bims-mascan 2023-04-23 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2023–04–23
sixteen papers selected by
Giovanny Rodriguez Blanco, University of Edinburgh

Increasing Proteome Depth While Maintaining Quantitative Precision in Short-Gradient Data-Independent Acquisition Proteomics.
Isolation and Mass Spectrometry-Based Profiling of Major Lipids in Brown Adipose Tissue.
Quantification of bulk lipid species in human platelets and their thrombin-induced release.
Structural elucidation of 3-nitrophenylhydrazine derivatives of tricarboxylic acid cycle acids and optimization of their fragmentation to boost sensitivity in liquid chromatography-mass spectrometry.
Uncoupled glycerol-3-phosphate shuttle in kidney cancer reveals that cytosolic GPD is essential to support lipid synthesis.
Non-alcoholic Fatty Liver Disease is characterised by a reduced polyunsaturated fatty acid transport via free fatty acids and high-density lipoproteins (HDL).
Pharmacological induction of membrane lipid poly-unsaturation sensitizes melanoma to ROS inducers and overcomes acquired resistance to targeted therapy.
Atherosclerosis and lipid metabolism.
Ether phospholipids are required for mitochondrial reactive oxygen species homeostasis.
Comparison of chromatographic conditions for the targeted tandem mass spectrometric determination of 354 mammalian metabolites.
Efficient Sample Preparation System for Multi-Omics Analysis via Single Cell Mass Spectrometry.
LipidOz enables automated elucidation of lipid carbon-carbon double bond positions from ozone-induced dissociation mass spectrometry data.
Proteomic signature associated with chronic kidney disease (CKD) progression identified by data-independent acquisition mass spectrometry.
Depletion of abundant plasma proteins for extracellular vesicle proteome characterization: benefits and pitfalls.
The proteomic landscape of genome-wide genetic perturbations.
The identification of a potential plasma metabolite marker for Alzheimer's disease by LC-MS untargeted metabolomics.

J Proteome Res. 2023 Apr 18.

Increasing Proteome Depth While Maintaining Quantitative Precision in Short-Gradient Data-Independent Acquisition Proteomics.

Joerg Doellinger, Christian Blumenscheit, Andy Schneider, Peter Lasch.

  The combination of short liquid chromatography (LC) gradients and data-independent acquisition (DIA) by mass spectrometry (MS) has proven its huge potential for high-throughput proteomics. However, the optimization of isolation window schemes resulting in a certain number of data points per peak (DPPP) is understudied, although it is one of the most important parameters for the outcome of this methodology. In this study, we show that substantially reducing the number of DPPP for short-gradient DIA massively increases protein identifications while maintaining quantitative precision. This is due to a large increase in the number of precursors identified, which keeps the number of data points per protein almost constant even at long cycle times. When proteins are inferred from its precursors, quantitative precision is maintained at low DPPP while greatly increasing proteomic depth. This strategy enabled us to quantify 6018 HeLa proteins (>80 000 precursor identifications) with coefficients of variation below 20% in 30 min using a Q Exactive HF, which corresponds to a throughput of 29 samples per day. This indicates that the potential of high-throughput DIA-MS has not been fully exploited yet. Data are available via ProteomeXchange with identifier PXD036451.

Keywords:  SPEED; data points per peak; data-independent acquisition; isolation window; predicted spectral library; quantitative proteomics

DOI:  https://doi.org/10.1021/acs.jproteome.3c00078
Methods Mol Biol. 2023 ;2662 219-239

Isolation and Mass Spectrometry-Based Profiling of Major Lipids in Brown Adipose Tissue.

Dongliang Lu, Hideji Fujiwara, Irfan J Lodhi, Fong-Fu Hsu.

  Brown adipose tissue (BAT) is an important regulator of metabolic homeostasis through its role in adaptive thermogenesis and control of whole-body glucose metabolism. Lipids play multiple roles in BAT functions, including serving as a fuel source for thermogenesis, mediating inter-organelle cross talk, and acting as BAT-derived signaling molecules that influence systemic energy metabolism. Profiling of various lipids in BAT under distinct metabolic states could provide new insights into their roles in the biology of the thermogenic fat. In this chapter, we describe a step-by-step workflow starting from sample preparations to mass spectrometry-based analysis of fatty acids and phospholipids in BAT.

