bims-mascan 2024-04-21 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2024–04–21
fiveteen papers selected by
Giovanny Rodriguez Blanco, University of Edinburgh

LipiDex 2 Integrates MSn Tree-Based Fragmentation Methods and Quality Control Modules to Improve Discovery Lipidomics.
Bone Proteomics Method Optimization for Forensic Investigations.
Concomitant investigation of crustacean amphipods lipidome and metabolome during the molting cycle by Zeno SWATH data-independent acquisition coupled with electron activated dissociation and machine learning.
Interplatform comparison between three ion mobility techniques for human plasma lipid collision cross sections.
Mass Spectrometry-Based Proteomics of Poly(methylmethacrylate)-Embedded Bone.
Diet and Cancer Metabolism.
Synthetic Knockout Protein Standard for Evaluating Interference in Tandem Mass Tag-Based Proteomics.
How Much Storage Precision Can Be Lost: Guidance for Near-Lossless Compression of Untargeted Metabolomics Mass Spectrometry Data.
Ion Mobility Mass Spectrometry-based Disaccharide Analysis of Glycosaminoglycans.
Recharacterization of RSL3 reveals that the selenoproteome is a druggable target in colorectal cancer.
Improved lipid analysis using a 2D-LC-MS system with a novel injection procedure.
Applications of chromatographic methods in metabolomics: A review.
Global, site-resolved analysis of ubiquitylation occupancy and turnover rate reveals systems properties.
Quantification Quality Control Emerges as A Crucial Factor to Enhance Single Cell Proteomics Data Analysis.
ABCG2 and SLC1A5 functionally interact to rewire metabolism and confer a survival advantage to cancer cells under oxidative stress.

Anal Chem. 2024 Apr 19.

LipiDex 2 Integrates MSn Tree-Based Fragmentation Methods and Quality Control Modules to Improve Discovery Lipidomics.

Benton J Anderson, Dain R Brademan, Yuchen He, Katherine A Overmyer, Joshua J Coon.

As lipidomics experiments increase in scale and complexity, data processing tools must support workflows for new liquid chromatography-mass spectrometry (LC-MS) methods while simultaneously supporting quality controls to maximize the confidence in lipid identifications. LipiDex 2 improves lipidomics data processing algorithms from LipiDex 1 and introduces new tools for spectral matching and peak annotation functions, with improvements in speed and user-friendliness. In silico spectral library generation now supports tandem mass spectral (MSn) tree-based fragmentation methods, and the LipiDex 2 workflow fully integrates the fragmentation logic into the data processing steps to enable lipid identification at the appropriate level of structural resolution. Finally, LipiDex 2 features new modules for automated quality control checks that also allow users to visualize data quality in a data dashboard user interface.

DOI: https://doi.org/10.1021/acs.analchem.4c00359
J Proteome Res. 2024 Apr 15.

Bone Proteomics Method Optimization for Forensic Investigations.

Luke Gent, Maria Elena Chiappetta, Stuart Hesketh, Pawel Palmowski, Andrew Porter, Andrea Bonicelli, Edward C Schwalbe, Noemi Procopio.

  The application of proteomic analysis to forensic skeletal remains has gained significant interest in improving biological and chronological estimations in medico-legal investigations. To enhance the applicability of these analyses to forensic casework, it is crucial to maximize throughput and proteome recovery while minimizing interoperator variability and laboratory-induced post-translational protein modifications (PTMs). This work compared different workflows for extracting, purifying, and analyzing bone proteins using liquid chromatography with tandem mass spectrometry (LC-MS)/MS including an in-StageTip protocol previously optimized for forensic applications and two protocols using novel suspension-trap technology (S-Trap) and different lysis solutions. This study also compared data-dependent acquisition (DDA) with data-independent acquisition (DIA). By testing all of the workflows on 30 human cortical tibiae samples, S-Trap workflows resulted in increased proteome recovery with both lysis solutions tested and in decreased levels of induced deamidations, and the DIA mode resulted in greater sensitivity and window of identification for the identification of lower-abundance proteins, especially when open-source software was utilized for data processing in both modes. The newly developed S-Trap protocol is, therefore, suitable for forensic bone proteomic workflows and, particularly when paired with DIA mode, can offer improved proteomic outcomes and increased reproducibility, showcasing its potential in forensic proteomics and contributing to achieving standardization in bone proteomic analyses for forensic applications.

