Regulation of alternative polyadenylation by the C2H2-zinc-finger protein Sp1.

Alternative polyadenylation (APA) enhances gene regulatory potential by increasing the diversity of mRNA transcripts. 3' UTR shortening through APA correlates with enhanced cellular proliferation and is a widespread phenomenon in tumor cells. Here, we show that the ubiquitously expressed transcription factor Sp1 binds RNA in vivo and is a common repressor of distal poly(A) site usage. RNA sequencing identified 2,344 genes (36% of the total mapped mRNA transcripts) with lengthened 3' UTRs upon Sp1 depletion. Sp1 preferentially binds the 3' UTRs of such lengthened transcripts and inhibits cleavage at distal sites by interacting with the subunits of the core cleavage and polyadenylation (CPA) machinery. The 3' UTR lengths of Sp1 target genes in breast cancer patient RNA-seq data correlate with Sp1 expression levels, implicating Sp1-mediated APA regulation in modulating tumorigenic properties. Taken together, our findings provide insights into the mechanism for dynamic APA regulation by unraveling a previously unknown function of the DNA-binding transcription factor Sp1.

Crosstalk between RNA Pol II C-Terminal Domain Acetylation and Phosphorylation via RPRD Proteins.

Post-translational modifications of the RNA polymerase II C-terminal domain (CTD) coordinate the transcription cycle. Crosstalk between different modifications is poorly understood. Here, we show how acetylation of lysine residues at position 7 of characteristic heptad repeats (K7ac)-only found in higher eukaryotes-regulates phosphorylation of serines at position 5 (S5p), a conserved mark of polymerases initiating transcription. We identified the regulator of pre-mRNA-domain-containing (RPRD) proteins as reader proteins of K7ac. K7ac enhanced CTD peptide binding to the CTD-interacting domain (CID) of RPRD1A and RPRD1B proteins in isothermal calorimetry and molecular modeling experiments. Deacetylase inhibitors increased K7ac- and decreased S5-phosphorylated polymerases, consistent with acetylation-dependent S5 dephosphorylation by an RPRD-associated S5 phosphatase. Consistent with this model, RPRD1B knockdown increased S5p but enhanced K7ac, indicating that RPRD proteins recruit K7 deacetylases, including HDAC1. We also report autoregulatory crosstalk between K7ac and S5p via RPRD proteins and their interactions with acetyl- and phospho-eraser proteins.

Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens using a dual fluorescence readthrough reporter.

Messenger RNA precursors (pre-mRNA) generally undergo 3' end processing by cleavage and polyadenylation (CPA), which is specified by a polyadenylation site (PAS) and adjacent RNA sequences and regulated by a large variety of core and auxiliary CPA factors. To date, most of the human CPA factors have been discovered through biochemical and proteomic studies. However, genetic identification of the human CPA factors has been hampered by the lack of a reliable genome-wide screening method. We describe here a dual fluorescence readthrough reporter system with a PAS inserted between two fluorescent reporters. This system enables measurement of the efficiency of 3' end processing in living cells. Using this system in combination with a human genome-wide CRISPR/Cas9 library, we conducted a screen for CPA factors. The screens identified most components of the known core CPA complexes and other known CPA factors. The screens also identified CCNK/CDK12 as a potential core CPA factor, and RPRD1B as a CPA factor that binds RNA and regulates the release of RNA polymerase II at the 3' ends of genes. Thus, this dual fluorescence reporter coupled with CRISPR/Cas9 screens reliably identifies bona fide CPA factors and provides a platform for investigating the requirements for CPA in various contexts.

Novel Metabolites to Improve Glomerular Filtration Rate Estimation.

