Filter-aided Sample Preparation Research Articles

Metabolomics and proteomics insights into hepatic responses of weaned piglets to dietary Spirulina inclusion and lysozyme supplementation

BackgroundStudying the effect of dietary Spirulina and lysozyme supplementation on the metabolome and proteome of liver tissue contributes to understanding potential hepatic adaptations of piglets to these novel diets. This study aimed to understand the influence of including 10% Spirulina on the metabolome and proteome of piglet liver tissue. Three groups of 10 post-weaned piglets, housed in pairs, were fed for 28 days with one of three experimental diets: a cereal and soybean meal-based diet (Control), a base diet with 10% Spirulina (SP), and an SP diet supplemented with 0.01% lysozyme (SP + L). At the end of the trial, animals were sacrificed and liver tissue was collected. Metabolomics analysis (n = 10) was performed using NMR data analysed with PCA and PLS-DA. Proteomics analysis (n = 5) was conducted using a filter aided sample preparation (FASP) protocol and Tandem Mass Tag (TMT)-based quantitative approach with an Orbitrap mass spectrometer.ResultsGrowth performance showed an average daily gain reduction of 9.5% and a feed conversion ratio increase of 10.6% in groups fed Spirulina compared to the control group. Metabolomic analysis revealed no significant differences among the groups and identified 60 metabolites in the liver tissue. Proteomics analysis identified 2,560 proteins, with 132, 11, and 52 differentially expressed in the Control vs. SP, Control vs. SP + L and SP vs. SP + L comparisons, respectively. This study demonstrated that Spirulina enhances liver energy conversion efficiency, detoxification and cellular secretion. It improves hepatic metabolic efficiency through alterations in fatty acid oxidation (e.g., upregulation of enzymes like fatty acid synthase and increased acetyl-CoA levels), carbohydrate catabolism (e.g., increased glucose and glucose-6-phosphate), pyruvate metabolism (e.g., higher levels of pyruvate and phosphoenolpyruvate carboxykinase), and cellular defence mechanisms (e.g., upregulation of glutathione and metallothionein). Lysozyme supplementation mitigates some adverse effects of Spirulina, bringing physiological responses closer to control levels. This includes fewer differentially expressed proteins and improved dry matter, organic matter and energy digestibility. Lysozyme also enhances coenzyme availability, skeletal myofibril assembly, actin-mediated cell contraction, tissue regeneration and development through mesenchymal migration and nucleic acid synthesis pathways.ConclusionsWhile Spirulina inclusion had some adverse effects on growth performance, it also enhanced hepatic metabolic efficiency by improving fatty acid oxidation, carbohydrate catabolism and cellular defence mechanisms. The addition of lysozyme further improved these benefits by reducing some of the negative impacts on growth and enhancing nutrient digestibility, tissue regeneration, and overall metabolic balance. Together, Spirulina and lysozyme demonstrate potential as functional dietary components, but further optimization is needed to fully realize their benefits without compromising growth performance.

Proteomic analysis of the human amniotic mesenchymal stromal cell secretome by integrated approaches via filter-aided sample preparation

The immunomodulatory, anti-inflammatory and regenerative properties of the human amniotic mesenchymal stromal cells (hAMSCs) secretome are acknowledged but the understanding of the specific bioactive components remains incomplete. To address these limitations, the present investigation aimed to profile the proteins and peptides content of the hAMSC secretome through sample pretreatment and fractionation on 10 kDa molecular cut-off FASP (Filter Aided Sample Preparation) device and LC-MS analysis. The filter retained protein fraction underwent trypsin digestion, while the unretained was collected unchanged for intact small proteins and peptides analysis. This combined approach (C-FASP) collects in a single step two complementary fractions, advantageously saving sample volume and time of analysis. The bottom-up analysis of the C-FASP proteins fraction >10 kDa confirmed our previous findings, establishing a set of proteins consistently characterizing the hAMSC secretome. The analysis of the fraction <10 kDa, never been investigated to our knowledge, identified peptide fragments of thymosin beta 4 and beta 10, collagen alpha 1 chains I and III, alpha-enolase, and glyceraldehyde-3-phosphate dehydrogenase, involved in wound healing, anti-inflammatory response, tissue repair and regeneration, key biological activities of the secretome. C-FASP provided a comprehensive molecular profile of the hAMSC secretome offering new insights for enhanced therapeutic applications in regenerative medicine. SignificanceIn this investigation we originally present the comprehensive proteomic investigation of the human amniotic mesenchymal stromal cell secretome by combining the analysis of the proteome and of the peptidome following sample pretreatment and fractionation by Filter Aided Sample Preparation (FASP) with 10 kDa molecular cut-off in coupling with LC-MS analysis. The proteome fraction retained by FASP filter was analyzed after enzymatic digestion, while the unretained fraction, below 10 kDa molecular mass, was analyzed unchanged in its intact form. This dual approach provides novel insights, previously unexplored, into the molecular components potentially responsible for the immunomodulatory and anti-inflammatory properties of the hAMSC secretome. These findings could significantly enhance the therapeutic potential of hAMSCs in regenerative medicine.