Keywords:  acquisition modes; bone proteomics; forensic science; mass spectrometry; protein extraction

DOI:  https://doi.org/10.1021/acs.jproteome.4c00151
Anal Chim Acta. 2024 May 22. pii: S0003-2670(24)00334-9. [Epub ahead of print]1304 342533

Concomitant investigation of crustacean amphipods lipidome and metabolome during the molting cycle by Zeno SWATH data-independent acquisition coupled with electron activated dissociation and machine learning.

Thomas Alexandre Brunet, Yohann Clément, Valentina Calabrese, Jérôme Lemoine, Olivier Geffard, Arnaud Chaumot, Davide Degli-Esposti, Arnaud Salvador, Sophie Ayciriex.

   BACKGROUND: DIA (Data-Independent Acquisition) is a powerful technique in Liquid Chromatography coupled with high-resolution tandem Mass Spectrometry (LC-MS/MS) initially developed for proteomics studies and recently emerging in metabolomics and lipidomics. It provides a comprehensive and unbiased coverage of molecules with improved reproducibility and quantitative accuracy compared to Data-Dependent Acquisition (DDA). Combined with the Zeno trap and Electron-Activated Dissociation (EAD), DIA enhances data quality and structural elucidation compared to conventional fragmentation under CID. These tools were applied to study the lipidome and metabolome of the freshwater amphipod Gammarus fossarum, successfully discriminating stages and highlighting significant biological features. Despite being underused, DIA, along with the Zeno trap and EAD, holds great potential for advancing research in the omics field.
RESULTS: DIA combined with the Zeno trap enhances detection reproducibility compared to conventional DDA, improving fragmentation spectra quality and putative identifications. LC coupled with Zeno-SWATH-DIA methods were used to characterize molecular changes in reproductive cycle of female gammarids. Multivariate data analysis including Principal Component Analysis and Partial Least Square Discriminant Analysis successfully identified significant features. EAD fragmentation helped to identify unknown features and to confirm their molecular structure using fragmentation spectra database annotation or machine learning. EAD database matching accurately annotated five glycerophospholipids, including the position of double bonds on fatty acid chain moieties. SIRIUS database predicted structures of unknown features based on experimental fragmentation spectra to compensate for database incompleteness.
SIGNIFICANCE: Reproducible detection of features and confident identification of putative compounds are pivotal stages within analytical pipelines. The DIA approach combined with Zeno pulsing enhances detection sensitivity and targeted fragmentation with EAD in positive polarity provides orthogonal fragmentation information. In our study, Zeno-DIA and EAD thereby facilitated a comprehensive and insightful exploration of pertinent biological molecules associated with the reproductive cycle of gammarids. The developed methodology holds great promises for identifying informative biomarkers on the health status of an environmental sentinel species.

Keywords:  Data-Independent acquisition; Electron activated dissociation; Gammarid; Lipidomics; Machine learning; Metabolomics; Sequential window acquisition of all theoretical mass spectra; Zeno pulsing

DOI:  https://doi.org/10.1016/j.aca.2024.342533
Anal Chim Acta. 2024 May 22. pii: S0003-2670(24)00336-2. [Epub ahead of print]1304 342535

Interplatform comparison between three ion mobility techniques for human plasma lipid collision cross sections.

Anaïs C George, Isabelle Schmitz, Florent Rouvière, Sandra Alves, Benoit Colsch, Sabine Heinisch, Carlos Afonso, François Fenaille, Corinne Loutelier-Bourhis.