INTRODUCTION: The glomerular filtration rate (GFR) is crucial for chronic kidney disease (CKD) diagnosis and therapy. Various studies have sought to recognize ideal endogenous markers to improve the estimated GFR for clinical practice. To screen out potential novel metabolites related to GFR (mGFR) measurement in CKD patients from the Chinese population, we identified more biomarkers for improving GFR estimation. METHODS: Fifty-three CKD participants were recruited from the Third Affiliated Hospital of Sun Yat-sen University in 2020. For each participant, mGFR was evaluated by utilizing the plasma clearance of iohexol and collecting serum samples for untargeted metabolomics analyses by ultrahigh-performance liquid chromatography-tandem mass spectroscopy. All participants were divided into four groups according to mGFR. The metabolite peak area data were uploaded to MetaboAnalyst 5.0 for one-way analysis of variance, principal component analysis, and partial least squares-discriminant analysis and confirmed the metabolites whose levels increased or decreased with mGFR and variable importance in projection (VIP) values >1. Metabolites were ranked by correlation with the original values of mGFR, and metabolites with a correlation coefficient >0.8 and VIP >2 were identified. RESULTS: We screened out 198 metabolites that increased or decreased with mGFR decline. After ranking by correlation with mGFR, the top 50 metabolites were confirmed. Further studies confirmed the 10 most highly correlated metabolites. CONCLUSION: We screened out the metabolites that increased or decreased with mGFR decline in CKD patients from the Chinese population, and 10 of them were highly correlated. They are potential novel metabolites to improve GFR estimation.

Synthesis of Chitosan Oligosaccharide-Loaded Glycyrrhetinic Acid Functionalized Mesoporous Silica Nanoparticles and In Vitro Verification of the Treatment of APAP-Induced Liver Injury.

OBJECTIVE: the study was to find a suitable treatment for acute drug-induced liver injury. The use of nanocarriers can improve the therapeutic effect of natural drugs by targeting hepatocytes and higher loads. METHODS: firstly, uniformly dispersed three-dimensional dendritic mesoporous silica nanospheres (MSNs) were synthesized. Glycyrrhetinic acid (GA) was covalently modified on MSN surfaces through amide bond and then loaded with COSM to form drug-loaded nanoparticles (COSM@MSN-NH2-GA). The constructed drug-loaded nano-delivery system was determined by characterization analysis. Finally, the effect of nano-drug particles on cell viability was evaluated and the cell uptake in vitro was observed. RESULTS: GA was successfully modified to obtain the spherical nano-carrier MSN-NH2-GA (≤200 nm). The neutral surface charge improves its biocompatibility. MSN-NH2-GA has high drug loading (28.36% ± 1.00) because of its suitable specific surface area and pore volume. In vitro cell experiments showed that COSM@MSN-NH2-GA significantly enhanced the uptake of liver cells (LO2) and decreased the AST and ALT indexes. CONCLUSION: this study demonstrated for the first time that formulation and delivery schemes using natural drug COSM and nanocarrier MSN have a protective effect on APAP-induced hepatocyte injury. This result provides a potential nano-delivery scheme for the targeted therapy of acute drug-induced liver injury.

Panorama of ancient metazoan macromolecular complexes.

Macromolecular complexes are essential to conserved biological processes, but their prevalence across animals is unclear. By combining extensive biochemical fractionation with quantitative mass spectrometry, here we directly examined the composition of soluble multiprotein complexes among diverse metazoan models. Using an integrative approach, we generated a draft conservation map consisting of more than one million putative high-confidence co-complex interactions for species with fully sequenced genomes that encompasses functional modules present broadly across all extant animals. Clustering reveals a spectrum of conservation, ranging from ancient eukaryotic assemblies that have probably served cellular housekeeping roles for at least one billion years, ancestral complexes that have accrued contemporary components, and rarer metazoan innovations linked to multicellularity. We validated these projections by independent co-fractionation experiments in evolutionarily distant species, affinity purification and functional analyses. The comprehensiveness, centrality and modularity of these reconstructed interactomes reflect their fundamental mechanistic importance and adaptive value to animal cell systems.

Magnetic Resonance Features of Lower-grade Gliomas in Prediction of the Reverse Phase Protein A.