Keywords:  Brown adipose tissue; Folch method; Free fatty acid; Mass spectrometry; Phospholipid; Solid phase extraction; Sphingolipid

DOI:  https://doi.org/10.1007/978-1-0716-3167-6_20
Sci Rep. 2023 Apr 15. 13(1): 6154

Quantification of bulk lipid species in human platelets and their thrombin-induced release.

Susanne Heimerl, Marcus Höring, Dominik Kopczynski, Alexander Sigruener, Christina Hart, Ralph Burkhardt, Anne Black, Robert Ahrends, Gerhard Liebisch.

Lipids play a central role in platelet physiology. Changes in the lipidome have already been described for basal and activated platelets. However, quantitative lipidomic data of platelet activation, including the released complex lipids, are unavailable. Here we describe an easy-to-use protocol based on flow-injection mass spectrometry for the quantitative analysis of bulk lipid species in basal and activated human platelets and their lipid release after thrombin activation. We provide lipid species concentrations of 12 healthy human donors, including cholesteryl ester (CE), ceramide (Cer), free cholesterol (FC), hexosylceramide (HexCer), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SM) and triglycerides (TG). The assay exhibited good technical repeatability (CVs < 5% for major lipid species in platelets). Except for CE and TG, the inter-donor variability of the majority of lipid species concentrations in platelets was < 30% CV. Balancing of concentrations revealed the generation of LPC and loss of TG. Changes in lipid species concentrations indicate phospholipase-mediated release of arachidonic acid mainly from PC, PI, and PE but not from PS. Thrombin induced lipid release was mainly composed of FC, PS, PC, LPC, CE, and TG. The similarity of the released lipidome with that of plasma implicates that lipid release may originate from the open-canalicular system (OCS). The repository of lipid species concentrations determined with this standardized platelet release assay contribute to elucidating the physiological role of platelet lipids and provide a basis for investigating the platelet lipidome in patients with hemorrhagic or thrombotic disorders.

DOI: https://doi.org/10.1038/s41598-023-33076-4
J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Apr 11. pii: S1570-0232(23)00129-0. [Epub ahead of print]1222 123719

Structural elucidation of 3-nitrophenylhydrazine derivatives of tricarboxylic acid cycle acids and optimization of their fragmentation to boost sensitivity in liquid chromatography-mass spectrometry.

Ondřej Hodek, John Henderson, Lidia Argemi-Muntadas, Adnan Khan, Thomas Moritz.

  Carboxylic acids participate in many metabolic pathways including tricarboxylic acid (TCA) cycle. Therefore, there have been ongoing attempts to develop sensitive liquid chromatography-mass spectrometry methods over the last decades. Derivatization of the carboxylic acids with 3-nitrophenylhydrazine presents a well-established methodology, and yet the derivatized species of polycarboxylic acids and their fragmentation in collision-induced dissociation have not been fully studied before. In our study, we elucidated how annotation of most abundant 3-nitrophenylhydrazine derivatives and optimization of their fragmentation in multiple reaction monitoring can boost the sensitivity, especially for polycarboxylic acids. Finally, the optimized liquid chromatography-tandem mass spectrometry method allowed for low detection limits ranging from 10 pM for 2-oxoglutaric acid to 800 pM for pyruvic acid. All TCA carboxylates were quantified in 20 µL of human plasma and the targeted method was validated in the same matrix. The same methodology with a modified gradient elution was also applied to untargeted screening of fatty acids by using high-resolution mass spectrometry enabling identification of 29 medium- to long-chain fatty acids in human plasma. The TCA carboxylates were also quantified in 105 of C2C12 mouse myuotube cells grown under different treatments to proof applicability of the methodology to biological studies in a wider sense. However, unfortunately all the TCA carboxylates were also found in the derivatized blanks in substantial amounts, which prevents from using the methodology for quantification of the carboxylates in less than 105 cells.

Keywords:  3-nitrophenylhydrazine; Carboxylic acid; Derivatization; Liquid chromatography-mass spectrometry

DOI:  https://doi.org/10.1016/j.jchromb.2023.123719
Mol Cell. 2023 Apr 20. pii: S1097-2765(23)00213-7. [Epub ahead of print]83(8): 1340-1349.e7

Uncoupled glycerol-3-phosphate shuttle in kidney cancer reveals that cytosolic GPD is essential to support lipid synthesis.

Cong-Hui Yao, Joon Seok Park, Kiran Kurmi, Song-Hua Hu, Giulia Notarangelo, Joseph Crowley, Heidi Jacobson, Sheng Hui, Arlene H Sharpe, Marcia C Haigis.