  The implementation of ion mobility spectrometry (IMS) in liquid chromatography-high-resolution mass spectrometry (LC-HRMS) workflows has become a valuable tool for improving compound annotation in metabolomics analyses by increasing peak capacity and by adding a new molecular descriptor, the collision cross section (CCS). Although some studies reported high repeatability and reproducibility of CCS determination and only few studies reported good interplatform agreement for small molecules, standardized protocols are still missing due to the lack of reference CCS values and reference materials. We present a comparison of CCS values of approximatively one hundred lipid species either commercially available or extracted from human plasma. We used three different commercial ion mobility technologies from different laboratories, drift tube IMS (DTIMS), travelling wave IMS (TWIMS) and trapped IMS (TIMS), to evaluate both instrument repeatability and interlaboratory reproducibility. We showed that CCS discrepancies of 0.3% (average) could occur depending on the data processing software tools. Moreover, eleven CCS calibrants were evaluated yielding mean RSD below 2% for eight calibrants, ESI Low concentration tuning mix (Tune Mix) showing the lowest RSD (< 0.5%) in both ion modes. Tune Mix calibrated CCS from the three different IMS instruments proved to be well correlated and highly reproducible (R2 > 0.995 and mean RSD ≤ 1%). More than 90% of the lipid CCS had deviations of less than 1%, demonstrating high comparability between techniques, and the possibility to use the CCS as molecular descriptor. We highlighted the need of standardized procedures for calibration, data acquisition, and data processing. This work demonstrates that using harmonized analytical conditions are required for interplatform reproducibility for CCS determination of human plasma lipids.

Keywords:  Collision cross section; Drift tube ion mobility spectrometry; Inter-laboratory comparison; Lipidomics; Trapped ion mobility spectrometry; Travelling wave ion mobility spectrometry

DOI:  https://doi.org/10.1016/j.aca.2024.342535
J Proteome Res. 2024 Apr 18.

Mass Spectrometry-Based Proteomics of Poly(methylmethacrylate)-Embedded Bone.

Annika Topitsch, Tim Halstenbach, René Rothweiler, Tobias Fretwurst, Katja Nelson, Oliver Schilling.

  Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a widely employed technique in proteomics research for studying the proteome biology of various clinical samples. Hard tissues, such as bone and teeth, are routinely preserved using synthetic poly(methyl methacrylate) (PMMA) embedding resins that enable histological, immunohistochemical, and morphological examination. However, the suitability of PMMA-embedded hard tissues for large-scale proteomic analysis remained unexplored. This study is the first to report on the feasibility of PMMA-embedded bone samples for LC-MS/MS analysis. Conventional workflows yielded merely limited coverage of the bone proteome. Using advanced strategies of prefractionation by high-pH reversed-phase liquid chromatography in combination with isobaric tandem mass tag labeling resulted in proteome coverage exceeding 1000 protein identifications. The quantitative comparison with cryopreserved samples revealed that each sample preparation workflow had a distinct impact on the proteomic profile. However, workflow replicates exhibited a high reproducibility for PMMA-embedded samples. Our findings further demonstrate that decalcification prior to protein extraction, along with the analysis of solubilization fractions, is not preferred for PMMA-embedded bone. The biological applicability of the proposed workflow was demonstrated using samples of human PMMA-embedded alveolar bone and the iliac crest, which revealed anatomical site-specific proteomic profiles. Overall, these results establish a crucial foundation for large-scale proteomics studies contributing to our knowledge of bone biology.

Keywords:  LC−MS/MS; PMMA embedding; TMT labeling; bone preservation; bone proteomics; fractionation

DOI:  https://doi.org/10.1021/acs.jproteome.4c00046
Cold Spring Harb Perspect Med. 2024 Apr 15. pii: a041549. [Epub ahead of print]

Diet and Cancer Metabolism.

Jason W Locasale, Marcus D Goncalves, Maira Di Tano, Guillermo Burgos-Barragan.

Diet and exercise are modifiable lifestyle factors known to have a major influence on metabolism. Clinical practice addresses diseases of altered metabolism such as diabetes or hypertension by altering these factors. Despite enormous public interest, there are limited defined diet and exercise regimens for cancer patients. Nevertheless, the molecular basis of cancer has converged over the past 15 years on an essential role for altered metabolism in cancer. However, our understanding of the molecular mechanisms that underlie the impact of diet and exercise on cancer metabolism is in its very early stages. In this work, we propose conceptual frameworks for understanding the consequences of diet and exercise on cancer cell metabolism and tumor biology and also highlight recent developments. By advancing our mechanistic understanding, we also discuss actionable ways that such interventions could eventually reach the mainstay of both medical oncology and cancer control and prevention.

