Program Official

Principal Investigator

Alexander R.
Awardee Organization

Northeastern University
United States

Fiscal Year
Activity Code
Early Stage Investigator Grants (ESI)
Not Applicable
Project End Date

Effect of methodological and biological variability on molecular profiling of extracellular vesicles in cancer detection

A novel paradigm in paracrine signaling has recently emerged based on the findings identifying extracellular vesicles (EVs) as intercellular conveyors of biological information both in normal and pathological conditions such as cancer. EVs and their cargo have been shown by us and others to regulate gene expression and alter cell function in various cell types. Moreover, during pathological conditions such as cancer, the number and compositions of EVs alter the host immune response as well as synchronize the behavior of secondary tumors. Isolation and molecular profiling of EVs (i.e. RNAs, proteins, post-translational modifications, lipids, metabolites) both in health and disease are critical for understanding EVs' biogenesis and effector functions. Currently, the study of EVs as biological entities relevant for intracellular signaling and disease diagnosis is based on the assumption that the biogenesis and removal of EVs happen at a steady state rate, being modified mostly by the healthy/diseased status of the host. Our published results show that that is not the case. Our data indicate that the tissue-origin, number, size distribution, as well as protein, lipid, metabolite and RNA composition of EVs isolated by standard techniques depend not just on the blood collection methods, but also on the time of the day the blood samples were collected. Therefore, systematic assessment of these factors and other sources of variability in EV profiles are important for enabling basic biology, clinical and personalized medicine applications. Although it is beyond the purpose of this grant, the long-term goal of our team is to establish reproducible methods for blood collection and sample processing that would allow us to identify the specific molecular EV signatures during the day/night cycle, with the aim of pinpointing the ideal, organ-specific times for blood collection, which would increase the reliability and specificity of early cancer detection. We believe that establishing biological and methodological baselines are absolutely vital for correctly comparing proteomics and glycomics data obtained in various studies, as well as interpreting the data obtained from patients’ samples.


