Enrichment Of Membrane Proteins Research Articles

Clinical Implications and Molecular Features of Extracellular Matrix Networks in Soft Tissue Sarcomas.

The landscape of extracellular matrix (ECM) alterations in soft tissue sarcomas (STS) remains poorly characterized. We aimed to investigate the tumor ECM and adhesion signaling networks present in STS and their clinical implications. Proteomic and clinical data from 321 patients across 11 histological subtypes were analyzed to define ECM and integrin adhesion networks. Subgroup analysis was performed in leiomyosarcomas (LMS), dedifferentiated liposarcomas (DDLPS), and undifferentiated pleomorphic sarcomas (UPS). This analysis defined subtype-specific ECM profiles including enrichment of basement membrane proteins in LMS and ECM proteases in UPS. Across the cohort, we identified three distinct coregulated ECM networks which are associated with tumor malignancy grade and histological subtype. Comparative analysis of LMS cell line and patient proteomic data identified the lymphocyte cytosolic protein 1 cytoskeletal protein as a prognostic factor in LMS. Characterization of ECM network events in DDLPS revealed three subtypes with distinct oncogenic signaling pathways and survival outcomes. Evaluation of the DDLPS subtype with the poorest prognosis nominates ECM remodeling proteins as candidate antistromal therapeutic targets. Finally, we define a proteoglycan signature that is an independent prognostic factor for overall survival in DDLPS and UPS. STS comprise heterogeneous ECM signaling networks and matrix-specific features that have utility for risk stratification and therapy selection, which could in future guide precision medicine in these rare cancers.

REMEMProt: a resource of membrane-enriched proteome profiles, their disease associations, and biomarker status.

The differential expression of plasma membrane proteins is integrally analyzed for their diagnosis, prognosis, and therapeutic applications in diverse clinical manifestations. Necessarily, distinct membrane protein enrichment methods and mass spectrometry platforms are employed for their global and relative quantitation. First of its kind to explore, we compiled membrane-associated proteomes in human and mouse systems into a database named, Resource of Experimental Membrane-Enriched Mass spectrometry-derived Proteome (REMEMProt). It currently hosts 14,626 proteins (9,507 proteins in Homo sapiens; 5,119 proteins in Mus musculus) with information on their membrane-protein enrichment methods, experimental/physiological context of detection in cells or tissues, transmembrane domain analysis, and their current attribution as biomarkers. Based on these annotations and the transmembrane domain analysis in proteins or their binary/complex protein-protein interactors, REMEMProt facilitates the assessment of the plasma membrane localization potential of proteins through batch query. A cross-study enrichment analysis platform is enabled in REMEMProt for comparative analysis of proteomes using novel/modified membrane enrichment methods and evaluation of methods for targeted enrichment of membrane proteins. REMEMProt data are made freely accessible to explore and download at https://rememprot.ciods.in/.

DnaK duplication and specialization in bacteria correlates with increased proteome complexity.

The chaperone 70 kDa heat shock protein (Hsp70) is important for cells from bacteria to humans to maintain proteostasis, and all eukaryotes and several prokaryotes encode Hsp70 paralogs. Although the mechanisms of Hsp70 function have been clearly illuminated, the function and evolution of Hsp70 paralogs is not well studied. DnaK is a highly conserved bacterial Hsp70 family. Here, we show that dnaK is present in 98.9% of bacterial genomes, and 6.4% of them possess two or more DnaK paralogs. We found that the duplication of dnaK is positively correlated with an increase in proteomic complexity (proteome size, number of domains). We identified the interactomes of the two DnaK paralogs of Myxococcus xanthus DK1622 (MxDnaKs), which revealed that they are mostly nonoverlapping, although both prefer α and β domain proteins. Consistent with the entire M. xanthus proteome, MxDnaK substrates have both significantly more multi-domain proteins and a higher isoelectric point than that of Escherichia coli, which encodes a single DnaK homolog. MxDnaK1 is transcriptionally upregulated in response to heat shock and prefers to bind cytosolic proteins, while MxDnaK2 is downregulated by heat shock and is more associated with membrane proteins. Using domain swapping, we show that the nucleotide-binding domain and the substrate-binding β domain are responsible for the significant differences in DnaK interactomes, and the nucleotide binding domain also determines the dimerization of MxDnaK2, but not MxDnaK1. Our work suggests that bacterial DnaK has been duplicated in order to deal with a more complex proteome, and that this allows evolution of distinct domains to deal with different subsets of target proteins.IMPORTANCEAll eukaryotic and ~40% of prokaryotic species encode multiple 70 kDa heat shock protein (Hsp70) homologs with similar but diversified functions. Here, we show that duplication of canonical Hsp70 (DnaK in prokaryotes) correlates with increasing proteomic complexity and evolution of particular regions of the protein. Using the Myxococcus xanthus DnaK duplicates as a case, we found that their substrate spectrums are mostly nonoverlapping, and are both consistent to that of Escherichia coli DnaK in structural and molecular characteristics, but show differential enrichment of membrane proteins. Domain/region swapping demonstrated that the nucleotide-binding domain and the β substrate-binding domain (SBDβ), but not the SBDα or disordered C-terminal tail region, are responsible for this functional divergence. This work provides the first direct evidence for regional evolution of DnaK paralogs.