DOI: https://doi.org/10.1101/cshperspect.a041549
Anal Chem. 2024 Apr 19.

Synthetic Knockout Protein Standard for Evaluating Interference in Tandem Mass Tag-Based Proteomics.

Jose Navarrete-Perea, Jiaming Li, Dylan Charles Mitchell, An Chi.

Isobaric labeling is widely used for unbiased, proteome-wide studies, and it provides several advantages, such as fewer missing values among samples and higher quantitative precision. However, ion interference may lead to compressed or distorted observed ratios due to the coelution and coanalysis of peptides. Here, we introduced a synthetic KnockOut standard (sKO) for evaluating interference in tandem mass tags-based proteomics. sKO is made by mixing TMTpro-labeled tryptic peptides derived from four nonhuman proteins and a whole human proteome as background at different proportions. We showcased the utility of the sKO standard by exploring ion interference at different peptide concentrations (up to a 30-fold change in abundance) and using a variety of mass spectrometer data acquisition strategies. We also demonstrated that the sKO standard could provide valuable information for the rational design of acquisition strategies to achieve optimal data quality and discussed its potential applications for high-throughput proteomics workflows development.

DOI: https://doi.org/10.1021/acs.analchem.4c00871
J Proteome Res. 2024 Apr 19.

How Much Storage Precision Can Be Lost: Guidance for Near-Lossless Compression of Untargeted Metabolomics Mass Spectrometry Data.

Junjie Tong, Miaoshan Lu, Ruimin Wang, Shaowei An, Jinyin Wang, Tong Wang, Cong Xie, Changbin Yu.

  Several lossy compressors have achieved superior compression rates for mass spectrometry (MS) data at the cost of storage precision. Currently, the impacts of precision losses on MS data processing have not been thoroughly evaluated, which is critical for the future development of lossy compressors. We first evaluated different storage precision (32 bit and 64 bit) in lossless mzML files. We then applied 10 truncation transformations to generate precision-lossy files: five relative errors for intensities and five absolute errors for m/z values. MZmine3 and XCMS were used for feature detection and GNPS for compound annotation. Lastly, we compared Precision, Recall, F1 - score, and file sizes between lossy files and lossless files under different conditions. Overall, we revealed that the discrepancy between 32 and 64 bit precision was under 1%. We proposed an absolute m/z error of 10-4 and a relative intensity error of 2 × 10-2, adhering to a 5% error threshold (F1 - scores above 95%). For a stricter 1% error threshold (F1 - scores above 99%), an absolute m/z error of 2 × 10-5 and a relative intensity error of 2 × 10-3 were advised. This guidance aims to help researchers improve lossy compression algorithms and minimize the negative effects of precision losses on downstream data processing.

Keywords:  GNPS; MZmine3; XCMS; compound annotation; feature detection; lossy compressors; mass spectrometry; precision losses; truncation transformations; untargeted metabolomics

DOI:  https://doi.org/10.1021/acs.jproteome.3c00851
Chemistry. 2024 Apr 17. e202400783

Ion Mobility Mass Spectrometry-based Disaccharide Analysis of Glycosaminoglycans.

Lukasz Polewski, Eunjin Moon, Andreas Zappe, Michael Götze, Gergő Péter Szekeres, Christian Roth, Kevin Pagel.