  The glycerol-3-phosphate shuttle (G3PS) is a major NADH shuttle that regenerates reducing equivalents in the cytosol and produces energy in the mitochondria. Here, we demonstrate that G3PS is uncoupled in kidney cancer cells where the cytosolic reaction is ∼4.5 times faster than the mitochondrial reaction. The high flux through cytosolic glycerol-3-phosphate dehydrogenase (GPD) is required to maintain redox balance and support lipid synthesis. Interestingly, inhibition of G3PS by knocking down mitochondrial GPD (GPD2) has no effect on mitochondrial respiration. Instead, loss of GPD2 upregulates cytosolic GPD on a transcriptional level and promotes cancer cell proliferation by increasing glycerol-3-phosphate supply. The proliferative advantage of GPD2 knockdown tumor can be abolished by pharmacologic inhibition of lipid synthesis. Taken together, our results suggest that G3PS is not required to run as an intact NADH shuttle but is instead truncated to support complex lipid synthesis in kidney cancer.

Keywords:  GPD; NAD; glycerol; glycerol-3-phosphate dehydrogenase; glycerol-3-phosphate shuttle; kidney cancer; lipids; metabolism; mitochondria

DOI:  https://doi.org/10.1016/j.molcel.2023.03.023
Mol Metab. 2023 Apr 19. pii: S2212-8778(23)00062-5. [Epub ahead of print] 101728

Non-alcoholic Fatty Liver Disease is characterised by a reduced polyunsaturated fatty acid transport via free fatty acids and high-density lipoproteins (HDL).

Gabriele Mocciaro, Michael Allison, Benjamin Jenkins, Vian Azzu, Isabel Huang-Doran, Luis Vicente Herrera-Marcos, Zoe Hall, Antonio Murgia, Davies Susan, Mattia Frontini, Antonio Vidal-Puig, Albert Koulman, Julian L Griffin, Michele Vacca.

   BACKGROUND AND OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) develops due to impaired hepatic lipid fluxes and is a risk factor for chronic liver disease and atherosclerosis. Lipidomic studies consistently reported characteristic hepatic/VLDL "lipid signatures" in NAFLD; whole plasma traits are more debated. Surprisingly, the HDL lipid composition by mass spectrometry has not been characterised across the NAFLD spectrum, despite HDL being a possible source of hepatic lipids delivered from peripheral tissues alongside free fatty acids (FFA). This study characterises the HDL lipidomic signature in NAFLD, and its correlation with metabolic and liver disease markers.
METHODS: We used liquid chromatography-mass spectrometry to determine the whole serum and HDL lipidomic profile in 89 biopsy-proven NAFLD patients and 20 sex and age-matched controls.
RESULTS: In the whole serum of NAFLD versus controls, we report a depletion in polyunsaturated (PUFA) phospholipids (PL) and FFA; with PUFA PL being also lower in HDL, and negatively correlated with BMI, insulin resistance, triglycerides, and hepatocyte ballooning. In the HDL of the NAFLD group we also describe higher saturated ceramides, which positively correlate with insulin resistance and transaminases.
CONCLUSION: NAFLD features lower serum lipid species containing polyunsaturated fatty acids; the most affected lipid fractions are FFA and (HDL) phospholipids; our data suggest a possible defect in the transfer of PUFA from peripheral tissues to the liver in NAFLD. Mechanistic studies are required to explore the biological implications of our findings addressing if HDL composition can influence liver composition and damage, thus contributing to NAFLD pathophysiology.

Keywords:  LC-MS; Non-alcoholic fatty liver disease (NAFLD); lipidomics; lipoprotein metabolism; obesity

DOI:  https://doi.org/10.1016/j.molmet.2023.101728
J Exp Clin Cancer Res. 2023 Apr 19. 42(1): 92

Pharmacological induction of membrane lipid poly-unsaturation sensitizes melanoma to ROS inducers and overcomes acquired resistance to targeted therapy.

Ali Talebi, Vincent de Laat, Xander Spotbeen, Jonas Dehairs, Florian Rambow, Aljosja Rogiers, Frank Vanderhoydonc, Lara Rizotto, Mélanie Planque, Ginevra Doglioni, Sahar Motamedi, David Nittner, Tania Roskams, Patrizia Agostinis, Oliver Bechter, Veerle Boecxstaens, Marjan Garmyn, Marie O'Farrell, Alan Wagman, George Kemble, Eleonora Leucci, Sarah-Maria Fendt, Jean-Christophe Marine, Johannes V Swinnen.