Decoding of the surfaceome and endocytome in primary glioblastoma cells identifies potential target antigens in the hypoxic tumor niche

Immunotherapies with antibody–drug-conjugates (ADC) and CAR-T cells, targeted at tumor surface antigens (surfaceome), currently revolutionize clinical oncology. However, target identification warrants a better understanding of the surfaceome and how it is modulated by the tumor microenvironment. Here, we decode the surfaceome and endocytome and its remodeling by hypoxic stress in glioblastoma (GBM), the most common and aggressive brain tumor in adults. We employed a comprehensive approach for global and dynamic profiling of the surfaceome and endocytosed (endocytome) proteins and their regulation by hypoxia in patient-derived GBM cultures. We found a heterogeneous surface-endocytome profile and a divergent response to hypoxia across GBM cultures. We provide a quantitative ranking of more than 600 surface resident and endocytosed proteins, and their regulation by hypoxia, serving as a resource to the cancer research community. As proof-of-concept, the established target antigen CD44 was identified as a commonly and abundantly expressed surface protein with high endocytic activity. Among hypoxia induced proteins, we reveal CXADR, CD47, CD81, BSG, and FXYD6 as potential targets of the stressed GBM niche. We could validate these findings by immunofluorescence analyses in patient tumors and by increased expression in the hypoxic core of GBM spheroids. Selected candidates were finally confronted by treatment studies, showing their high capacity for internalization and ADC delivery. Importantly, we highlight the limited correlation between transcriptomics and proteomics, emphasizing the critical role of membrane protein enrichment strategies and quantitative mass spectrometry. Our findings provide a comprehensive understanding of the surface-endocytome and its remodeling by hypoxia in GBM as a resource for exploration of targets for immunotherapeutic approaches in GBM.

E and M SARS-CoV-2 membrane protein expression and enrichment with plant lipid droplets.

Plants are gaining traction as a cost-effective and scalable platform for producing recombinant proteins. However, expressing integral membrane proteins in plants is challenging due to their hydrophobic nature. In our study, we used transient and stable expression systems in Nicotiana benthamiana and Camelina sativa respectively to express SARS-CoV-2 E and M integral proteins, and target them to lipid droplets (LDs). LDs offer an ideal environment for folding hydrophobic proteins and aid in their purification through flotation. We tested various protein fusions with different linkers and tags and used three dimensional structure predictions to assess their effects. E and M mostly localized in the ER in N. benthamiana leaves but E could be targeted to LDs in oil accumulating tobacco when fused with oleosin, a LD integral protein. In Camelina sativa seeds, E and M were however found associated with purified LDs. By enhancing the accumulation of E and M within LDs through oleosin, we enriched these proteins in the purified floating fraction. This strategy provides an alternative approach for efficiently producing and purifying hydrophobic pharmaceuticals and vaccines using plant systems.

Targeted Proteomics Reveals Quantitative Differences in Low-Abundance Glycosyltransferases of Patients with Congenital Disorders of Glycosylation.

Protein glycosylation is an essential post-translational modification in all domains of life. Its impairment in humans can result in severe diseases named congenital disorders of glycosylation (CDGs). Most of the glycosyltransferases (GTs) responsible for proper glycosylation are polytopic membrane proteins that represent challenging targets in proteomics. We established a multiple reaction monitoring (MRM) assay to comprehensively quantify GTs involved in the processes of N-glycosylation and O- and C-mannosylation in the endoplasmic reticulum. High robustness was achieved by using an enriched membrane protein fraction of isotopically labeled HEK 293T cells as an internal protein standard. The analysis of primary skin fibroblasts from eight CDG type I patients with impaired ALG1, ALG2, and ALG11 genes, respectively, revealed a substantial reduction in the corresponding protein levels. The abundance of the other GTs, however, remained unchanged at the transcript and protein levels, indicating that there is no fail-safe mechanism for the early steps of glycosylation in the endoplasmic reticulum. The established MRM assay was shared with the scientific community via the commonly used open source Skyline software environment, including Skyline Batch for automated data analysis. We demonstrate that another research group could easily reproduce all analysis steps, even while using different LC-MS hardware.