  Glycosaminoglycans (GAGs) are linear and acidic polysaccharides. They are ubiquitous molecules, which are involved in a wide range of biological processes. Despite being structurally simple at first glance, with a repeating backbone of alternating hexuronic acid and hexosamine dimers, GAGs display a highly complex structure, which predominantly results from their heterogeneous sulfation patterns. The commonly applied method for compositional analysis of all GAGs is "disaccharide analysis." In this process, GAGs are enzymatically depolymerized into disaccharides, derivatized with a fluorescent label, and then analysed through liquid chromatography. The limiting factor in the high throughput analysis of GAG disaccharides is the time-consuming liquid chromatography. To address this limitation, we here utilized trapped ion mobility-mass spectrometry (TIM-MS) for the separation of isomeric GAG disaccharides, which reduces the measurement time from hours to a few minutes. A full set of disaccharides comprises twelve structures, with eight possessing isomers. Most disaccharides cannot be differentiated by TIM-MS in underivatized form. Therefore, we developed chemical modifications to reduce sample complexity and enhance differentiability. Quantification is performed using stable isotope labelled standards, which are easily available due to the nature of the performed modifications.

Keywords:  disaccharide analysis; glycosaminoglycans; heparin; quantification; trapped ion mobility spectrometry

DOI:  https://doi.org/10.1002/chem.202400783
bioRxiv. 2024 Apr 01. pii: 2024.03.29.587381. [Epub ahead of print]

Recharacterization of RSL3 reveals that the selenoproteome is a druggable target in colorectal cancer.

Stephen L DeAngelo, Sofia Dziechciarz, Sumeet Solanki, Myungsun Shin, Liang Zhao, Andrii Balia, Marwa O El-Derany, Nupur K Das, Cristina Castillo, Hannah N Bell, Joao A Paulo, Yuezhong Zhang, Nicholas J Rossiter, Elizabeth C McCulla, Jianping He, Indrani Talukder, Zachary T Schafer, Nouri Neamati, Joseph D Mancias, Markos Koutmos, Yatrik M Shah.

Ferroptosis is an iron-dependent, non-apoptotic form of cell death resulting from the accumulation of lipid peroxides. Colorectal cancer (CRC) accumulates high levels of intracellular iron and reactive oxygen species (ROS), thereby sensitizing cells to ferroptosis. The selenoprotein glutathione peroxidase (GPx4) is a key enzyme in the detoxification of lipid peroxides and can be inhibited by the compound (S)-RSL3 ([1S,3R]-RSL3). However, the stereoisomer (R)-RSL3 ([1R,3R]-RSL3), which does not inhibit GPx4, exhibits equipotent activity to (S)-RSL3 across a panel of CRC cell lines. Utilizing CRC cell lines with an inducible knockdown of GPx4, we demonstrate that (S)-RSL3 sensitivity does not align with GPx4 dependency. Subsequently, a biotinylated (S)-RSL3 was then synthesized to perform affinity purification-mass spectrometry (AP-MS), revealing that (S)-RSL3 acts as a pan-inhibitor of the selenoproteome, targeting both the glutathione and thioredoxin peroxidase systems as well as multiple additional selenoproteins. To investigate the therapeutic potential of broadly disrupting the selenoproteome as a therapeutic strategy in CRC, we employed further chemical and genetic approaches to disrupt selenoprotein function. The findings demonstrate that the selenoprotein inhibitor Auranofin can induce ferroptosis and/or oxidative cell death both in-vitro and in-vivo . Consistent with this data we observe that AlkBH8, a tRNA-selenocysteine methyltransferase required for the translational incorporation of selenocysteine, is essential for CRC growth. In summary, our research elucidates the complex mechanisms underlying ferroptosis in CRC and reveals that modulation of the selenoproteome provides multiple new therapeutic targets and opportunities in CRC.

DOI: https://doi.org/10.1101/2024.03.29.587381
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Apr 15. pii: S1570-0232(24)00137-5. [Epub ahead of print]1239 124129

Improved lipid analysis using a 2D-LC-MS system with a novel injection procedure.

József Pánczél, Vilmos Kertesz, Matthias Schiell.