OBJECTIVES: The Cancer Genome Atlas Research Network identified 4 novel protein expression-defined subgroups in patients with lower-grade gliomas (LGGs). The RPPA3 subtype had high levels of Epidermal Growth Factor Receptor and Human epidermal growth factor receptor-2, further increasing the chances for targeted therapy. In this study, we aimed to explore the relationships between magnetic resonance features and reverse phase protein array (RPPA) subtypes (R1-R4). METHODS: Survival estimates for the Cancer Genome Atlas cohort were generated using the Kaplan-Meier method and time-dependent receiver operating characteristic curves. A total of 153 patients with LGG with brain magnetic resonance imaging from The Cancer Imaging Archive were retrospectively analyzed. Least absolute shrinkage and selection operator algorithm was used to reduce the feature dimensions of the RPPA3 subtype. RESULTS: A total of 51 (33.3%) RPPA1 subtype, 42 (27.4) RPPA2 subtype, 19 (12.4%) RPPA3 subtype, and 38 (24.8%) RPPA4 subtype were identified. On multivariate logistic regression analysis, subventricular zone involvement [odds ratio (OR), 0.370; P = 0.006; 95% confidence interval (CI), 0.181-0.757) was associated with RPPA1 subtype [area under the curve (AUC), 0.598]. Volume of 60 cm3 or greater (OR, 5.174; P < 0.001; 95% CI, 2.182-12.267) was associated with RPPA2 subtype (AUC, 0.684). Proportion contrast-enhanced tumor greater than 5% (OR, 4.722; P = 0.010; 95% CI, 1.456-15.317), extranodular growth (OR, 5.524; P = 0.010; 95% CI, 1.509-20.215), and L/CS ratio equal to or greater than median (OR, 0.132; P = 0.003; 95% CI, 0.035-0.500) were associated with RPPA3 subtype (AUC, 0.825). Proportion contrast-enhanced tumor greater than 5% (OR, 0.206; P = 0.005; 95% CI, 0.068-0.625) was associated with RPPA4 subtype (AUC, 0.638). For the prediction of RPPA3 subtype, the nomogram showed good discrimination, with an AUC of 0.825 (95% CI, 0.711-0.939) and was well calibrated. The RPPA3 subtype was associated with shortest mean overall survival (RPPA3 subtype vs other: 613 vs 873 days; P < 0.05). The time-dependent receiver operating characteristic curves for the RPPA3 subtype was 0.72 (95% CI, 0.60-0.84) for survival at 1 year. Decision curve analysis indicated that prediction for the RPPA3 model was clinically useful. CONCLUSIONS: The RPPA3 subtype is an unfavorable prognostic biomarker for overall survival in patients with LGG. Radiogenomics analysis of magnetic resonance features can predict the RPPA subtype preoperatively and may be of clinical value in tailoring the management strategies in patients with LGG.

Construction of Flexible Amine-linked Covalent Organic Frameworks by Catalysis and Reduction of Formic Acid via the Eschweiler-Clarke Reaction.

Compared to the current mainstream rigid covalent organic frameworks (COFs) linked by imine bonds, flexible COFs have certain advantages of elasticity and self-adaptability, but their construction and application are greatly limited by the complexity in synthesis and difficulty in obtaining regular structure. Herein, we reported for the first time a series of flexible amine-linked COFs with high crystallinity synthesized by formic acid with unique catalytic and reductive bifunctional properties, rather than acetic acid, the most common catalyst for COF synthesis. The reaction mechanism was demonstrated to be a synchronous in situ reduction during the formation of imine bond. The flexibilities of the products endow them with accommodative adaptability to guest molecules, thus increasing the adsorption capacities for nitrogen and iodine by 27 % and 22 %, respectively. Impressively, a novel concept of flexibilization degree was proposed firstly, which provides an effective approach to rationally measure the flexibility of COFs.

Colyliform Crystalline 2D Covalent Organic Frameworks (COFs) with Quasi-3D Topologies for Rapid I2 Adsorption.

Constructing three-dimensional (3D) structural characteristics on two-dimensional (2D) covalent organic frameworks (COFs) is a good approach to effectively improve the permeability and mass transfer rate of the materials and realize the rapid adsorption for guest molecules, while avoiding the high cost and monomer scarcity in preparing 3D COFs. Herein, we report for the first time a series of colyliform crystalline 2D COFs with quasi-three-dimensional (Q-3D) topologies, consisting of unique "stereoscopic" triangular pores, large interlayer spacings and flexible constitutional units which makes the pores elastic and self-adaptable for the guest transmission. The as-prepared QTD-COFs have a faster adsorption rate (2.51 g h-1 ) for iodine than traditional 2D COFs, with an unprecedented maximum adsorption capacity of 6.29 g g-1 . The excellent adsorption performance, as well as the prominent irradiation stability allow the QTD-COFs to be applied for the rapid removal of radioactive iodine.

Redox-Active Two-Dimensional Covalent Organic Frameworks (COFs) for Selective Reductive Separation of Valence-Variable, Redox-Sensitive and Long-Lived Radionuclides.