Low-Invasive Sampling Method with Tape-Disc Sampling for the Taxonomic Identification of Archeological and Paleontological Bones by Proteomics.

Collagen from paleontological bones is an important organic material for isotopic measurement, radiocarbon analysis, and paleoproteomic analysis to provide information on diet, dating, taxonomy, and phylogeny. Current paleoproteomic methods are destructive and require from a few milligrams to several tens of milligrams of bone for analysis. In many cultures, bones are raw materials for artifacts that are conserved in museums, which hampers damage to these precious objects during sampling. Here, we describe a low-invasive sampling method that identifies collagen, taxonomy, and post-translational modifications from Holocene and Upper Pleistocene bones dated to 130,000 and 150 BC using dermatological skin tape discs for sampling. The sampled bone micropowders were digested following our highly optimized enhanced filter-aided sample preparation protocol and then analyzed by MALDI FTICR MS and LC-MS/MS for identifying the genus taxa of the bones. We show that this low-invasive sampling does not deteriorate the bones and achieves results similar to those obtained by more destructive sampling. Moreover, this sampling method can be carried out at archeological sites or in museums.

An Optimized Miniaturized Filter-Aided Sample Preparation Method for Sensitive Cross-Linking Mass Spectrometry Analysis of Microscale Samples.

Cross-linking mass spectrometry (XL-MS) is a powerful tool for elucidating protein structures and protein-protein interactions (PPIs) at the global scale. However, sensitive XL-MS analysis of mass-limited samples remains challenging, due to serious sample loss during sample preparation of the low-abundance cross-linked peptides. Herein, an optimized miniaturized filter-aided sample preparation (O-MICROFASP) method was presented for sensitive XL-MS analysis of microscale samples. By systematically investigating and optimizing crucial experimental factors, this approach dramatically improves the XL identification of low and submicrogram samples. Compared with the conventional FASP method, more than 7.4 times cross-linked peptides were identified from single-shot analysis of 1 μg DSS cross-linked HeLa cell lysates (440 vs 59). The number of cross-linked peptides identified from 0.5 μg HeLa cell lysates was increased by 58% when further reducing the surface area of the filter to 0.058 mm2 in the microreactor. To deepen the identification coverage of XL-proteome, five different types of cross-linkers were used and each μg of cross-linked HeLa cell lysates was processed by O-MICROFASP integrated with tip-based strong cation exchange (SCX) fractionation. Up to 2741 unique cross-linked peptides were identified from the 5 μg HeLa cell lysates, representing 2579 unique K-K linkages on 1092 proteins. About 96% of intraprotein cross-links were within the maximal distance restraints of 26 Å, and 75% of the identified PPIs reported by the STRING database were with high confidence (scores ≥0.9), confirming the high validity of the identified cross-links for protein structural mapping and PPI analysis. This study demonstrates that O-MICROFASP is a universal and efficient method for proteome-wide XL-MS analysis of microscale samples with high sensitivity and reliability.

#1978 Proteomic analysis of exosomes derived from mesangial cells treated with galactose-deficient IgA1