   BACKGROUND: One of the key limitations of targeted cancer therapies is the rapid onset of therapy resistance. Taking BRAF-mutant melanoma as paradigm, we previously identified the lipogenic regulator SREBP-1 as a central mediator of resistance to MAPK-targeted therapy. Reasoning that lipogenesis-mediated alterations in membrane lipid poly-unsaturation lie at the basis of therapy resistance, we targeted fatty acid synthase (FASN) as key player in this pathway to evoke an exquisite vulnerability to clinical inducers of reactive oxygen species (ROS), thereby rationalizing a novel clinically actionable combination therapy to overcome therapy resistance.
METHODS: Using gene expression analysis and mass spectrometry-based lipidomics of BRAF-mutant melanoma cell lines, melanoma PDX and clinical data sets, we explored the association of FASN expression with membrane lipid poly-unsaturation and therapy-resistance. Next, we treated therapy-resistant models with a preclinical FASN inhibitor TVB-3664 and a panel of ROS inducers and performed ROS analysis, lipid peroxidation tests and real-time cell proliferation assays. Finally, we explored the combination of MAPK inhibitors, TVB-3664 and arsenic trioxide (ATO, as a clinically used ROS-inducer) in Mel006 BRAF mutant PDX as a gold model of therapy resistance and assessed the effect on tumor growth, survival and systemic toxicity.
RESULTS: We found that FASN expression is consistently increased upon the onset of therapy resistance in clinical melanoma samples, in cell lines and in Mel006 PDX and is associated with decreased lipid poly-unsaturation. Forcing lipid poly-unsaturation in therapy-resistant models by combining MAPK inhibition with FASN inhibition attenuated cell proliferation and rendered cells exquisitely sensitive to a host of ROS inducers. In particular, the triple combination of MAPK inhibition, FASN inhibition, and the clinical ROS-inducing compound ATO dramatically increased survival of Mel006 PDX models from 15 to 72% with no associated signs of toxicity.
CONCLUSIONS: We conclude that under MAPK inhibition the direct pharmacological inhibition of FASN evokes an exquisite vulnerability to inducers of ROS by increasing membrane lipid poly-unsaturation. The exploitation of this vulnerability by combining MAPK and/or FASN inhibitors with inducers of ROS greatly delays the onset of therapy resistance and increases survival. Our work identifies a clinically actionable combinatorial treatment for therapy-resistant cancer.

Keywords:  Lipid metabolism; Melanoma; Therapy resistance

DOI:  https://doi.org/10.1186/s13046-023-02664-7
Clin Chem Lab Med. 2023 May 25. 61(s1): s359-s399

Atherosclerosis and lipid metabolism.

DOI: https://doi.org/10.1515/cclm-2023-7038
Nat Commun. 2023 04 17. 14(1): 2194

Ether phospholipids are required for mitochondrial reactive oxygen species homeostasis.

Ziheng Chen, I-Lin Ho, Melinda Soeung, Er-Yen Yen, Jintan Liu, Liang Yan, Johnathon L Rose, Sanjana Srinivasan, Shan Jiang, Q Edward Chang, Ningping Feng, Jason P Gay, Qi Wang, Jing Wang, Philip L Lorenzi, Lucas J Veillon, Bo Wei, John N Weinstein, Angela K Deem, Sisi Gao, Giannicola Genovese, Andrea Viale, Wantong Yao, Costas A Lyssiotis, Joseph R Marszalek, Giulio F Draetta, Haoqiang Ying.

Mitochondria are hubs where bioenergetics, redox homeostasis, and anabolic metabolism pathways integrate through a tightly coordinated flux of metabolites. The contributions of mitochondrial metabolism to tumor growth and therapy resistance are evident, but drugs targeting mitochondrial metabolism have repeatedly failed in the clinic. Our study in pancreatic ductal adenocarcinoma (PDAC) finds that cellular and mitochondrial lipid composition influence cancer cell sensitivity to pharmacological inhibition of electron transport chain complex I. Profiling of patient-derived PDAC models revealed that monounsaturated fatty acids (MUFAs) and MUFA-linked ether phospholipids play a critical role in maintaining ROS homeostasis. We show that ether phospholipids support mitochondrial supercomplex assembly and ROS production; accordingly, blocking de novo ether phospholipid biosynthesis sensitized PDAC cells to complex I inhibition by inducing mitochondrial ROS and lipid peroxidation. These data identify ether phospholipids as a regulator of mitochondrial redox control that contributes to the sensitivity of PDAC cells to complex I inhibition.