We report the first example of 2D covalent organic framework nanosheets (Redox-COF1) for the selective reduction and in situ loading of valence-variable, redox-sensitive and long-lived radionuclides (abbreviated as VRL nuclides). Compared with sorbents based on chemical adsorption and physical adsorption, the redox adsorption mechanism of Redox-COF1 can effectively reduce the impact of functional group protonation under the usual high-acidity conditions in chemisorption, and raise the adsorption efficiency from the monotonous capture by pores in physisorption. The adsorption selectivity for UO2 2+ reaches up to unprecedented ca. 97 % at pH 3, more than for any analogous adsorbing material.

Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation.

Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.

Interaction landscape of membrane-protein complexes in Saccharomyces cerevisiae.

Macromolecular assemblies involving membrane proteins (MPs) serve vital biological roles and are prime drug targets in a variety of diseases. Large-scale affinity purification studies of soluble-protein complexes have been accomplished for diverse model organisms, but no global characterization of MP-complex membership has been described so far. Here we report a complete survey of 1,590 putative integral, peripheral and lipid-anchored MPs from Saccharomyces cerevisiae, which were affinity purified in the presence of non-denaturing detergents. The identities of the co-purifying proteins were determined by tandem mass spectrometry and subsequently used to derive a high-confidence physical interaction map encompassing 1,726 membrane protein-protein interactions and 501 putative heteromeric complexes associated with the various cellular membrane systems. Our analysis reveals unexpected physical associations underlying the membrane biology of eukaryotes and delineates the global topological landscape of the membrane interactome.

Proteomic characterization of the acid tolerance response in Lactobacillus delbrueckii subsp. bulgaricus†CAUH1 and functional identification of a novel acid stress-related transcriptional regulator Ldb0677.

To overcome the deleterious effects of acid stress, Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) elicits an adaptive response to acid stress. In this study, proteomics approach complemented by transcriptional analysis revealed some cellular changes in L. bulgaricus CAUH1 during acid adaptation. We observed an increase of glycolysis-associated proteins, promoting an optimal utilization of carbohydrates. Also, rerouting of the pyruvate metabolism to fatty acid biosynthesis was observed, indicating a possible modification of the cell membrane rigidity and impermeability. In addition, expression of ribosomal protein S1 (RpsA) was repressed; however, the expression of EF-Tu, EF-G and TypA was up-regulated at both protein and transcript levels. This suggests a reduction of protein synthesis in response to acid stress along with possible enhancement of the translational accuracy and protein folding. It is noteworthy that the putative transcriptional regulator Ldb0677 was 1.84-fold up-regulated. Heterologous expression of Ldb0677 was shown to significantly enhance acid resistance in host strain Lactococcus lactis. To clarify its role in transcriptional regulation network, the DNA-binding specificity of Ldb0677 was determined using bacterial one-hybrid and electrophoretic mobility shift assay. The identification of a binding motif (SSTAGACR) present in the promoter regions of 22 genes indicates that it might function as a major regulator in acid stress response in L. bulgaricus.

Value of magnetic resonance imaging in brucellar spondylodiscitis.

PURPOSE: The aim of this retrospective study was to evaluate magnetic resonance (MR) imaging in patients with brucellar spondylodiscitis. MATERIALS AND METHODS: Sixty-three patients with spondylodiscitis were diagnosed based on positive clinical findings, ≥1/160 titres of brucella agglutination tests and/or positive blood cultures. MR imaging was performed in all of the patients with spondylodiscitis. Signal changes and enhancement of vertebral bodies, involvement of paravertebral soft tissues and epidural spaces, nerve root and cord compression and abscess formation were assessed. RESULTS: Of 63 patients with spinal brucellosis, eight had thoracic, 35 had lumbar, ten had cervical vertebral, seven had both thoracic and lumbar, and three had both lumbar and sacral vertebral involvement. Thirteen patients had cord compression and six had root compression. Four patients had facet-joint involvement, and one had erector spinae muscle involvement. Twenty-four had intervertebral disc narrowing. Seventeen patients were in the acute stage, 32 in the subacute stage and 14 in the chronic stage. Vertebral bodies, vertebral end plates and intervertebral disc spaces were hypointense and hyperintense in the acute stage, whereas they were hypointense and heterogeneous in the subacute and chronic stages on T1- and T2-weighted images, respectively. CONCLUSION: Brucella is still a public health problem in endemic areas. MR imaging is a highly sensitive and noninvasive imaging technique which should be first choice of imaging in the early diagnosis of spondylodiscitis.