Abstract Background and Aims IgA nephropathy (IgAN) is an autoimmune disease characterized by IgA deposits that build up in the kidneys, inflame and damage glomeruli causing hematuria and proteinuria. It is a leading cause of chronic kidney disease, which often results in end-stage kidney disease. There is no disease specific treatment for IgAN. The glomerulus, in which IgA1 lacking galactose residues (gdIgA1) are deposited, consists of mesangial cells, podocytes, and endothelial cells. Crosstalk between the cell types allows the glomerulus to act as a unit and react to its dynamic environment. There is very little evidence on mesangial cell derived exosomes and their cargo to understand the pathological crosstalk signaling in the glomerulus of IgAN. We isolated exosomes from mesangial cells that were treated with patient-derived gdIgA1 and IgA1 complexes. Proteomic analysis was performed to identify the exosome cargo and delineate the signaling pathways leading to maladaptive paracrine crosstalk. From our study, we aim to understand the molecular targets for therapeutic strategies in IgAN. Method Human mesangial cells were treated with/without IgA1, gdIgA1 or IgG for 24 h. Cell culture supernatant was collected for exosome isolations. Exosomes were analyzed by size, morphology and number using TEM and NanoFCM. Exosome protein extracts were processed for LC-Tandem mass spectrometry analysis using filter-aided sample preparation protocol. Proteomic analysis was performed using Qlucore omics explorer and pathway analysis using Ingenuity Pathway Analysis (IPA). Exosome marker expressions such as CD63, CD81 and FLOT1 and proteome validation was performed using Western blotting. Statistical analysis was performed using GraphPad Prism and values &amp;lt;0.05 were considered significant. Results We confirmed the release of exosomes by mesangial cells in response to IgA1/gdIgA1 complexes. Exosomes were fewer in unstimulated cells as compared to IgA1/gdIgA1 treated cells. They showed round morphology with a dense core and uniform size distribution. Exosomes from treated cells showed a mean size of about 75 nm and concentration in the range of 1-2 × 10^10 particles/ml. While untreated cells showed a mean size of about 100 nm and concentration of about 1 × 10^9 particles/ml. We confirmed the protein expression of exosome markers such as CD63, CD81 and FLOT1. For the first time, we demonstrated global proteomics on exosomes from mesangial cells followed by pathway analysis. We have identified 89 unique proteins and pathways in exosomes derived from gdIgA1 treated mesangial cells that could mediate pathological signaling in the glomerulus in comparison to exosomes derived from IgA1 treated mesangial cells. Conclusion Our study provides an understanding of the pathological crosstalk signaling through exosomes and key molecular targets for IgAN.

Evaluation of PAC and FASP Performance: DIA-Based Quantitative Proteomic Analysis.

The aim of this study was to compare filter-aided sample preparation (FASP) and protein aggregation capture (PAC) starting from a three-species protein mix (Human, Soybean and Pisum sativum) and two different starting amounts (1 and 10 µg). Peptide mixtures were analyzed by data-independent acquisition (DIA) and raw files were processed by three commonly used software: Spectronaut, MaxDIA and DIA-NN. Overall, the highest number of proteins (mean value of 5491) were identified by PAC (10 µg), while the lowest number (4855) was identified by FASP (1 µg). The latter experiment displayed the worst performance in terms of both specificity (0.73) and precision (0.24). Other tested conditions showed better diagnostic accuracy, with specificity values of 0.95-0.99 and precision values between 0.61 and 0.86. In order to provide guidance on the data analysis pipeline, the accuracy diagnostic of three software was investigated: (i) the highest sensitivity was obtained with Spectronaut (median of 0.67) highlighting the ability of Spectronaut to quantify low-abundance proteins, (ii) the best precision value was obtained by MaxDIA (median of 0.84), but with a reduced number of identifications compared to Spectronaut and DIA-NN data, and (iii) the specificity values were similar (between 0.93 and 0.99). The data are available on ProteomeXchange with the identifier PXD044349.

From bottom-up to cell surface proteomics: detergents or no detergents, that is the question.

Measuring the expression levels of membrane proteins (MPs) is crucial for understanding cell differentiation and tissue specificity, defining disease characteristics, identifying biomarkers, and developing therapeutics. While bottom-up proteomics addresses the need for accurately surveying the membrane proteome, the lower abundance and hydrophobic nature of MPs pose challenges in sample preparation. As MPs normally reside in the lipid bilayer, conventional extraction methods rely on detergents, introducing here a paradox - detergents prevent aggregation and facilitate protein processing, but themselves become contaminants that interfere with downstream analytical applications. Various detergent removal methods exist to mitigate this issue, including filter-aided sample preparation, SP3, suspension trapping, and membrane mimetics. This review delves into the fundamentals of each strategy, applications, merits, and limitations, providing insights into their effectiveness in MP research.

Proteomic Analysis of Salivary Secretions from the Tea Green Leafhopper, Empoasca flavescens Fabrecius.

Saliva plays a crucial role in shaping the compatibility of piercing-sucking insects with their host plants. Understanding the complex composition of leafhopper saliva is important for developing effective and eco-friendly control strategies for the tea green leafhopper, Empoasca flavescens Fabrecius, a major piercing-sucking pest in Chinese tea plantations. This study explored the saliva proteins of tea green leafhopper adults using a custom collection device, consisting of two layers of Parafilm stretched over a sucrose diet. A total of 152 proteins were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) following the filter-aided sample preparation (FASP). These proteins were categorized into six groups based on their functions, including enzymes, transport proteins, regulatory proteins, cell structure proteins, other proteins, and unknown proteins. Bioinformatics analyses predicted 16 secreted proteins, which were successfully cloned and transcriptionally analyzed across various tissues and developmental stages. Genes encoding putative salivary secretory proteins, including Efmucin1, EfOBP1, EfOBP2, EfOBP3, Efmucin2, low-density lipoprotein receptor-related protein (EfLRP), EFVg1, and EFVg2, exhibited high expressions in salivary gland (SG) tissues and feeding-associated expressions at different developmental stages. These findings shed light on the potential elicitors or effectors mediating the leafhopper feeding and defense responses in tea plants, providing insights into the coevolution of tea plants and leafhoppers. The study's conclusions open avenues for the development of innovative leafhopper control technologies that reduce the reliance on pesticides in the tea industry.