DOI: https://doi.org/10.1038/s41467-023-37924-9
J Chromatogr A. 2023 Apr 09. pii: S0021-9673(23)00211-X. [Epub ahead of print]1697 463985

Comparison of chromatographic conditions for the targeted tandem mass spectrometric determination of 354 mammalian metabolites.

Dimitrios J Floros, Kangkang Xu, Franz Berthiller, Heidi Schwartz-Zimmermann.

  Metabolomics is becoming increasingly popular in livestock research, but no single analytical method can cover the entire metabolome. As such, we compared similar and complementary chromatographic methods with respect to analyte coverage and chromatographic properties of mammalian metabolites. We investigated 354 biologically relevant primary metabolites from 19 compound classes including amino acids, bile acids, biogenic amines, carboxylic acids, lipids, nucleotides and sugars. A total of 2063 selected reaction monitoring transitions were optimized on a triple quadrupole mass spectrometer. We then determined the retention profiles and peak parameters of our compounds using an anion exchange chromatography (AIC), three reversed-phase (RP) and three hydrophilic interaction liquid chromatography (HILIC) methods. On average, HILIC methods covered 54% of all metabolites with retention factors >1, while average RP coverage was 41%. In contrast to RP, HILIC methods could also retain polar metabolites such as amino acids and biogenic amines. Carboxylic acids, nucleotides, and sugar related compounds were best separated by AIC or zwitterionic pHILIC with alkaline eluents. Combining two complementary HILIC and RP methods increased the library coverage to 92%. By further including important short chain fatty acids, a combination of HILIC, RP and AIC methods achieved a coverage of 97%. The resulting dataset of LC and MS/MS parameters will facilitate the development of tailor-made quantitative targeted LC-MS/MS methods to investigate the mammalian metabolome.

Keywords:  Anion-exchange chromatography; HILIC; LC-MS/MS; Metabolomics; Reversed phase chromatography

DOI:  https://doi.org/10.1016/j.chroma.2023.463985
Anal Chem. 2023 Apr 20.

Efficient Sample Preparation System for Multi-Omics Analysis via Single Cell Mass Spectrometry.

Peng Zhao, Yongxiang Feng, Junhan Wu, Junwen Zhu, Jinlei Yang, Xiaoxiao Ma, Zheng Ouyang, Xinrong Zhang, Wenpeng Zhang, Wenhui Wang.

Mass spectrometry (MS) has become a powerful tool for metabolome, lipidome, and proteome analyses. The efficient analysis of multi-omics in single cells, however, is still challenging in the manipulation of single cells and lack of in-fly cellular digestion and extraction approaches. Here, we present a streamlined strategy for highly efficient and automatic single-cell multi-omics analysis by MS. We developed a 10-pL-level microwell chip for housing individual single cells, whose proteins were found to be digested in 5 min, which is 144 times shorter than traditional bulk digestion. Besides, an automated picoliter extraction system was developed for sampling of metabolites, phospholipids, and proteins in tandem from the same single cell. Also, 2 min MS2 spectra were obtained from 700 pL solution of a single cell sample. In addition, 1391 proteins, phospholipids, and metabolites were detected from one single cell within 10 min. We further analyzed cells digested from cancer tissue samples, achieving up to 40% increase in cell classification accuracy using multi-omics analysis in comparison with single-omics analysis. This automated single-cell MS strategy is highly efficient in analyzing multi-omics information for investigation of cell heterogeneity and phenotyping for biomedical applications.

DOI: https://doi.org/10.1021/acs.analchem.2c05728
Commun Chem. 2023 Apr 19. 6(1): 74

LipidOz enables automated elucidation of lipid carbon-carbon double bond positions from ozone-induced dissociation mass spectrometry data.

Dylan H Ross, Joon-Yong Lee, Aivett Bilbao, Daniel J Orton, Josie G Eder, Meagan C Burnet, Brooke L Deatherage Kaiser, Jennifer E Kyle, Xueyun Zheng.