Abstract B052: Defining the lysosome proteome during tumor evolution

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a 5-year survival rate of 12%. PDAC tumors are highly reliant on nutrient scavenging pathways such as autophagy, and the lysosome – a degradative organelle which plays an essential role in the digestion and recycling of diverse cellular material. Lysosome and autophagy processes are regulated by the MiT/TFE family of transcription factors. In PDAC, MiT/TFE are uncoupled from normal regulatory mechanisms and have been shown to be constitutively nuclear, and therefore active. The function of lysosomes and the status of MiT/TFE over the course of tumor evolution and metastasis remains unknown. To fully understand the functions of the lysosome in cancer, our lab uses biochemical approaches to isolate intact lysosomes (termed LysoIP) from cultured PDAC cells, followed by mass spectrometry-based proteomics. Using this strategy, we have uncovered unique features and functions of PDAC lysosomes that promote cellular adaptation to stress in support of tumor growth. A limitation of lysosome isolation from 2D cultured cells is that it does not recapitulate lysosomal features associated with tumors growing in vivo. To address this limitation, we generated a genetically engineered mouse model of PDAC incorporating conditional expression of our ‘LysoTag’ (TMEM192-mRFP-3xHA) that enables capture and profiling of lysosomes at different stages of tumor evolution, including early stage, late stage, and metastatic disease. I have now established purification protocols for isolation of intact lysosomes with high efficiency and purity from both normal pancreas and diseased tissue. My preliminary analysis of pancreatic tumors, liver and lung metastases shows enrichment of bonafide resident lysosomal membrane proteins (Lamp1, Cln3, Npc1), and lumenal proteins (Ctsb, Lipa, Neu1), confirming successful isolation of intact lysosomes from these complex tissues. Further analysis highlights pathways and proteins that are unique to lysosomes of each tissue. For instance, lysosomes from the primary tumor show enrichment for pathways involved in ‘antigen processing and presentation of peptide antigen’ and ‘cellular response to oxidative stress’, while the liver and lung metastasis lysosomes show enrichment for ‘cholesterol metabolism’ and ‘mitochondrial gene expression’, respectively. Our analysis of lysosomes from the primary tumor, lung and liver metastasis highlights unique differences in the lysosome proteome of tumor cells growing in different tissues. Functional validation of our proteomics datasets will be needed to uncover novel roles for lysosomes in helping tumor cells adapt and grow in distinct tumor microenvironments. My ongoing studies will provide the first comprehensive atlas of lysosomal cargo and resident proteins that co-evolve with increasing tumor stage and help to inform novel therapeutic targets for treatment of PDAC. Citation Format: Grace A. Hernandez, Thuy Nguyen, Matthew Luy, Alexander Li, Longhui Qiu, Joseph D. Mancias, Rushika M. Perera. Defining the lysosome proteome during tumor evolution [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B052.

TGFBR3L is associated with gonadotropin production in non-functioning gonadotroph pituitary neuroendocrine tumours