The effect of feeding on different hosts on the egg proteins in Haemaphysalis qinghaiensis tick.

The majority of ixodid ticks display host-specificity to varying extents. Feeding on different hosts affects their development and reproduction. Consequences can be analyzed at the level of the egg, as it is the initial stage of tick development. Tick egg proteins are abundant and diverse, providing nutrients for embryonic development. However, studies on tick egg profiles are scarce. In this study, we aimed to analyze whether feeding Haemaphysalis qinghaiensis ticks on the yaks (Bos grunniens) and domestic sheep (Ovis aries) has an impact on the variety and variability of the egg proteome. Detached engorged females were used to lay eggs, which were then collected, dewaxed, and subjected to protein extraction. The extracted egg proteins were enzymatically digested using Filter-Aided Sample Preparation (FASP), and the unique peptides were separated and detected by Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS). The MS data were searched against the previously constructed whole tick transcriptome library of H. qinghaiensis, and the UniProt database for the identification of tick-derived egg proteins. The analysis revealed 49 and 53 high-confidence proteins identified in eggs collected from B. grunniens (EggBg) and O. aries (EggOa), respectively. Of these, 46 high-confidence proteins were common to both egg types, while three were unique to EggBg and seven to EggOa. All the identified proteins mainly belonged to enzymes, enzyme inhibitors, transporters, and proteins with unknown functions. The differential abundance analysis showed that nine proteins were significantly more present in EggBg, while six were significantly more present in EggOa. Overall, enzymes were the most diverse group, while vitellogenin (Vg) was the most abundant. Blood meal uptake on different hosts has a certain effect on the egg proteome composition and the abundance of some proteins, but it may also lead to compensation of protein roles.

A Comparison of Bottom-Up Proteomic Sample Preparation Methods for the Human Parasite Trichomonas vaginalis.

Bottom-up proteomic approaches depend on the efficient digestion of proteins into peptides for mass spectrometric analysis. Sample preparation strategies, based on magnetic beads, filter-aided systems, or in-solution digests, are commonly used for proteomic analysis. Time-intensive methods like filter-aided sample preparation (FASP) have led to the development of new, more time-efficient filter-based strategies like suspension trappings (S-Traps) or magnetic bead-based strategies like SP3. S-Traps have been reported as an alternative proteomic sample preparation method as they allow for high sodium dodecyl sulfate (SDS) concentrations to be present in the sample. In this study, we compare the efficiency of different protocols for FASP, SP3, and S-Trap-based digestion of proteins after extraction from Trichomonas vaginalis. Overall, we found a high number of protein IDs for all tested methods and a high degree of reproducibility within each method type. However, FASP with a 3 kDa cutoff filter unit outperformed the other methods analyzed, referring to the number of protein IDs. This is the first work providing the direct comparison of four different bottom-up proteomic approaches regarding the most efficient proteomic sample preparation protocol for the human parasite T. vaginalis.

The High Resolution MASS spectrometry in Personalised Medicine: Retinol-Binding Protein 4 as a Candidate Biomarker Predictor of Progression in Bladder Urothelial Carcinoma

Introduction: Bladder cancer (BC) is the seventh most commonly diagnosed cancer in males. A large proportion of T1 cases and some Ta cases are under-staged and the significant risk of residual tumour after initial TURB of TaT1 lesions has been demonstrated. A second TURB is recommended in T1 tumours because it can increase recurrence-free survival (RFS) providing prognostic information. Non-invasive methods differentiating bladder cancer stages would be essential for diagnosis of under staged cases. Previously we demonstrated that a panel of urinary proteins measured by high resolution mass spectrometry could predict bladder cancer stages namely Ta, T1 and T2 cases. Our aim is to increase the accuracy of the biomarker panel for BC stage differentiation using a more patients, also intending to identify proteins that could be a signature to bladder cancer progression. Methods: Forty-eight urine samples were collected from volun- teers of these groups: 20 patients with bladder cancer stage Ta; 19 patients with BC stage T1 and 9 patients with BC stage T2+ (T2, T3 and T4). Urinary proteome was cleaned and digested using the Filter-Aided Sample Preparation methodology and analysed by liquid chromatography-mass spectrometry. For protein identification and label-free quantification we used MaxQuant, the data was further interrogated with the bioinformatics platform Perseus. Results: A biomarker panel was developed which consists of 87 proteins which were down-regulated between three different urothelial bladder carcinoma stages evaluated. Retinol-binding protein 4 (RBP4) consistently increases from Ta to T1, to T2+ (p &lt;0.001) and in high grade tumours (p = 0.006). Conclusion: Our results showed a proteomic biomarker panel capable to differentiate bladder cancer stages. Besides, retinolbinding protein 4 can be a candidate signature of progression.