Lipids play essential roles in many biological processes and disease pathology, but unambiguous identification of lipids is complicated by the presence of multiple isomeric species differing by fatty acyl chain length, stereospecifically numbered (sn) position, and position/stereochemistry of double bonds. Conventional liquid chromatography-mass spectrometry (LC-MS/MS) analyses enable the determination of fatty acyl chain lengths (and in some cases sn position) and number of double bonds, but not carbon-carbon double bond positions. Ozone-induced dissociation (OzID) is a gas-phase oxidation reaction that produces characteristic fragments from lipids containing double bonds. OzID can be incorporated into ion mobility spectrometry (IMS)-MS instruments for the structural characterization of lipids, including additional isomer separation and confident assignment of double bond positions. The complexity and repetitive nature of OzID data analysis and lack of software tool support have limited the application of OzID for routine lipidomics studies. Here, we present an open-source Python tool, LipidOz, for the automated determination of lipid double bond positions from OzID-IMS-MS data, which employs a combination of traditional automation and deep learning approaches. Our results demonstrate the ability of LipidOz to robustly assign double bond positions for lipid standard mixtures and complex lipid extracts, enabling practical application of OzID for future lipidomics.

DOI: https://doi.org/10.1038/s42004-023-00867-9
Clin Proteomics. 2023 Apr 20. 20(1): 19

Proteomic signature associated with chronic kidney disease (CKD) progression identified by data-independent acquisition mass spectrometry.

Carlos R Ramírez Medina, Ibrahim Ali, Ivona Baricevic-Jones, Aghogho Odudu, Moin A Saleem, Anthony D Whetton, Philip A Kalra, Nophar Geifman.

   BACKGROUND: Halting progression of chronic kidney disease (CKD) to established end stage kidney disease is a major goal of global health research. The mechanism of CKD progression involves pro-inflammatory, pro-fibrotic, and vascular pathways, but pathophysiological differentiation is currently lacking.
METHODS: Plasma samples of 414 non-dialysis CKD patients, 170 fast progressors (with ∂ eGFR-3 ml/min/1.73 m2/year or worse) and 244 stable patients (∂ eGFR of - 0.5 to + 1 ml/min/1.73 m2/year) with a broad range of kidney disease aetiologies, were obtained and interrogated for proteomic signals with SWATH-MS. We applied a machine learning approach to feature selection of proteins quantifiable in at least 20% of the samples, using the Boruta algorithm. Biological pathways enriched by these proteins were identified using ClueGo pathway analyses.
RESULTS: The resulting digitised proteomic maps inclusive of 626 proteins were investigated in tandem with available clinical data to identify biomarkers of progression. The machine learning model using Boruta Feature Selection identified 25 biomarkers as being important to progression type classification (Area Under the Curve = 0.81, Accuracy = 0.72). Our functional enrichment analysis revealed associations with the complement cascade pathway, which is relevant to CKD as the kidney is particularly vulnerable to complement overactivation. This provides further evidence to target complement inhibition as a potential approach to modulating the progression of diabetic nephropathy. Proteins involved in the ubiquitin-proteasome pathway, a crucial protein degradation system, were also found to be significantly enriched.
CONCLUSIONS: The in-depth proteomic characterisation of this large-scale CKD cohort is a step toward generating mechanism-based hypotheses that might lend themselves to future drug targeting. Candidate biomarkers will be validated in samples from selected patients in other large non-dialysis CKD cohorts using a targeted mass spectrometric analysis.

Keywords:  Biomarkers; Chronic kidney disease (CKD) Progression; Complement cascade pathway; Proteasome pathway; Proteomics; SWATH-MS

DOI:  https://doi.org/10.1186/s12014-023-09405-0
Anal Bioanal Chem. 2023 Apr 18.

Depletion of abundant plasma proteins for extracellular vesicle proteome characterization: benefits and pitfalls.

Sandrine Reymond, Lyssia Gruaz, Jean-Charles Sanchez.