  The aim of this study was to improve analysis of nonpolar lipidomics sample extracts using reversed phase (RP) chromatography. A 4/3/3 (v/v/v) mixture of methanol/methyl tert-butyl ether/chloroform (MeOH/MTBE/CHCl3, MMC) was chosen for sample extraction solvent based on its proven extraction capability for several lipid classes. To avoid carry over, loss of analytes and peak distortion the loops and all capillaries of the presented LC system were flushed and filled up with methanol until the analytical column. The choice of methanol was due to its weak elution strength and being infinitely miscible with MMC and several other nonpolar solvents. This allowed injection of a 100 μl sample that was 20 μl nonpolar extraction solvent diluted fivefold with methanol. All lipids of 25 lipid classes were transferred quantitatively to the column head where the online dilution of methanol was carried out with aqueous eluent for focusing the lipid analytes. The weak elution strength of methanol prevented peak distortions. The consecutive reversed phase elution resulted in remarkably narrow peaks (full width at half maximum was 0.07-0.08 min typically) and enhanced sensitivity (limit of detection usually in sub nM region) because of increased sample injection volume and narrow peaks. Calibration and quality control samples made by diluting commercial lipid standards 200-50000 times confirmed the applicability of this approach both for targeted lipid quantification and for untargeted quantitative comparison of lipids from different sources.

Keywords:  Lipidomics; Mass spectrometry; Reversed phase (RP) chromatography; Signal enhancement

DOI:  https://doi.org/10.1016/j.jchromb.2024.124129
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Apr 16. pii: S1570-0232(24)00132-6. [Epub ahead of print]1239 124124

Applications of chromatographic methods in metabolomics: A review.

Susan T Ovbude, Sadia Sharmeen, Isaac Kyei, Harshana Olupathage, Jacob Jones, Richard J Bell, Robert Powers, David S Hage.

  Chromatography is a robust and reliable separation method that can use various stationary phases to separate complex mixtures commonly seen in metabolomics. This review examines the types of chromatography and stationary phases that have been used in targeted or untargeted metabolomics with methods such as mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. General considerations for sample pretreatment and separations in metabolomics are considered, along with the various supports and separation formats for chromatography that have been used in such work. The types of liquid chromatography (LC) that have been most extensively used in metabolomics will be examined, such as reversed-phase liquid chromatography and hydrophilic liquid interaction chromatography. In addition, other forms of LC that have been used in more limited applications for metabolomics (e.g., ion-exchange, size-exclusion, and affinity methods) will be discussed to illustrate how these techniques may be utilized for new and future research in this field. Multidimensional LC methods are also discussed, as well as the use of gas chromatography and supercritical fluid chromatography in metabolomics. In addition, the roles of chromatography in NMR- vs. MS-based metabolomics are considered. Applications are given within the field of metabolomics for each type of chromatography, along with potential advantages or limitations of these separation methods.

Keywords:  Gas chromatography; Liquid chromatography; Mass spectrometry; Metabolomics; Nuclear magnetic resonance spectroscopy; Supercritical fluid chromatography

DOI:  https://doi.org/10.1016/j.jchromb.2024.124124
Cell. 2024 Apr 08. pii: S0092-8674(24)00315-5. [Epub ahead of print]

Global, site-resolved analysis of ubiquitylation occupancy and turnover rate reveals systems properties.

Gabriela Prus, Shankha Satpathy, Brian T Weinert, Takeo Narita, Chunaram Choudhary.

  Ubiquitylation regulates most proteins and biological processes in a eukaryotic cell. However, the site-specific occupancy (stoichiometry) and turnover rate of ubiquitylation have not been quantified. Here we present an integrated picture of the global ubiquitylation site occupancy and half-life. Ubiquitylation site occupancy spans over four orders of magnitude, but the median ubiquitylation site occupancy is three orders of magnitude lower than that of phosphorylation. The occupancy, turnover rate, and regulation of sites by proteasome inhibitors are strongly interrelated, and these attributes distinguish sites involved in proteasomal degradation and cellular signaling. Sites in structured protein regions exhibit longer half-lives and stronger upregulation by proteasome inhibitors than sites in unstructured regions. Importantly, we discovered a surveillance mechanism that rapidly and site-indiscriminately deubiquitylates all ubiquitin-specific E1 and E2 enzymes, protecting them against accumulation of bystander ubiquitylation. The work provides a systems-scale, quantitative view of ubiquitylation properties and reveals general principles of ubiquitylation-dependent governance.