Optimizing MS-Based Multi-Omics: Comparative Analysis of Protein, Metabolite, and Lipid Extraction Techniques.

Multi-omics integrates diverse types of biological information from genomic, proteomic, and metabolomics experiments to achieve a comprehensive understanding of complex cellular mechanisms. However, this approach is also challenging due to technical issues such as limited sample quantities, the complexity of data pre-processing, and reproducibility concerns. Furthermore, existing studies have primarily focused on technical performance assessment and the presentation of modified protocols through quantitative comparisons of the identified protein counts. Nevertheless, the specific differences in these comparisons have been minimally investigated. Here, findings obtained from various omics approaches were profiled using various extraction methods (methanol extraction, the Folch method, and Matyash methods for metabolites and lipids) and two digestion methods (filter-aided sample preparation (FASP) and suspension traps (S-Trap)) for resuspended proteins. FASP was found to be more effective for the identification of membrane-related proteins, whereas S-Trap excelled in isolating nuclear-related and RNA-processing proteins. Thus, FASP may be suitable for investigating the immune response and bacterial infection pathways, whereas S-Trap may be more effective for studies focused on the mechanisms of neurodegenerative diseases. Moreover, regarding the choice of extraction method, the single-phase method identified organic compounds and compounds related to fatty acids, whereas the two-phase extraction method identified more hydrophilic compounds such as nucleotides. Lipids with strong hydrophobicity, such as ChE and TG, were identified in the two-phase extraction results. These findings highlight that significant differences among small molecules are primarily identified due to the varying polarities of extraction solvents. These results, obtained by considering variables such as human error and batch effects in the sample preparation step, offer comprehensive and detailed results not previously provided by existing studies, thereby aiding in the selection of the most suitable pre-processing approach.

A Fast-Tracking Sample Preparation Protocol for Proteomics of Formalin-Fixed Paraffin-Embedded Tumor Tissues.

Archived tumor specimens are routinely preserved by formalin fixation and paraffin embedding. Despite the conventional wisdom that proteomics might be ineffective due to the cross-linking and pre-analytical variables, these samples have utility for both discovery and targeted proteomics. Building on this capability, proteomics approaches can be used to maximize our understanding of cancer biology and clinical relevance by studying preserved tumor tissues annotated with the patients' medical histories. Proteomics of formalin-fixed paraffin-embedded (FFPE) tissues also integrates with histological evaluation and molecular pathology strategies, so that additional collection of research biopsies or resected tumor aliquots is not needed. The acquisition of data from the same tumor sample also overcomes concerns about biological variation between samples due to intratumoral heterogeneity. However, the protein extraction and proteomics sample preparation from FFPE samples can be onerous, particularly for small (i.e., limited or precious) samples. Therefore, we provide a protocol for a recently introduced kit-based EasyPep method with benchmarking against a modified version of the well-established filter-aided sample preparation strategy using laser-capture microdissected lung adenocarcinoma tissues from a genetically engineered mouse model. This model system allows control over the tumor preparation and pre-analytical variables while also supporting the development of methods for spatial proteomics to examine intratumoral heterogeneity. Data are posted in ProteomeXchange (PXD045879).

Reduced Proliferation and Cell Death of Acute Myeloid Leukemia Cell Lines after Acalabrutinib Treatment