Combinative predictive effect of left ventricular mass index, ratio of HDL and CRP for progression of chronic kidney disease in non-dialysis patient.

PURPOSE: This current study scrutinized the association among left ventricular mass index (LVMI), ratio of high-density lipoprotein (HDL) and C-reactive protein (CRP), and renal function. Furthermore, we examined the predictive effects of left ventricular mass index and HDL/CRP on progression of non-dialysis chronic kidney disease. METHODS: We enrolled adult patients with chronic kidney disease (CKD) who were not receiving dialysis and obtained follow-up data on them. We extracted and compared data between different groups. To investigate the relationship between left ventricular mass index (LVMI), high-density lipoprotein (HDL)/C-reactive protein (CRP) levels, and CKD, we employed linear regression analysis, Kaplan-Meier analysis, and Cox proportional hazards regression analysis. RESULTS: Our study enrolled a total of 2351 patients. Compared with those in the non-progression group, subjects in the CKD progression group had lower ln(HDL/CRP) levels (- 1.56 ± 1.78 vs. - 1.14 ± 1.77, P < 0.001) but higher left ventricular mass index (LVMI) values (115.45 ± 29.8 vs. 102.8 ± 26.31 g/m2, P < 0.001). Moreover, after adjusting for demographic factors, ln(HDL/CRP) was found to be positively associated with estimated glomerular filtration rate (eGFR) (B = 1.18, P < 0.001), while LVMI was negatively associated with eGFR (B = - 0.15, P < 0.001). In the end, we found that both LVH (HR = 1.53, 95% CI 1.15 to 2.05, P = 0.004) and lower ln(HDL/CRP) (HR = 1.46, 95% CI 1.08 to 1.96, P = 0.013) independently predicted CKD progression. Notably, the combined predictive power of these variables was stronger than either variable alone (HR = 1.98, 95% CI 1.5 to 2.62, P < 0.001). CONCLUSION: Our study findings indicate that in pre-dialysis patients, both HDL/CRP and LVMI are associated with basic renal function and are independently correlated with CKD progression. These variables may serve as predictors for CKD progression, and their combined predictive power is stronger than that of either variable alone.

From Synthesis to Mechanisms: In-Depth Exploration of the Dual-Atom Catalytic Mechanisms Toward Oxygen Electrocatalysis.

Dual-atom catalysts (DACs) hold a higher metal atom loading and provide greater flexibility in terms of the structural characteristics of their active sites in comparison to single-atom catalysts. Consequently, DACs hold great promise for achieving improved catalytic performance. This article aims to provide a focused overview of the latest advancements in DACs, covering their synthesis and mechanisms in reversible oxygen electrocatalysis, which plays a key role in sustainable energy conversion and storage technologies. The discussion starts by highlighting the structures of DACs and the differences in diatomic coordination induced by various substrates. Subsequently, the state-of-the-art fabrication strategies of DACs for oxygen electrocatalysis are discussed from several different perspectives. It particularly highlights the challenges of increasing the diatomic loading capacity. More importantly, the main focus of this overview is to investigate the correlation between the configuration and activity in DACs in order to gain a deeper understanding of their active roles in oxygen electrocatalysis. This will be achieved through density functional theory calculations and sophisticated in situ characterization technologies. The aim is to provide guidelines for optimizing and upgrading DACs in oxygen electrocatalysis. Additionally, the overview discusses the current challenges and future prospects in this rapidly evolving area of research.

Metabolic implications of amino acid metabolites in chronic kidney disease progression: a metabolomics analysis using OPLS-DA and MBRole2.0 database.