Keywords:  cell signaling; dynamics; mass spectrometry; occupancy; posttranslational modification; proteomics; turnover rate; ubiquitin signaling; ubiquitylation

DOI:  https://doi.org/10.1016/j.cell.2024.03.024
Mol Cell Proteomics. 2024 Apr 13. pii: S1535-9476(24)00058-6. [Epub ahead of print] 100768

Quantification Quality Control Emerges as A Crucial Factor to Enhance Single Cell Proteomics Data Analysis.

Sung-Huan Yu, Shiau-Ching Chen, Pei-Shan Wu, Pei-I Kuo, Ting-An Chen, Hsiang-Ying Lee, Miao-Hsia Lin.

  Mass spectrometry (MS)-based single-cell proteomics (SCP) provides us the opportunity to unbiasedly explore biological variability within cells without the limitation of antibody availability. This field is rapidly developed with the main focuses on instrument advancement, sample preparation refinement, and signal boosting methods; however, the optimal data processing and analysis are rarely investigated which holds an arduous challenge because of the high proportion of missing values and batch effect. Here, we introduced a quantification quality control to intensify the identification of differentially expressed proteins (DEPs) by considering both within and across SCP data. Combining quantification quality control with isobaric matching between runs (IMBR) and PSM-level normalization, an additional 12% and 19% of proteins and peptides, with more than 90% of proteins/peptides containing valid values, were quantified. Clearly, quantification quality control was able to reduce quantification variations and q-values with the more apparent cell type separations. In addition, we found that PSM-level normalization performed similarly to other protein-level normalizations but kept the original data profiles without the additional requirement of data manipulation. In proof of concept of our refined pipeline, six uniquely identified DEPs exhibiting varied fold-changes and playing critical roles for melanoma and monocyte functionalities were selected for validation using immunoblotting. Five out of six validated DEPs showed an identical trend with the SCP dataset, emphasizing the feasibility of combining the IMBR, cell quality control, and PSM-level normalization in SCP analysis, which is beneficial for future SCP studies.

Keywords:  PSM-level normalization; Single-cell proteomics; differential expression analysis; isobaric labeling; matching between runs

DOI:  https://doi.org/10.1016/j.mcpro.2024.100768
J Biol Chem. 2024 Apr 17. pii: S0021-9258(24)01800-3. [Epub ahead of print] 107299

ABCG2 and SLC1A5 functionally interact to rewire metabolism and confer a survival advantage to cancer cells under oxidative stress.

Jia Shi, Kirk Pabon, Rui Ding, Kathleen W Scotto.

ABCG2, a member of the ABC transporter superfamily, is overexpressed in many human tumors and has long been studied for its ability to export a variety of chemotherapeutic agents, thereby conferring a multidrug resistance (MDR) phenotype. However, several studies have shown that ABCG2 can also confer an MDR-independent survival advantage to tumor cells exposed to stress. While investigating the mechanism by which ABCG2 enhances survival in stressful milieus, we have identified a physical and functional interaction between ABCG2 and SLC1A5, a member of the solute transporter superfamily and the primary transporter of glutamine in cancer cells. This interaction was accompanied by increased glutamine uptake, increased glutaminolysis and rewired cellular metabolism, as evidenced by an increase in key metabolic enzymes and alteration of glutamine-dependent metabolic pathways. Specifically, we observed an increase in glutamine metabolites shuttled to the TCA cycle, an increase in the synthesis of glutathione, accompanied by a decrease in basal levels of reactive oxygen species and a marked increase in cell survival in the face of oxidative stress. Notably, knockdown of SLC1A5 or depletion of exogenous glutamine diminished ABCG2-enhanced autophagy flux, further implicating this solute transporter in ABCG2-mediated cell survival. This is, to our knowledge, the first report of a functionally significant physical interaction between members of the two major transporter superfamilies. Moreover, these observations may underlie the protective role of ABCG2 in cancer cells under duress and suggest a novel role for ABCG2 in the regulation of metabolism in normal and diseased states.

DOI: https://doi.org/10.1016/j.jbc.2024.107299