Background Acute myeloid leukemia (AML) still remains a significant challenge, with intensive chemotherapy treatments showing limited efficacy and lack of druggable targets in contrast to other hematologic malignancies. Exosomes, nanomolecules derived from cells reflect the parent cell's state, present opportunities for disease biomarker discovery and targeted therapy. Proteomic analysis of AML-derived exosomes may identify druggable targets despite limited prior research. In this study we investigated the potential of exosomes as a tool for target discovery in AML. Methods We conducted a proteomic analysis of AML-derived exosomes from HL-60, KG-1, and THP-1 cell lines, using fibroblast and mesenchymal stem cells as controls. Exosomes of each cell line were isolated, and digested via filter-aided sample preparation (FASP), followed by isobaric tagging using tandem mass tag (TMT) reagents. The samples were then fractionated using high-pH reversed-phase liquid chromatography and analyzed using Q-Exactive Orbitrap Hybrid Mass Spectrometry. Proteomic data were evaluated using SEQUEST in Proteome Discoverer 2.1 with a false discovery rate set to 1%. Differentially expressed proteins (DEPs) were identified and visualized in an interaction network and was analyzed for the gene ontology biological process (GOBP) using DAVID software. Protein-protein interactions were analyzed using the STRING software. Based on proteomic analysis, we identified the BTK-related pathway as potential druggable target for AML cells. AML cells were incubated with ACT, a BTK inhibitor, in various exposure time and density. Cell viability and proliferation rate was assessed using CCK-8 assay. BTK activity and signaling were assessed via Western blot analysis and immunocytochemistry of AML-derived exosomes. Cell death was evaluated using Annexin-V &amp; PI apoptosis kit and FACS analysis. Statistical analysis was performed to ascertain significance. Results Proteomics of exosomes from three AML cell types revealed distinct protein profiles via LC-MS, with 2450 unique proteins identified (Figure 1A). For KG1, HL60, and THP1, the number of up-regulated DEPs were 438, 434, and 408, and the number of down-regulated DEPs was 126, 118, and 89 (fold change&amp;gt;1.5 or &amp;lt;0.67, P&amp;lt;0.05). Gene ontology analysis identified four main categories of biological processes, including cell migration, T-cell pathways, cell damage, and vesicle formation pathways. Investigation into protein-protein interactions using the STRING software allowed selection of tyrosine kinases SYK and LYN as biomarker candidates. Both kinases regulate BTK, a critical molecule in B-cell antigen receptor signaling. SYK and LYN were found to be enriched in exosomes, promoting phosphorylation of BTK in recipient cells. (Figure 1B) Cell viability experiments with AML cell lines revealed reduced viability upon treatment with ACT. Viability decreased with increased exposure time and density, with the highest effects observed at 1,000nM ACT concentration for 48 hours (Figure 2A). This was accompanied by decreased cell numbers and proliferation rate which was significant in the HL-60 and KG-1 cell lines (Figure 2B). Western blot analysis demonstrated significant decrease of the BTK downstream signaling (P-BTK, PKC and p-ERK) by acalabrutinib treatment (1,000nM) at 48 hours (Figure 2C). Proliferative markers, such as PCNA, and Cyclin D1 were also significantly decreased. LYN and SYK was also decreased in the exosomes of HL-60 and THP-1 cell line. The inhibition of P-BTK and Cyclin D1 by ACT was further confirmed through Immunofluorescence assay. Finally, ACT was shown to promote cell death in AML cell lines, as evidenced by an apoptosis assay, with the most significant increases observed in cells treated exposed to 1,000nM ACT for 72 h (Figure 2D). Conclusion Overall, the findings of this study revealed that acalabrutinib reduced proliferation and induced cell death in AML cells suggesting the potential of BTK inhibitors in AML treatment. This was explained through proteomic analysis of AML-derived exosomes. However, further studies are required to validate the clinical use of exosomes as biomarkers and druggable target.

Disclosing the molecular profile of the human amniotic mesenchymal stromal cell secretome by filter-aided sample preparation proteomic characterization

BackgroundThe secretome of mesenchymal stromal cells isolated from the amniotic membrane (hAMSCs) has been extensively studied for its in vitro immunomodulatory activity as well as for the treatment of several preclinical models of immune-related disorders. The bioactive molecules within the hAMSCs secretome are capable of modulating the immune response and thus contribute to stimulating regenerative processes. At present, only a few studies have attempted to define the composition of the secretome, and several approaches, including multi-omics, are underway in an attempt to precisely define its composition and possibly identify key factors responsible for the therapeutic effect.MethodsIn this study, we characterized the protein composition of the hAMSCs secretome by a filter-aided sample preparation (FASP) digestion and liquid chromatography-high resolution mass spectrometry (LC–MS) approach. Data were processed for gene ontology classification and functional protein interaction analysis by bioinformatics tools.ResultsProteomic analysis of the hAMSCs secretome resulted in the identification of 1521 total proteins, including 662 unique elements. A number of 157 elements, corresponding to 23.7%, were found as repeatedly characterizing the hAMSCs secretome, and those that resulted as significantly over-represented were involved in immunomodulation, hemostasis, development and remodeling of the extracellular matrix molecular pathways.ConclusionsOverall, our characterization enriches the landscape of hAMSCs with new information that could enable a better understanding of the mechanisms of action underlying the therapeutic efficacy of the hAMSCs secretome while also providing a basis for its therapeutic translation.