PurposeTransforming growth factor-beta receptor 3-like (TGFBR3L) is a pituitary enriched membrane protein selectively detected in gonadotroph cells. TGFBR3L is named after transforming growth factor-beta receptor 3 (TGFBR3), an inhibin A co-receptor in mice, due to sequence identity to the C-terminal region. We aimed to characterize TGFBR3L detection in a well-characterized, prospectively collected cohort of non-functioning pituitary neuroendocrine tumours (NF-PitNETs) and correlate it to clinical data.Methods144 patients operated for clinically NF-PitNETs were included. Clinical, radiological and biochemical data were recorded. Immunohistochemical (IHC) staining for FSHβ and LHβ was scored using the immunoreactive score (IRS), TGFBR3L and TGFBR3 were scored by the percentage of positive stained cells.ResultsTGFBR3L staining was selectively present in 52% of gonadotroph tumours. TGFBR3L was associated to IRS of LHβ (median 2 [IQR 0–3] in TGFBR3L negative and median 6 [IQR 3–9] in TGFBR3L positive tumours, p < 0.001), but not to the IRS of FSHβ (p = 0.32). The presence of TGFBR3L was negatively associated with plasma gonadotropin concentrations in males (P-FSH median 5.5 IU/L [IQR 2.9–9.6] and median 3.0 [IQR 1.8–5.6] in TGFBR3L negative and positive tumours respectively, p = 0.008) and P-LH (median 2.8 IU/L [IQR 1.9–3.7] and median 1.8 [IQR 1.1-3.0] in TGFBR3L negative and positive tumours respectively, p = 0.03). TGFBR3 stained positive in 22% (n = 25) of gonadotroph tumours with no correlation to TGFBR3L.ConclusionTGFBR3L was selectively detected in half (52%) of gonadotroph NF-PitNETs. The association to LHβ staining and plasma gonadotropins suggests that TGFBR3L may be involved in hormone production in gonadotroph NF-PitNETs.

Enrichment of Membrane Proteins for Downstream Analysis Using Styrene Maleic Acid Lipid Particles (SMALPs) Extraction.

Integral membrane proteins are an important class of cellular proteins. These take part in key cellular processes such as signaling transducing receptors to transporters, many operating within the plasma membrane. More than half of the FDA-approved protein-targeting drugs operate via interaction with proteins that contain at least one membrane-spanning region, yet the characterization and study of their native interactions with therapeutic agents remains a significant challenge. This challenge is due in part to such proteins often being present in small quantities within a cell. Effective solubilization of membrane proteins is also problematic, with the detergents typically employed in solubilizing membranes leading to a loss of functional activity and key interacting partners. In recent years, alternative methods to extract membrane proteins within their native lipid environment have been investigated, with the aim of producing functional nanodiscs, maintaining protein-protein and protein-lipid interactions. A promising approach involves extracting membrane proteins in the form of styrene maleic acid lipid particles (SMALPs) that allow the retention of their native conformation. This extraction method offers many advantages for further protein analysis and allows the study of the protein interactions with other molecules, such as drugs. Here, we describe a protocol for efficient SMALP extraction of functionally active membrane protein complexes within nanodiscs. We showcase the method on the isolation of a low copy number plasma membrane receptor complex, the nicotinic acetylcholine receptor (nAChR), from adult Drosophila melanogaster heads. We demonstrate that these nanodiscs can be used to study native receptor-ligand interactions. This protocol can be applied across many biological scenarios to extract the native conformations of low copy number integral membrane proteins.

Huc-MSC-derived exosomes modified with the targeting peptide of aHSCs for liver fibrosis therapy

BackgroundEffective therapeutics to stop or reverse liver fibrosis have not emerged, because these potential agents cannot specifically target activated hepatic stellate cells (aHSCs) or are frequently toxic to parenchymal cells. Human umbilical cord mesenchymal stem cell (Huc-MSC)-derived exosomes show promise in nanomedicine for the treatment of liver fibrosis. However, systemic injection showed that unmodified exosomes were mainly taken up by the mononuclear phagocyte system. The discovery of ligands that selectively bind to a specific target plays a crucial role in clinically relevant diagnostics and therapeutics. Herein, we aimed to identify the targeting peptide of aHSCs by screening a phage-displayed peptide library, and modify Huc-MSC-derived exosomes with the targeting peptide.ResultsIn this study, we screened a phage-displayed peptide library by biopanning for peptides preferentially bound to HSC-T6 cells. The identified peptide, HSTP1, also exhibited better targeting ability to aHSCs in pathological sections of fibrotic liver tissues. Then, HSTP1 was fused with exosomal enriched membrane protein (Lamp2b) and was displayed on the surface of exosomes through genetic engineering technology. The engineered exosomes (HSTP1-Exos) could be more efficiently internalized by HSC-T6 cells and outperformed both unmodified exosomes (Blank-Exos) and Lamp2b protein overexpressed exosomes (Lamp2b + Exos) in enhancing the ability of exosomes to promote HSC-T6 reversion to a quiescent phenotype. In vivo results showed HSTP1-Exos could specifically target to the aHSC region after intravenous administration, as demonstrated by coimmunofluorescence with the typical aHSCs marker α-SMA, and enhance the therapeutic effect on liver fibrosis.ConclusionThese results suggest that HSTP1 is a reliable targeting peptide that can specifically bind to aHSCs and that HSTP1-modified exosomes realize the precise treatment for aHSCs in complex liver tissue. We provide a novel strategy for clinical liver fibrosis therapy.Graphical

Proteomic Analysis of HCC-1954 and MCF-7 Cell Lines Highlights Crosstalk between αv and β1 Integrins, E-Cadherin and HER-2.