  Blood extracellular vesicles (EVs) play essential roles in cell-cell communication and their molecular cargo is a promising source of disease biomarkers. However, proteomic characterization of plasma-derived EVs is challenged by the presence of highly abundant plasma proteins, which limits the detection of less abundant proteins, and by the low number of EVs in biological fluids. The aim of this study was to investigate if the removal of abundant plasma proteins prior to EV isolation could improve plasma-derived EV characterization by LC-MS/MS and expand the proteome coverage. Plasma depletion was performed using a single-use spin column and EVs were isolated from only 100 µL of non-depleted and depleted plasma by size exclusion chromatography. Afterwards, EVs were characterized by nanoparticle tracking analysis and mass spectrometry-based proteomics using a data-independent acquisition approach. Depleted plasma-derived EVs had higher particle concentrations and particle-to-protein ratios. Depletion did increase the protein coverage with a higher number of identifications in EVs from depleted plasma (474 proteins) than from non-depleted (386 proteins). However, EVs derived from non-depleted plasma carried a slightly higher number of common EV markers. Overall, our findings suggest that plasma depletion prior to EV isolation by size exclusion chromatography provides higher yield and protein coverage, but slightly lower identification of EV markers. This study also showed the possibility to characterize the proteome of EVs derived from small plasma volumes, encouraging the clinical feasibility of the discovery of EV biomarkers.

Keywords:  Extracellular vesicles; Plasma depletion; Proteomics; Size exclusion chromatography

DOI:  https://doi.org/10.1007/s00216-023-04684-w
Cell. 2023 Apr 17. pii: S0092-8674(23)00300-8. [Epub ahead of print]

The proteomic landscape of genome-wide genetic perturbations.

Christoph B Messner, Vadim Demichev, Julia Muenzner, Simran K Aulakh, Natalie Barthel, Annika Röhl, Lucía Herrera-Domínguez, Anna-Sophia Egger, Stephan Kamrad, Jing Hou, Guihong Tan, Oliver Lemke, Enrica Calvani, Lukasz Szyrwiel, Michael Mülleder, Kathryn S Lilley, Charles Boone, Georg Kustatscher, Markus Ralser.

  Functional genomic strategies have become fundamental for annotating gene function and regulatory networks. Here, we combined functional genomics with proteomics by quantifying protein abundances in a genome-scale knockout library in Saccharomyces cerevisiae, using data-independent acquisition mass spectrometry. We find that global protein expression is driven by a complex interplay of (1) general biological properties, including translation rate, protein turnover, the formation of protein complexes, growth rate, and genome architecture, followed by (2) functional properties, such as the connectivity of a protein in genetic, metabolic, and physical interaction networks. Moreover, we show that functional proteomics complements current gene annotation strategies through the assessment of proteome profile similarity, protein covariation, and reverse proteome profiling. Thus, our study reveals principles that govern protein expression and provides a genome-spanning resource for functional annotation.

Keywords:  Saccharomyces cerevisiae; data-independent acquisition; deletion; functional genomics; functional proteomics; gene annotation; high throughput; knockout; quantitative proteomics; systems biology

DOI:  https://doi.org/10.1016/j.cell.2023.03.026
J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Mar 30. pii: S1570-0232(23)00096-X. [Epub ahead of print]1222 123686

The identification of a potential plasma metabolite marker for Alzheimer's disease by LC-MS untargeted metabolomics.

Chieh-Hsin Lin, Yu-Ning Lin, Hsien-Yuan Lane, Chao-Jung Chen.

   BACKGROUND AND AIMS: Alzheimer's disease (AD), the most common type of dementia, is hard to recognize early, resulting in delayed treatment and poor outcome. At present, there is neither reliable, non-invasive methods to diagnose it accurately and nor effective drugs to recover it. Discovery and quantification of novel metabolite markers in plasma of AD patients and investigation of the correlation between the markers and AD assessment scores.
MATERIALS AND METHODS: Untargeted liquid chromatography-mass spectrometry (LC-MS)-based metabolomics with LC-quadrupole- time-of-flight (Q-TOF) was performed in plasma samples of age-matched AD patients and healthy controls. The potential markers were further quantified with targeted multiple reaction monitoring (MRM) approach.
RESULTS: Among the candidates, progesterone, and 3-indoleacetic acid (3-IAA) were successfully identified and then validated in 50 plasma samples from 25 AD patients and 25 matched normal controls with MRM approach. As a result, 3-IAA was significantly altered in AD patients and correlated with some AD assessment scores.
CONCLUSION: By using untargeted LC-MS metabolomic and LC-MRM approaches to analyze plasma metabolites of AD patients and normal subjects, 3-IAA was discovered and quantified to be significantly altered in AD patients and correlated with several AD assessment scores.

Keywords:  Alzheimer’s disease; Biomarkers; Blood; Diagnosis; Metabolite; Plasma

DOI:  https://doi.org/10.1016/j.jchromb.2023.123686