An ionic liquid-assisted sample preparation method for sensitive integral-membrane proteome analysis

Many ion channels and receptor proteins are potential targets for new drugs. However, standard methods for profiling these integral membrane proteins (IMPs) have not been fully established, especially when applied to rare and quantity-limited biological samples. We previously demonstrated that a mixture containing 1-butyl-3-methylimidazolium cyanate, an ionic liquid (IL), and NaOH (termed i-soln) is an excellent solubilizer for insoluble aggregates. In this study, we present a combined i-soln-assisted proteomic sample preparation platform (termed pTRUST), which is compatible with starting materials in the sub-microgram range, using our previously reported i-soln-based sample preparation strategy (iBOPs) and an in-StageTip technique. This novel and straightforward approach allows for the rapid solubilization and processing of a variety of IMPs from human samples to support highly sensitive mass spectrometry analysis. We also demonstrated that the performance of this technology surpasses that of conventional methods such as filter-aided sample preparation methods, FASP and i-FASP. The convenience and availability of pTRUST technology using the IL system have great potential for proteomic identification and characterization of novel drug targets and disease biology in research and clinical settings.

OR33-03 Circadian Analysis Of Nuclear Receptor Protein Expression In Mouse Liver Reveals Peak COUP-TFII, EAR2, LXRa, LXRb And TR4 Expression At The End Of The Mouse Active Cycle ZT0

Abstract Disclosure: M.F. Saikali: None. J. Li: None. C.L. Cummins: None. Nuclear Receptors (NRs) are a family of ligand-mediated transcription factors that control the expression of genes involved in a wide range of physiological processes. The ability to study NRs quantitatively at the protein level has been hindered by the poor quality of available antibodies, as well as the lack of available high throughput methods to study more than a few receptors at a time. To address this need, we have developed a mass spectrometry based targeted proteomic assay to quantify the absolute amounts of NR protein in mouse livers over a circadian period. C57Bl6 mice were sacrificed at 12 weeks every 4 hours from zeitgeber time ZT0 to ZT20. Their livers were processed using a transcription factor extraction protocol1. For each sample, 100 µg nuclear protein was processed using the NR assay described previously2. Briefly, the lysate is digested following the Multi-Enzyme Digestion Filter Aided Sample Preparation protocol. The sample is then spiked with 50 fmol of our internal standard mix. This mix is composed of 66 carefully selected peptides that account for 49 NRs, 11 histone peptides, and 6 circadian clock proteins. The resulting digest that contains the internal standards is desalted on C18 tips, followed by separation on an EASY-Spray C18 column (75 um x 50 cm, 3Å), and analyzed on a Thermo QExactive HF mass spectrometer in parallel reaction monitoring mode. We used this assay to quantify the changes in NR protein expression in mouse livers for 9 receptors; RORα, CAR, COUP-TFII, EAR2, GR, LXRα, LXRβ, PPARα, and TR4. RORα showed peak nuclear expression at ZT0, with a peak-to-trough change of 2.2-fold. COUP-TFII, EAR2, LXRα, LXRβ, and TR4 also showed peak expression at ZT0 (the end of the active phase) with changes of 3.3, 2.4, 1.7, 2.0, and 1.6-fold respectively. GR showed peak nuclear expression at ZT12 with a 1.8-fold change. CAR showed 2.3-fold peak expression at ZT16. COUP-TFII, EAR2, and TR4 are considered orphan receptors with no known endogenous ligands. These data will be used to uncover endogenous tissue-specific ligands by analyzing extracts isolated at the zeitgeber time that the receptor is maximally expressed. Sources of Research Support: This project has been supported by the J. P. Bickell Foundation and the Canadian Institutes of Health Research New Frontiers Research Fund and International Development Research Fund. 1.Gillespie, M. A. et al. Absolute Quantification of Transcription Factors Reveals Principles of Gene Regulation in Erythropoiesis. Mol Cell (2020) doi:10.1016/j.molcel.2020.03.031.2.Saikali, M. F. &amp; Cummins, C. L. Quantifying the Protein Levels of All Nuclear Hormone Receptors by Mass Spectrometry. J Endocr Soc5, (2021). Presentation: Sunday, June 18, 2023

Development of a sample preparation method for micro-proteomics analysis of the formaldehyde-fixed paraffin-embedded liver tissue samples