Overexpression of human epidermal growth factor receptor-2 (HER-2) occurs in 20% of all breast cancer subtypes, especially those that present the worst prognostic outcome through a very invasive and aggressive tumour. HCC-1954 (HER-2+) is a highly invasive, metastatic cell line, whereas MCF-7 is mildly aggressive and non-invasive. We investigated membrane proteins from both cell lines that could have a pivotal biological significance in metastasis. Membrane protein enrichment for HCC-1954 and MCF-7 proteomic analysis was performed. The samples were analysed and quantified by mass spectrometry. High abundance membrane proteins were confirmed by Western blot, immunofluorescence, and flow cytometry. Protein interaction prediction and correlations with the Cancer Genome Atlas (TCGA) patient data were conducted by bioinformatic analysis. In addition, β1 integrin expression was analysed by Western blot in cells upon trastuzumab treatment. The comparison between HCC-1954 and MCF-7 membrane-enriched proteins revealed that proteins involved in cytoskeleton organisation, such as HER-2, αv and β1 integrins, E-cadherin, and CD166 were more abundant in HCC-1954. β1 integrin membrane expression was higher in the HCC-1954 cell line resistant after trastuzumab treatment. TCGA data analysis showed a trend toward a positive correlation between HER-2 and β1 integrin in HER-2+ breast cancer patients. Differences in protein profile and abundance reflected distinctive capabilities for aggressiveness and invasiveness between HCC-1954 and MCF-7 cell line phenotypes. The higher membrane β1 integrin expression after trastuzumab treatment in the HCC-1954 cell line emphasised the need for investigating the contribution of β1 integrin modulation and its effect on the mechanism of trastuzumab resistance.

TMT-based quantitative membrane proteomics identified PRRs potentially involved in the perception of MSP1 in rice leaves

Pathogen-associated molecular patterns (PAMPs) play a key role in triggering PAMPs triggered immunity (PTI) in plants. In the case of the rice-Magnaporthe oryzae pathosystem, fewer PAMPs and their pattern recognition receptors (PRRs) have been characterized. Recently, a M. oryzae snodprot1 homolog protein (MSP1) has been identified that functions as PAMP and triggering the PTI responses in rice. However, the molecular mechanism underlying MSP1-induced PTI is currently elusive. Therefore, we generated MSP1 overexpressed transgenic lines of rice, and a tandem mass tag (TMT)-based quantitative membrane proteomic analysis was employed to decipher the potential MSP1-induced signaling in rice using total cytosolic as well as membrane protein fractions. This approach led to the identification of 8033 proteins of which 1826 were differentially modulated in response to overexpression of MSP1 and/or exogenous jasmonic acid treatment. Of these, 20 plasma membrane-localized receptor-like kinases (RLKs) showed increased abundance in MSP1 overexpression lines. Moreover, activation of proteins related to the protein degradation and modification, calcium signaling, redox, and MAPK signaling was observed in transgenic lines expressing MSP1 in the apoplast. Taken together, our results identified potential PRR candidates involved in MSP1 recognition and suggested the overview mechanism of the MSP1-induced PTI signaling in rice leaves. SignificanceIn plants, recognition of pathogen pathogen-derived molecules, such as PAMPs, by plant plant-derived PRRs has an essential role for in the activation of PTI against pathogen invasion. Typically, PAMPs are recognized by plasma membrane (PM) localized PRRs, however, identifying the PM-localized PRR proteins is challenging due to their low abundance. In this study, we performed an integrated membrane protein enrichment by microsomal membrane extraction (MME) method and subsequent TMT-labeling-based quantitative proteomic analysis using MSP1 overexpressed rice. Based on these results, we successfully identified various intracellular and membrane membrane-localized proteins that participated in the MSP1-induced immune response and characterized the potential PM-localized PRR candidates in rice.