  • Kostas JC, Greguš M, Schejbal J, Ray S, Ivanov AR. Simple and Efficient Microsolid-Phase Extraction Tip-Based Sample Preparation Workflow to Enable Sensitive Proteomic Profiling of Limited Samples (200 to 10,000 Cells). Journal of proteome research. 2021 Mar 5;20(3):1676-1688. Epub 2021 Feb 24. PMID: 33625864
  • de Oliveira Junior GP, Welsh JA, Pinckney B, Palu CC, Lu S, Zimmerman A, Barbosa RH, Sahu P, Noshin M, Gummuluru S, Tigges J, Jones JC, Ivanov AR, Ghiran IC. Human red blood cells release microvesicles with distinct sizes and protein composition that alter neutrophil phagocytosis. Journal of extracellular biology. 2023 Nov;2. (11). Epub 2023 Oct 25. PMID: 37942280
  • Marie AL, Ray S, Lu S, Jones J, Ghiran I, Ivanov AR. High-Sensitivity Glycan Profiling of Blood-Derived Immunoglobulin G, Plasma, and Extracellular Vesicle Isolates with Capillary Zone Electrophoresis-Mass Spectrometry. Analytical chemistry. 2021 Feb 2;93(4):1991-2002. Epub 2021 Jan 12. PMID: 33433994
  • Bagwe K, Gould N, Johnson KR, Ivanov AR. Single-cell omic molecular profiling using capillary electrophoresis-mass spectrometry. Trends in analytical chemistry : TRAC. 2023 Aug;165. Epub 2023 Jun 1. PMID: 37388554
  • Johnson KR, Greguš M, Kostas JC, Ivanov AR. Capillary Electrophoresis Coupled to Electrospray Ionization Tandem Mass Spectrometry for Ultra-Sensitive Proteomic Analysis of Limited Samples. Analytical chemistry. 2022 Jan 18;94(2):704-713. Epub 2022 Jan 4. PMID: 34983182
  • Chatterjee S, Yabaji SM, Rukhlenko OS, Bhattacharya B, Waligurski E, Vallavoju N, Ray S, Kholodenko BN, Brown LE, Beeler AB, Ivanov AR, Kobzik L, Porco JA Jr, Kramnik I. Channeling macrophage polarization by rocaglates increases macrophage resistance to Mycobacterium tuberculosis. iScience. 2021 Jul 10;24(8):102845. doi: 10.1016/j.isci.2021.102845. eCollection 2021 Aug 20. PMID: 34381970
  • Gandham S, Su X, Wood J, Nocera AL, Alli SC, Milane L, Zimmerman A, Amiji M, Ivanov AR. Technologies and Standardization in Research on Extracellular Vesicles. Trends in biotechnology. 2020 Oct;38(10):1066-1098. Epub 2020 Jun 18. PMID: 32564882
  • Donnelly DP, Rawlins CM, DeHart CJ, Fornelli L, Schachner LF, Lin Z, Lippens JL, Aluri KC, Sarin R, Chen B, Lantz C, Jung W, Johnson KR, Koller A, Wolff JJ, Campuzano IDG, Auclair JR, Ivanov AR, Whitelegge JP, Paša-Tolić L, Chamot-Rooke J, Danis PO, Smith LM, Tsybin YO, Loo JA, Ge Y, Kelleher NL, Agar JN. Best practices and benchmarks for intact protein analysis for top-down mass spectrometry. Nature methods. 2019 Jul;16(7):587-594. Epub 2019 Jun 27. PMID: 31249407
  • Sarkar S, Kang W, Jiang S, Li K, Ray S, Luther E, Ivanov AR, Fu Y, Konry T. Machine learning-aided quantification of antibody-based cancer immunotherapy by natural killer cells in microfluidic droplets. Lab on a chip. 2020 Jun 30;20(13):2317-2327. PMID: 32458907
  • Greguš M, Ivanov AR, Wilson SR. Ultralow flow liquid chromatography and related approaches: A focus on recent bioanalytical applications. Journal of separation science. 2023 Sep;46(18):e2300440. Epub 2023 Aug 1. PMID: 37528733
  • Johnson KR, Greguš M, Ivanov AR. Coupling High-Field Asymmetric Ion Mobility Spectrometry with Capillary Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry Improves Protein Identifications in Bottom-Up Proteomic Analysis of Low Nanogram Samples. Journal of proteome research. 2022 Oct 7;21(10):2453-2461. Epub 2022 Sep 16. PMID: 36112031
  • Schmitt ND, Rawlins CM, Randall EC, Wang X, Koller A, Auclair JR, Kowalski JM, Kowalski PJ, Luther E, Ivanov AR, Agar NYR, Agar JN. Genetically Encoded Fluorescent Proteins Enable High-Throughput Assignment of Cell Cohorts Directly from MALDI-MS Images. Analytical chemistry. 2019 Mar 19;91(6):3810-3817. Epub 2019 Mar 6. PMID: 30839199
  • Berger Fridman I, Kostas J, Gregus M, Ray S, Sullivan MR, Ivanov AR, Cohen S, Konry T. High-throughput microfluidic 3D biomimetic model enabling quantitative description of the human breast tumor microenvironment. Acta biomaterialia. 2021 Sep 15;132:473-488. Epub 2021 Jun 18. PMID: 34153511
  • Welsh JA, Killingsworth B, Kepley J, Traynor T, McKinnon K, Savage J, Appel D, Aldape K, Camphausen K, Berzofsky JA, Ivanov AR, Ghiran IH, Jones JC. A simple, high-throughput method of protein and label removal from extracellular vesicle samples. Nanoscale. 2021 Feb 18;13(6):3737-3745. PMID: 33544111
