins significantly enriched by at least twofold in EVs released from hMSCs as compared to parental hMSCs, where neuropilin 1 (NRP1) was identified among 21 membrane-bound proteins regulating the migration and invasion of cells, as well as chemotaxis and vasculogenesis. Validation by western blot of multiple batches of EVs confirmed consistent enrichment of NRP1 in the nanovesicles released from all five hMSC donors. CONCLUSION: The identification and verification of NRP1 as a novel enriched surface marker from multiple batches of EVs derived from multiple hMSC donors may serve as a biomarker for the assessment and measurement of EVs for therapeutic uses.

PD-1 of Sigmodon hispidus: Gene identification, characterization and preliminary evaluation of expression in inactivated RSV vaccine-induced enhanced respiratory disease.

Sigmodon hispidus or cotton rat is an excellent animal model for studying human infections of respiratory viruses including respiratory syncytial virus (RSV), which is the leading cause of hospitalization in infants and causes high rates of infection in the elderly and immunocompromised patient populations. Despite several decades of research, no vaccine has been licensed whereas inactivated vaccines have been shown to induce severe adverse reaction in a clinical trial, with other forms of RSV vaccine also found to induce enhanced disease in preclinical animal studies. While arguably the cotton rat is the best small animal model for evaluation of RSV vaccines and antivirals, many important genes of the immune system remain to be isolated. Programmed cell death-1 (PD-1) plays an integral role in regulating many aspects of immunity by inducing suppressive signals. In this study, we report the isolation of mRNA encoding the cotton rat PD-1 (crPD-1) and characterization of the PD-1 protein. crPD-1 bound to its cognate ligand on dendritic cells and effectively suppressed cytokine secretion. Moreover, using the newly acquired gene sequence, we observed a decreased level of crPD-1 levels in cotton rats with enhanced respiratory disease induced by inactivated RSV vaccine, unraveling a new facet of vaccine-induced disease.

Unveiling Integrated Functional Pathways Leading to Enhanced Respiratory Disease Associated With Inactivated Respiratory Syncytial Viral Vaccine.

Respiratory syncytial virus (RSV) infection is a severe threat to young children and the elderly. Despite decades of research, no vaccine has been approved. Notably, instead of affording protection, a formalin-inactivated RSV vaccine induced severe respiratory disease including deaths in vaccinated children in a 1960s clinical trial; however, recent studies indicate that other forms of experimental vaccines can also induce pulmonary pathology in pre-clinical studies. These findings suggest that multiple factors/pathways could be involved in the development of enhanced respiratory diseases. Clearly, a better understanding of the mechanisms underlying such adverse reactions is critically important for the development of safe and efficacious vaccines against RSV infection, given the exponential growth of RSV vaccine clinical trials in recent years. By employing an integrated systems biology approach in a pre-clinical cotton rat model, we unraveled a complex network of pulmonary canonical pathways leading to disease development in vaccinated animals upon subsequent RSV infections. Cytokines including IL-1, IL-6 GRO/IL-8, and IL-17 in conjunction with mobilized pulmonary inflammatory cells could play important roles in disease development, which involved a wide range of host responses including exacerbated pulmonary inflammation, oxidative stress, hyperreactivity, and homeostatic imbalance between coagulation and fibrinolysis. Moreover, the observed elevated levels of MyD88 implicate the involvement of this critical signal transduction module as the central node of the inflammatory pathways leading to exacerbated pulmonary pathology. Finally, the immunopathological consequences of inactivated vaccine immunization and subsequent RSV exposure were further substantiated by histological analyses of these key proteins along with inflammatory cytokines, while hypercoagulation was supported by increased pulmonary fibrinogen/fibrin accompanied by reduced levels of plasma D-dimers. Enhanced respiratory disease associated with inactivated RSV vaccine involves a complex network of host responses, resulting in significant pulmonary lesions and clinical manifestations such as tachypnea and airway obstruction. The mechanistic insight into the convergence of different signal pathways and identification of biomarkers could help facilitate the development of safe and effective RSV vaccine and formulation of new targeted interventions.

Brief Report: Elastin Microfibril Interface 1 and Integrin-Linked Protein Kinase Are Novel Markers of Islet Regenerative Function in Human Multipotent Mesenchymal Stromal Cells.

Multipotent mesenchymal stromal cell (MSC) transplantation is proposed as a novel therapy for treating diabetes by promoting the regeneration of damaged islets. The clinical promise of such treatments may be hampered by a high degree of donor-related variability in MSC function and a lack of standards for comparing potency. Here, we set out to identify markers of cultured human MSCs directly associated with islet regenerative function. Stromal cultures from nine separate bone marrow donors were demonstrated to have differing capacities to reduce hyperglycemia in the NOD/SCID streptozotocin-induced diabetic model. Regenerative (R) and non-regenerative (NR) MSC cultures were directly compared using isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics. A total of 1,410 proteins were quantified resulting in the identification of 612 upregulated proteins and 275 downregulated proteins by ± 1.2-fold in R-MSC cultures. Elastin microfibril interface 1 (EMILIN-1), integrin-linked protein kinase (ILK), and hepatoma-derived growth factor (HDGF) were differentially expressed in R-MSCs, and Ingenuity Pathway Analyses revealed each candidate as known regulators of integrin signaling. Western blot validation of EMILIN-1, ILK, and HDGF not only showed significantly higher abundance levels in R-MSCs, as compared with NR-MSCs, but also correlated with passage-induced loss of islet-regenerative potential. Generalized estimating equation modeling was applied to examine the association between each marker and blood glucose reduction. Both EMILIN-1 and ILK were significantly associated with blood glucose lowering function in vivo. Our study is the first to identify EMILIN-1 and ILK as prospective markers of islet regenerative function in human MSCs. Stem Cells 2016;34:2249-2255.