Optimization of a detergent-based protocol for membrane proteins purification from mammalian cells

Membrane proteins constitute around 20–30 % of the proteins encoded by mammalian genes, are involved in many cell functions, and represent the majority of drug targets. However, the isolation of membrane proteins is challenging because of their partial hydrophobicity, requiring detergents to extract them from cell membranes and stabilize them in solution. Many commercial kits use this principle, but they are expensive, and their chemical composition is not known. In this work, we propose a fast, detergent-based protocol for the purification of membrane proteins from murine and human cells. This protocol is based on three steps: cell washing to remove cell culture medium proteins, cells permeabilization using digitonin to remove the intracellular components, and cell membranes disruption using Triton X-100 to solubilize membrane proteins and keep them in solution. We measured the total protein yield using our protocol with two different detergent concentrations and compared it to a commercial kit. We further assessed membrane protein enrichment by comparing markers for specific cellular components using SDS-PAGE/western blot and identifying specific proteins by qualitative mass spectrometry. Our protocol led to a final protein yield analogous to the commercial kit and similar membrane protein purity, while resulting significantly cheaper compared to the commercial kit. Furthermore, this process can be applied to a different number and types of cells, resulting scalable, versatile, and robust. The possibility to perform downstream mass spectrometry analysis is of particular importance since it enables the use of “omics” techniques for protein discovery and characterization. Our approach could be used as a starting point for the isolation of membrane proteins for pharmacological and biochemical studies, or for the discovery of new druggable or prognostic markers.

Abstract 1233: A novel 14-gene signature to predict response to neoadjuvant chemotherapy and endocrine treatment in breast cancer patients

Abstract mRNA transcriptomic markers have become a key tool in the classification of breast cancer and deciding treatment regimens for patients. However, limited studies have evaluated markers that might be predictive of response to neoadjuvant treatment (chemotherapy and endocrine treatment). Defining such key markers that are associated with treatment response could offer new insights on the relative molecular mechanisms and provide more tailored treatment regimens to target pathways that might be over-represented. We aimed to identify mRNA expression markers associated with breast cancer patients’ response to neoadjuvant treatment from a pool of studies in which tumor samples were collected at pre-treatment and on-treatment timepoints. We collated 1194 pre- and on-treatment samples (721 unique patients) from 9 publicly available gene expression datasets that met our inclusion criteria. The standardized gene expression values from each study were merged in a global matrix of 1551 genes and 1194 samples. Differential Gene Expression Analysis adjusted for timepoint, pam50 subtype (as predicted by the genefu package in R), treatment and batch effects was conducted. 14 significantly (FDR = 0.05) differentially expressed were identified (CCNA2, RFC3, VRK1, PSMB2, ALDH7A1, RRM2, ADRM1, TSPAN4, ZNF473, RNH1, HDAC1, CDK1, SMC1A and TOR1AIP1) when responders and non-responders were compared, some of which are known drug targets (e.g. PSMB2/carfilzomib). Using the set of identified genes we performed Monte-Carlo consensus clustering on the full set (optimal number of clusters: 6). Clusters were significantly associated with response (p = 2·10-8), timepoint (p &amp;lt; 10-15) and pam50 subtype (p &amp;lt; 10-15), but not treatment (p = 0.35). We then split our data into a training, a validation and a test set (776, 299, 119 samples respectively) and used the 14 genes (alone or in combination with metadata) as predictors to fit three types of machine learning models (lasso-regularized Logistic Regression, Decision Trees and Support Vector Machines). Support Vector Machines demonstrated the best classification performance on the validation set (75% classification accuracy) and achieved accuracy of 80% on the test set. Pathway analysis based on the identified genes revealed enriched nuclear membrane organization, protein deubiquitination, DNA replication and histone modification pathways. Prediction of response to treatment at baseline or mid-treatment can aid in patient stratification in the neoadjuvant setting and separate patients who would benefit from treatment the most and could undergo a less extensive surgical operation from patients who are unlikely to respond and should be scheduled for surgery sooner. This kind of early intervention has the potential to lead to improved patient outcomes and reduced side effects from unnecessary treatment administration. Citation Format: Aristeidis Sionakidis, Jonine D. Figueroa, Timothy I. Cannings. A novel 14-gene signature to predict response to neoadjuvant chemotherapy and endocrine treatment in breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1233.