Liver micro-proteomics based on the routinely used formaldehyde-fixed paraffin-embedded (FFPE) samples is valuable for innovative research, but the technical approach for sample preparation is often challenging. In this study, we aimed to develop a method for sample preparation for micro-proteomics on using the FFPE liver samples. We collected 2000 individual cells per batch from FFPE liver slices with laser capture microdissection and used them as test samples. We used the microscale fresh-frozen liver samples or HepG2 cells as control samples. For the FFPE samples, we first established a procedure for protein extraction. 2 h incubation at 95 °C in alkaline amine buffer supplemented with 4% sodium dodecyl sulfate allows improved production, efficiency, and quality of protein extraction. Then, we developed a dedicated protocol HDMSP for the micro-concentrated (<0.05 μg/μL) protein preparation for mass spectrometry (MS) based analysis, in which 2 μg/μL carboxyl magnetic beads and 70% acetonitrile are used to induce protein precipitation. For the 0.01 μg/μL protein control samples, protein recovery rate (PRR) by HDMSP is 72.1%, while the PRR is 5.9% if using a standard method solid phase-enhanced sample preparation. For the FFPE samples, the HDMSP PRR is 88.8%, and the subsequent MS analysis demonstrates increased depth, robustness, and quantitation accuracy for HDMSP relative to the control of in-gel digestion. Moreover, the physicochemical properties and subcellular location of the FFPE liver micro-proteome are comparable to those of the fresh-frozen control samples processed with filter-aided sample preparation (FASP). HDMSP is also comparable to FASP in terms of reproducibility and physicochemical properties in liver subcellular proteomes, and meanwhile reduces the sample preparation time by 15.9% and the experimental cost by 30.8%. Overall, the new method is simple and highly effective for preparing the microscale FFPE liver protein samples for MS analysis. This study provides a useful solution for FFPE liver micro-proteomics.

A Rapid and Efficient Method for the Extraction of Histone Proteins.

Current protocols used to extract and purify histones are notoriously tedious, especially when using yeast cells. Here, we describe the use of a simple filter-aided sample preparation approach enabling histone extraction from yeast and mammalian cells using acidified ethanol, which not only improves extraction but also inactivates histone-modifying enzymes. We show that our improved method prevents N-terminal clipping of H3, an artifact frequently observed in yeast cells using standard histone extraction protocols. Our method is scalable and provides efficient recovery of histones when extracts are prepared from as few as two million yeast cells. We further demonstrate the application of this approach for the analysis of histone modifications in fungal clinical isolates available in a limited quantity. Compared with standard protocols, our method enables the study of histones and their modifications in a faster, simpler, and more robust manner.

Integrated Mass Spectrometry‐based Pipeline for Characterization of Tau in Human Alzheimer’s Brain: Development and Comparison of Sample Preparation Methods

AbstractBackgroundTau protein aggregation into neurofibrillary tangles in the brain is a pathological hallmark of Alzheimer’s disease (AD) and related tauopathies [1]. Though the mechanism of tau destabilization is not fully understood, tau protein exhibits several allele‐specific isoforms and post‐translational modifications (PTMs) specific to tau aggregates [2]. To explore in‐depth tau species in brain tissues of AD patients, we developed novel pipelines based on different tau fractions retrieved by buffer solubility, Filter Aided Sample Preparation (FASP) or immunoprecipitation, before high‐resolution mass spectrometry (HRMS) analysis.MethodAs a proof of concept, we used frozen brain samples from two AD patients (0.6‐1.2 g per replicate, middle frontal gyrus). The samples were divided and homogenized, ultracentrifuged at 180.000xg for 30min and solubilized in different buffers successively (2 replicates): (1) SarkMethodlow‐salt buffer, 10% Triton X‐100, sarkosyl buffer, and urea buffer; (2) RIPAMethodhigh‐salt buffer (twice), RIPA buffer and urea buffer; and, (3) GnHMethodguanidine hydrochloride and urea buffer. Then, we performed an antibody‐free approach by FASP or immunoprecipitation with a panel of commercial tau antibodies and analyzed on reversed‐phase capillary liquid chromatography, coupled with a qExactive HRMS.ResultHRMS demonstrated that the RIPA method performed better than the other methods for Tau analysis: coverage of 63% (full‐length, 2N4R) and 342 tau peptide‐spectrum matches (PSMs) (Sark: 49% and 183 PSMs; GnH: 41% and 169 PSMs). PTM‐modified tau species (phosphorylated, methylated, and acetylated) were primarily seen using the RIPA method (59 PSMs; Sark: 19 PSMs, GnH: 21 PSMs). Immunoprecipitation improved tau coverage and PTM detection compared to FASP.ConclusionThese findings highlight the best performance of the RIPA method followed by immunoprecipitation for the study of AD brain tau allele‐specific isoforms and PTMs using HRMS. Better characterization of Tau molecular profile in the brain of AD patients opens the gate for a better understanding of AD pathogenesis and represents an opportunity in the biomarker field.[1] Mroczko, B. et al. Int J Mol Sci 2019, 20, E4661, https://doi.org/10.3390/ijms20194661.[2] Boyarko, B.; Hook, V. Front. Neurosci. 2021, 15, 702788, https://doi.org/10.3389/fnins.2021.702788.