BACKGROUND: As chronic kidney disease (CKD) progresses, metabolites undergo diverse transformations. Nevertheless, the impact of these metabolic changes on the etiology, progression, and prognosis of CKD remains uncertain. Our objective is to conduct a metabolomics analysis to scrutinize metabolites and identify significant metabolic pathways implicated in CKD progression, thereby pinpointing potential therapeutic targets for CKD management. METHODS: We recruited 145 patients with CKD and determined their mGFR by measuring the plasma iohexol clearance, whereupon we partitioned them into four groups based on their mGFR values. Non-targeted metabolomics analysis was conducted using UPLC-MS/MS assays. Differential metabolites were identified via one-way ANOVA, PCA, PLS-DA, and OPLS-DA analyses employing the MetaboAnalyst 5.0 platform. Ultimately, we performed differential metabolite pathway enrichment analysis, using both the MetaboAnalyst 5.0 platform and the MBRole2.0 database. RESULTS: According to the findings of the MBRole2.0 and MetaboAnalyst 5.0 enrichment analysis, six amino acid metabolism pathways were discovered to have significant roles in the progression of CKD, with the glycine, serine, and threonine metabolism pathway being the most prominent. The latter enriched 14 differential metabolites, of which six decreased while two increased concomitantly with renal function deterioration. CONCLUSIONS: The metabolic analysis unveiled that glycine, serine, and threonine metabolism plays a pivotal role in the progression of CKD. Specifically, glycine was found to increase while serine decreased with the deterioration of CKD.

Mechanism of action of the bile acid receptor TGR5 in obesity.

G protein-coupled receptors (GPCRs) are a large family of membrane protein receptors, and Takeda G protein-coupled receptor 5 (TGR5) is a member of this family. As a membrane receptor, TGR5 is widely distributed in different parts of the human body and plays a vital role in regulating metabolism, including the processes of energy consumption, weight loss and blood glucose homeostasis. Recent studies have shown that TGR5 plays an important role in glucose and lipid metabolism disorders such as fatty liver, obesity and diabetes. With the global obesity situation becoming more and more serious, a comprehensive explanation of the mechanism of TGR5 and filling the gaps in knowledge concerning clinical ligand drugs are urgently needed. In this review, we mainly explain the anti-obesity mechanism of TGR5 to promote the further study of this target, and show the electron microscope structure of TGR5 and review recent studies on TGR5 ligands to illustrate the specific binding between TGR5 receptor binding sites and ligands, which can effectively provide new ideas for ligand research and promote drug research.

Heterogeneous ferroptosis susceptibility of macrophages caused by focal iron overload exacerbates rheumatoid arthritis.

Focal iron overload is frequently observed in patients with rheumatoid arthritis (RA), yet its functional significance remains elusive. Herein, we report that iron deposition in lesion aggravates arthritis by inducing macrophage ferroptosis. We show that excessive iron in synovial fluid positively correlates with RA disease severity as does lipid hyperoxidation of focal monocyte/macrophages. Further study reveals high susceptibility to iron induced ferroptosis of the anti-inflammatory macrophages M2, while pro-inflammatory M1 are less affected. Distinct glutathione peroxidase 4 (GPX4) degradation depending on p62/SQSTM1 in the two cell types make great contribution mechanically. Of note, ferroptosis inhibitor liproxstatin-1 (LPX-1) can alleviate the progression of K/BxN serum-transfer induced arthritis (STIA) mice accompanied with increasing M2 macrophages proportion. We thus propose that the heterogeneous ferroptosis susceptibility of macrophage subtypes as well as consequent inflammation and immune disorders are potential biomarkers and therapeutic targets in RA.

An improved SPE method for fractionation and identification of phospholipids.

This work reports an efficient and universal SPE method developed for separation and identification of phospholipids derived from complex biological samples. For the separation step, sequential combination of silica gel-aminopropyl-silica gel SPE cartridges is applied. This setup enables separation of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol, phosphatidylserine, cardiolipin, and sphingomyelin into four fractions according to the polarity of their headgroups. Sample acquisition of the SPE fractions is performed by a high-resolution LC-MS system consisting of a hybrid linear IT Fourier transform ion cyclotron resonance mass spectrometer coupled to RP-HPLC. The unequivocal advantage of our SPE sample preparation setup is avoidance of analyte peak overlapping in the determination step done by RP-HPLC. Overlapping phospholipid signals would otherwise exert adverse ion suppression effects. An additional benefit of this method is the elimination of polar and nonpolar (e.g. neutral lipids) contaminants from the phospholipid fractions, which highly reduces contamination of the LC-MS system. The method was validated with fermentation samples of organic waste, where 78 distinct phospholipid and sphingomyelin species belonging to six lipid classes were successfully identified.