Selective Enrichment of Cell Surface Proteins Using ProMTag to Detect O‐glycosylation‐Dependent Changes

O‐linked glycosylation, which is regulated by the enzyme GalNAc transferase, plays a vital role in cellular function by regulating membrane protein composition and sorting. Changes in O‐linked glycosylation are very common in diseases such as cancers, diabetes, and Alzheimer’s. It is important to study the effects of changes in O‐link glycosylation on cell membrane composition and function to understand how these changes affect the onset, phenotype, and progression of these diseases. However, membrane proteins are notoriously challenging to analyze at the proteomic level due to their hydrophobicity and the inability to separate the membrane proteins from other proteins in the cell.Here we introduce a new sample preparation workflow that enriches membrane proteins for mass spectrometry analysis to understand changes in protein composition of the plasma membrane as the result of changes in O‐linked glycosylation. To do this, we used the bifunctional tag ProMTag to label and capture plasma membrane proteins via their extracellular domains in cells treated with GalNAc Transferase inhibitors. One end of the ProMTag forms a reversible, covalent bond with protein the other end of the ProMTag is capable of forming an irreversible, covalent bond with a solid bead support via the click chemistry pair of methyltetrazine and trans‐Cyclooctene (TCO). The extracellular domains of proteins from intact wild type HEK cells and HEK cells treated with GalNAc Transferase inhibitors were ProMTagged, captured, cleaned up, and eluted for comparison by mass spectrometry (MS) analysis. MS confirmed enrichment of plasma membrane proteins after sample preparation and protein changes in the plasma membrane as a result of GalNAc Transferase inhibition were characterized. This work establishes a method for enrichment of plasma membrane proteins and reveals how changes in O‐linked glycosylation via GalNAc Transferase inhibition leads to changes in plasma membrane protein composition due to altered membrane protein stability and sorting.

Utilization of dairy beta stream to produce phospholipids products by salt precipitation and solvent fractionation

AbstractDairy phospholipids (PLs) have unique functional and nutraceutical properties over PLs obtained from other sources that typically lack phosphatidylserine (PS) and sphingomyelin (SM). In this study, an underutilized dairy by‐product, beta stream, was used for the production of enriched dairy PLs products and membrane proteins by solvent extractions. After calcium salt precipitation of the membrane components, various green (i.e., safer or bioderived) solvent systems were evaluated for their capabilities for PLs extraction from the PLs‐protein‐salt precipitate in comparison to the standard Folch extraction (chloroform: methanol, 2:1, v/v). Among the five solvent systems (Folch alternatives) evaluated, the combination of hexane and isopropanol (H:IP, 3:2, v/v) was identified as the best for total lipid and PLs extraction, resulting in a recovery of 74.4% and 65.7% (w/w), respectively. EDTA chelating of the calcium ions was applied to produce a salt‐free membrane protein product. A treatment with 0.2% EDTA was able to significantly reduce the calcium content to 0.20 mg/g protein compared to 58.81 mg/g in the initial calcium‐precipitated MFGM. EDTA chelating of cations also facilitated the extraction of lipids during H:IP (3:2) treatment, which increased the total recovery of total lipid and PLs to 90.7% and 82.5% (w/w), respectively. Overall, this study demonstrates a great potential for the utilization of beta stream and other low‐solids content aqueous dairy by‐products for PLs recovery and further fractionation. Value‐added dairy products (PLs and membrane proteins) can be obtained using green solvents identified in this study, and the process is industrially scalable.

Simple But Efficacious Enrichment of Integral Membrane Proteins and Their Interactions for In-Depth Membrane Proteomics

Membrane proteins play essential roles in various cellular processes, such as nutrient transport, bioenergetic processes, cell adhesion, and signal transduction. Proteomics is one of the key approaches to exploring membrane proteins comprehensively. Bottom–up proteomics using LC–MS/MS has been widely used in membrane proteomics. However, the low abundance and hydrophobic features of membrane proteins, especially integral membrane proteins, make it difficult to handle the proteins and are the bottleneck for identification by LC–MS/MS. Herein, to improve the identification and quantification of membrane proteins, we have stepwisely evaluated methods of membrane enrichment for the sample preparation. The enrichment methods of membranes consisted of precipitation by ultracentrifugation and treatment by urea or alkaline solutions. The best enrichment method in the study, washing with urea after isolation of the membranes, resulted in the identification of almost twice as many membrane proteins compared with samples without the enrichment. Notably, the method significantly enhances the identified numbers of multispanning transmembrane proteins, such as solute carrier transporters, ABC transporters, and G-protein–coupled receptors, by almost sixfold. Using this method, we revealed the profiles of amino acid transport systems with the validation by functional assays and found more protein–protein interactions, including membrane protein complexes and clusters. Our protocol uses standard procedures in biochemistry, but the method was efficient for the in-depth analysis of membrane proteome in a wide range of samples.