  • Welsh JA, Killingsworth B, Kepley J, Traynor T, Cook S, Savage J, Marte J, Lee MJ, Maeng HM, Pleet ML, Magana S, Gorgens A, Maire CL, Lamszus K, Ricklefs FL, Merino MJ, Linehan WM, Greten T, Cooks T, Harris CC, Apolo A, Abdel-Mageed A, Ivanov AR, Trepel JB, Roth M, Tkach M, Milosavljevic A, Théry C, LeBlanc A, Berzofsky JA, Ruppin E, Aldape K, Camphausen K, Gulley JL, Ghiran I, Jacobson S, Jones JC. MPAPASS software enables stitched multiplex, multidimensional EV repertoire analysis and a standard framework for reporting bead-based assays. Cell reports methods. 2022 Jan 24;2(1):100136. doi: 10.1016/j.crmeth.2021.100136. eCollection 2022 Jan 24. PMID: 35474866
  • Marie AL, Ray S, Ivanov AR. Highly-sensitive label-free deep profiling of N-glycans released from biomedically-relevant samples. Nature communications. 2023 Mar 23;14(1):1618. PMID: 36959283
  • Phaneuf CG, Aizikov K, Grinfeld D, Kreutzmann A, Mourad D, Lange O, Dai D, Zhang B, Belenky A, Makarov AA, Ivanov AR. Experimental strategies to improve drug-target identification in mass spectrometry-based thermal stability assays. Communications chemistry. 2023 Apr 6;6(1):64. PMID: 37024568
  • Ali AS, Chen R, Raju R, Kshirsagar R, Gilbert A, Zang L, Karger BL, Ivanov AR. Multi-Omics Reveals Impact of Cysteine Feed Concentration and Resulting Redox Imbalance on Cellular Energy Metabolism and Specific Productivity in CHO Cell Bioprocessing. Biotechnology journal. 2020 Aug;15(8):e1900565. Epub 2020 Apr 3. PMID: 32170810
  • Tošić I, Heppler LN, Egusquiaguirre SP, Boehnke N, Correa S, Costa DF, Moore EAG, Pal S, Richardson DS, Ivanov AR, Haas-Kogan DA, Nomura DK, Hammond PT, Frank DA. Lipidome-based Targeting of STAT3-driven Breast Cancer Cells Using Poly-l-glutamic Acid-coated Layer-by-Layer Nanoparticles. Molecular cancer therapeutics. 2021 Apr;20(4):726-738. Epub 2021 Feb 3. PMID: 33536189
  • Johnson KR, Gao Y, Greguš M, Ivanov AR. On-capillary Cell Lysis Enables Top-down Proteomic Analysis of Single Mammalian Cells by CE-MS/MS. Analytical chemistry. 2022 Oct 18;94(41):14358-14367. Epub 2022 Oct 4. PMID: 36194750
  • Toole EN, Dufresne C, Ray S, Schwann A, Cook K, Ivanov AR. Rapid Highly-Efficient Digestion and Peptide Mapping of Adeno-Associated Viruses. Analytical chemistry. 2021 Aug 3;93(30):10403-10410. Epub 2021 Jul 22. PMID: 34291903
  • Peng Y, Jain S, Li YF, Greguš M, Ivanov AR, Vitek O, Radivojac P. New mixture models for decoy-free false discovery rate estimation in mass spectrometry proteomics. Bioinformatics (Oxford, England). 2020 Dec 30;36(Suppl_2):i745-i753. PMID: 33381824
  • Welsh JA, Van Der Pol E, Arkesteijn GJA, Bremer M, Brisson A, Coumans F, Dignat-George F, Duggan E, Ghiran I, Giebel B, Görgens A, Hendrix A, Lacroix R, Lannigan J, Libregts SFWM, Lozano-Andrés E, Morales-Kastresana A, Robert S, De Rond L, Tertel T, Tigges J, De Wever O, Yan X, Nieuwland R, Wauben MHM, Nolan JP, Jones JC. MIFlowCyt-EV: a framework for standardized reporting of extracellular vesicle flow cytometry experiments. Journal of extracellular vesicles. 2020 Feb 3;9(1):1713526. doi: 10.1080/20013078.2020.1713526. eCollection 2020. PMID: 32128070
  • Toole EN, Dufresne C, Ray S, Schwann AB, Sadiki A, Zhou ZS, Cook K, Ivanov AR. Correction to Rapid Highly-Efficient Digestion and Peptide Mapping of Adeno-Associated Viruses. Analytical chemistry. 2021 Dec 14;93(49):16734. Epub 2021 Dec 1. PMID: 34850630
  • Gatto L, Aebersold R, Cox J, Demichev V, Derks J, Emmott E, Franks AM, Ivanov AR, Kelly RT, Khoury L, Leduc A, MacCoss MJ, Nemes P, Perlman DH, Petelski AA, Rose CM, Schoof EM, Van Eyk J, Vanderaa C, Yates JR 3rd, Slavov N. Initial recommendations for performing, benchmarking and reporting single-cell proteomics experiments. Nature methods. 2023 Mar;20(3):375-386. Epub 2023 Mar 2. PMID: 36864200
  • Belov AM, Zang L, Sebastiano R, Santos MR, Bush DR, Karger BL, Ivanov AR. Complementary middle-down and intact monoclonal antibody proteoform characterization by capillary zone electrophoresis - mass spectrometry. Electrophoresis. 2018 Aug;39(16):2069-2082. Epub 2018 Jun 5. PMID: 29749064
  • Shah A, Cui W, Harrahy J, Ivanov AR. Characterization of charge variants, including post-translational modifications and proteoforms, of bispecific antigen-binding protein by cation-exchange chromatography coupled to native mass spectrometry. Talanta. 2024 Jan 1;266(Pt 1):125062. Epub 2023 Aug 8. PMID: 37566926