Characterizing the surface-exposed proteome of Planococcus halocryophilus during cryophilic growth.

Planococcus halocryophilus OR1 is a bacterial isolate capable of growth at temperatures ranging from -15 to +37 °C. During sub-zero (cryophilic) growth, nodular features appear on its cell surface; however, the biochemical compositions of these features as well as any cold-adaptive benefits they may offer are not understood. This study aimed to identify differences in the cell surface proteome (surfaceome) of P. halocryophilus cells grown under optimal (24 °C, no added salt), low- and mid-salt (5 and 12 % NaCl, respectively) at 24 °C, and low- and mid-salt sub-zero (5 % NaCl at -5 °C and 12 % NaCl at -10 °C) culture conditions, for the purpose of gaining insight into cold-adapted proteomic traits at the cell surface. Mid-log cells were harvested, treated briefly with trypsin and the resultant peptides were purified followed by identification by LC-MS/MS analysis. One hundred and forty-four proteins were subsequently identified in at least one culture condition. Statistically significant differences in amino acid usage, a known indicator of cold adaptation, were identified through in silico analysis. Two proteins with roles in peptidoglycan (PG) metabolism, an N-acetyl-L-alanine amidase and a multimodular transpeptidase-transglycosylase, were detected, though each was only detected under optimal conditions, indicating that high-salt and high-cold stress each affect PG metabolism. Two iron transport-binding proteins, associated with two different iron transport strategies, were identified, indicating that P. halocryophilus uses a different iron acquisition strategy at very low temperatures. Here we present the first set of data that describes bacterial adaptations at the cellular surface that occur as a cryophilic bacterium is transitioned from optimal to near-inhibitory sub-zero culture conditions.

Proteomic identification of Listeria monocytogenes surface-associated proteins.

This study aimed to identify proteins exposed on the surface of Listeria monocytogenes cells for diagnostic reagent development. Brief trypsin treatment of L. monocytogenes cells followed by peptide separation and identification by nano-LC and online-MS/MS was performed. In parallel, as a negative control, proteins secreted into the digest buffer as well as proteins from cell lysis were identified. One hundred and seventy-four proteins were identified in at least two of three trials in either the negative control or during cell digest. Nineteen surface, 21 extracellularly secreted, 132 cytoplasmic, and two phage proteins were identified. Immunofluorescence microscopy of L. monocytogenes cells revealed the surface localization of two potential candidates for L. monocytogenes isolation and detection: lipoprotein LMOf2365_0546 and PBPD1 (LMOf2365_2742). In this report, we present the first data set of surface-exposed L. monocytogenes proteins currently available. The data have been deposited to the ProteomeXchange Consortium with identifier PXD000035.

Simultaneous quantification of the viral antigens hemagglutinin and neuraminidase in influenza vaccines by LC-MSE.

Current methods for quality control of inactivated influenza vaccines prior to regulatory approval include determining the hemagglutinin (HA) content by single radial immunodiffusion (SRID), verifying neuraminidase (NA) enzymatic activity, and demonstrating that the levels of the contaminant protein ovalbumin are below a set threshold of 1 µg/dose. The SRID assays require the availability of strain-specific reference HA antigens and antibodies, the production of which is a potential rate-limiting step in vaccine development and release, particularly during a pandemic. Immune responses induced by neuraminidase also contribute to protection from infection; however, the amounts of NA antigen in influenza vaccines are currently not quantified or standardized. Here, we report a method for vaccine analysis that yields simultaneous quantification of HA and NA levels much more rapidly than conventional HA quantification techniques, while providing additional valuable information on the total protein content. Enzymatically digested vaccine proteins were analyzed by LC-MS(E), a mass spectrometric technology that allows absolute quantification of analytes, including the HA and NA antigens, other structural influenza proteins and chicken egg proteins associated with the manufacturing process. This method has potential application for increasing the accuracy of reference antigen standards and for validating label claims for HA content in formulated vaccines. It can also be used to monitor NA and chicken egg protein content in order to monitor manufacturing consistency. While this is a useful methodology with potential for broad application, we also discuss herein some of the inherent limitations of this approach and the care and caution that must be taken in its use as a tool for absolute protein quantification. The variations in HA, NA and chicken egg protein concentrations in the vaccines analyzed in this study are indicative of the challenges associated with the current manufacturing and quality control testing procedures.

Strain identification of commercial influenza vaccines by mass spectrometry.

Current influenza vaccine manufacturing and testing timelines require that the constituent hemagglutinin (HA) and neuraminidase (NA) strains be selected each year approximately 10 months before the vaccine becomes available. The threat of a pandemic influenza outbreak requires that more rapid testing methods be found. We have developed a specialized on-filter sample preparation method that uses both trypsin and chymotrypsin to enzymatically digest peptide-N-glycosidase F (PNGase F)-deglycosylated proteins in vaccines. In tandem with replicate liquid chromatography-mass spectrometry (LC-MS) analyses, this approach yields sufficient protein sequencing data (>85% sequence coverage on average) for strain identification of HA and NA components. This has allowed the confirmation, and in some cases the correction, of the identity of the influenza strains in recent commercial vaccines as well as the correction of some ambiguous HA sequence annotations in available databases. This method also allows the identification of low-level contaminant egg proteins produced during the manufacturing process.