Hemodynamic Mimic Shear Stress for Platelet Membrane Nanobubbles Preparation and Integrin αIIbβ3 Conformation Regulation.

Platelet (PLT) membrane biomimetic nanomaterials have become promising theranostic platforms due to their good biocompatibility and effectiveness. However, in order to achieve precise regulation of cell membrane components, novel controllable construction approaches need to be developed. Inspired by the interaction mechanism among platelet production, activation, and dynamic biomechanical signals in blood circulation, here a platelet nanobubbles (PNBs) with reassembled platelet membrane with ideal echogenicity was fabricated using an adjustable pressure-induced shear stress method. The results demonstrate that the high shear stress during PNBs fabrication led to the enrichment of platelet membrane lipid rafts and proteins, as well as their reassembly on the gas-liquid interface. More importantly, the conformation of platelet integrin αIIbβ3 was transformed into a shear stress-induced intermediate affinity state, which gives PNBs enhanced adhesion ability to the vascular endothelial injury. Taken together, these PNBs have great application potential in the specifically targeted ultrasound diagnosis of vascular endothelial injury.

Comparison of the performance of secretome analysis based on metabolic labeling by three unnatural sugars

分泌蛋白质是调控细胞间信号转导的重要生物大分子。由于分泌蛋白的丰度相比于胞内蛋白以及培养基添加剂更低,因此分泌蛋白的高通量鉴定是目前蛋白质组学界研究的热点和难点。目前,基于生物质谱的分泌蛋白质组学分析一般均需要从无血清的条件培养基中获得分泌蛋白质,再对其进行富集和分析。该流程操作步骤繁琐,易造成分泌蛋白质的损失和降解。本工作采用基于生物正交化学生物学技术实现对分泌蛋白质的高选择性标记和高效富集。通过结合点击化学技术,综合评估了分泌蛋白质分析中用于代谢标记的不同糖类似物。采用3种最常用的商品化糖类似物,N-叠氮乙酰甘露糖胺(ManNAz)、N-叠氮乙酰半乳糖胺(GalNAz)和N-叠氮乙酰葡萄糖胺(GlcNAz)分别对HeLa细胞进行代谢标记,之后通过炔基生物素探针对条件培养基中的分泌蛋白进行富集,结合质谱分析来对比3种糖类似物对分泌蛋白的标记效率。最后通过无标定量蛋白质组学分析,系统评估了3种糖类似物用于分泌蛋白质组分析的性能。结果表明,基于ManNAz的分泌蛋白标记方法鉴定到了282个分泌蛋白、224个细胞质膜蛋白以及846个N-糖基化位点;对分泌蛋白的富集效率分别较GalNAz和GlcNAz提高了130%和67.2%;对细胞质膜蛋白的富集效率较GalNAz和GlcNAz分别提高了273.3%和148.7%,体现出了明显的优势。本研究的实验结果为分泌蛋白高选择性富集和系统分析提供了有益的对比分析和新技术策略。

An Integrated Approach for the Efficient Extraction and Solubilization of Rice Microsomal Membrane Proteins for High-Throughput Proteomics.

The preparation of microsomal membrane proteins (MPs) is critically important to microsomal proteomics. To date most research studies have utilized an ultracentrifugation-based approach for the isolation and solubilization of plant MPs. However, these approaches are labor-intensive, time-consuming, and unaffordable in certain cases. Furthermore, the use of sodium dodecyl sulfate (SDS) and its removal prior to a mass spectrometry (MS) analysis through multiple washing steps result in the loss of proteins. To address these limitations, this study introduced a simple micro-centrifugation-based MP extraction (MME) method from rice leaves, with the efficacy of this approach being compared with a commercially available plasma membrane extraction kit (PME). Moreover, this study assessed the subsequent solubilization of isolated MPs in an MS-compatible surfactant, namely, 4-hexylphenylazosulfonate (Azo) and SDS using a label-free proteomic approach. The results validated the effectiveness of the MME method, specifically in the enrichment of plasma membrane proteins as compared with the PME method. Furthermore, the findings showed that Azo demonstrated several advantages over SDS in solubilizing the MPs, which was reflected through a label-free quantitative proteome analysis. Altogether, this study provided a relatively simple and rapid workflow for the efficient extraction of MPs with an Azo-integrated MME approach for bottom-up proteomics.