  • Greguš M, Kostas JC, Ray S, Abbatiello SE, Ivanov AR. Improved Sensitivity of Ultralow Flow LC-MS-Based Proteomic Profiling of Limited Samples Using Monolithic Capillary Columns and FAIMS Technology. Analytical chemistry. 2020 Nov 3;92(21):14702-14712. Epub 2020 Oct 15. PMID: 33054160
  • MacDonald JA, Bothun AM, Annis SN, Sheehan H, Ray S, Gao Y, Ivanov AR, Khrapko K, Tilly JL, Woods DC. A nanoscale, multi-parametric flow cytometry-based platform to study mitochondrial heterogeneity and mitochondrial DNA dynamics. Communications biology. 2019 Jul 11;2:258. doi: 10.1038/s42003-019-0513-4. eCollection 2019. PMID: 31312727
  • Pleet ML, Cook S, Tang VA, Stack E, Ford VJ, Lannigan J, Do N, Wenger E, Fraikin JL, Jacobson S, Jones JC, Welsh JA. Extracellular Vesicle Refractive Index Derivation Utilizing Orthogonal Characterization. Nano letters. 2023 Oct 25;23(20):9195-9202. Epub 2023 Oct 3. PMID: 37788377
  • Geller S, Lieberman H, Kloss A, Ivanov AR. A systematic approach to development of analytical scale and microflow-based liquid chromatography coupled to mass spectrometry metabolomics methods to support drug discovery and development. Journal of chromatography. A. 2021 Apr 12;1642:462047. Epub 2021 Mar 9. PMID: 33744605
  • Huang T, Staniak M, Veiga Leprevost FD, Figueroa-Navedo AM, Ivanov AR, Nesvizhskii AI, Choi M, Vitek O. Statistical Detection of Differentially Abundant Proteins in Experiments with Repeated Measures Designs and Isobaric Labeling. Journal of proteome research. 2023 Aug 4;22(8):2641-2659. Epub 2023 Jul 19. PMID: 37467362
  • Valkov N, Das A, Tucker NR, Li G, Salvador AM, Chaffin MD, Pereira De Oliveira Junior G, Kur I, Gokulnath P, Ziegler O, Yeri A, Lu S, Khamesra A, Xiao C, Rodosthenous R, Srinivasan S, Toxavidis V, Tigges J, Laurent LC, Momma S, Kitchen R, Ellinor P, Ghiran I, Das S. SnRNA sequencing defines signaling by RBC-derived extracellular vesicles in the murine heart. Life science alliance. 2021 Oct 18;4. (12). Print 2021 Dec. PMID: 34663679
  • Oliveira GP Jr, Zigon E, Rogers G, Davodian D, Lu S, Jovanovic-Talisman T, Jones J, Tigges J, Tyagi S, Ghiran IC. Detection of Extracellular Vesicle RNA Using Molecular Beacons. iScience. 2020 Jan 24;23(1):100782. Epub 2019 Dec 19. PMID: 31958756
  • Thompson L, Pinckney B, Lu S, Gregory M, Tigges J, Ghiran I. Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation. Journal of visualized experiments : JoVE. 2021 May 17;(171). PMID: 34057455
  • Li J, Salvador AM, Li G, Valkov N, Ziegler O, Yeri A, Yang Xiao C, Meechoovet B, Alsop E, Rodosthenous RS, Kundu P, Huan T, Levy D, Tigges J, Pico AR, Ghiran I, Silverman MG, Meng X, Kitchen R, Xu J, Van Keuren-Jensen K, Shah R, Xiao J, Das S. Mir-30d Regulates Cardiac Remodeling by Intracellular and Paracrine Signaling. Circulation research. 2021 Jan 8;128(1):e1-e23. Epub 2020 Oct 22. PMID: 33092465
  • Geller S, Lieberman H, Belanger AJ, Yew NS, Kloss A, Ivanov AR. Comparison of Microflow and Analytical Flow Liquid Chromatography Coupled to Mass Spectrometry Global Metabolomics Methods Using a Urea Cycle Disorder Mouse Model. Journal of proteome research. 2022 Jan 7;21(1):151-163. Epub 2021 Nov 29. PMID: 34843255
  • Oliveira-Jr GP, Barbosa RH, Thompson L, Pinckney B, Murphy-Thornley M, Lu S, Jones J, Hansen CH, Tigges J, Wong WP, Ghiran IC. Electrophoretic mobility shift as a molecular beacon-based readout for miRNA detection. Biosensors & bioelectronics. 2021 Oct 1;189:113307. Epub 2021 May 15. PMID: 34062334
  • Shah A, Desai R, Cui W, Harrahy JJ, Ivanov AR. Characterization of bispecific antigen-binding biotherapeutic fragmentation sites using microfluidic capillary electrophoresis coupled to mass spectrometry (mCZE-MS). The Analyst. 2023 Jan 31;148(3):665-674. PMID: 36625279
  • Ivanov A, Marie AL, Gao Y. In-capillary sample processing coupled to label-free capillary electrophoresis-mass spectrometry to decipher the native N-glycome of single mammalian cells and ng-level blood isolates. Research square. 2023 Nov 14. PMID: 38014012
  • Marie AL, Georgescauld F, Johnson KR, Ray S, Engen JR, Ivanov AR. Native Capillary Electrophoresis-Mass Spectrometry of Near 1 MDa Non-Covalent GroEL/GroES/Substrate Protein Complexes. Advanced science (Weinheim, Baden-Wurttemberg, Germany). 2024 Jan 8: e2306824. Epub 2024 Jan 8. PMID: 38191978