Document Type : Original Article
Department of Genetic Engineering, School of Bioengineering, SRM Institute of Science and Technology, Kattankulathur, Chengalpattu-603203, India
Department of Paediatric Neurology, Apollo Children’s Hospital, Thousand lights, Chennai-600006, India
Department of Biological Sciences, SRM University – Andrapradesh, 522240, India
School of Bioengineering, SRM Institute of Science and Technology, Kattankulathur, Chengalpattu-603203, India
Introduction: Extracellular vesicles (EVs) are biomolecular messengers secreted by all types of cells. It passes messages through molecular cargoes such as proteins, nucleic acids, and lipids from the parent cell to the target cell. EVs derived from the plasma are used for minimally invasive biopsy and as a possible biomarker to monitor the progression and severity of the disease. In this study, the matrix-assisted laser desorption and ionization-time of flight mass spectrometry approach was used to characterize and study the protein fingerprint region of plasma-derived extracellular vesicles.
Materials and Methods: At first, 3 ml of blood sample was collected from five boys of 5-10 years in sodium citrate tubes, and the samples were centrifuged within an hour to extract plasma. The total exosome isolation (TEI) method was used to obtain high-yield plasma-derived EVs, and the EVs were stored at -80 °C for further analysis. The isolated intact EVs were mixed with an optimal concentration of sinapinic acid matrix (20 mg/ml) in a 1:1 ratio for fingerprint analysis using matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF MS). We found that the following operational conditions yielded good and high-resolution spectra: broad mass range (2000- 20000 m/z), detector gain (30x), and laser shot (50, 300, and 1000) with 100% laser intensity. The obtained MALDI TOF spectral peaks of plasma EVs (pEVs) matched the reported biomarkers.
Results: Based on the analyses, we improved the crucial experimental conditions and identified five distinct peaks at m/z = 3315, 6630, 9421, 8875, and 8917. C4A, C3, and apolipoproteins A-II, C-I, C-II, and C-III were identified by comparing MALDI-TOF MS data with existing reports.
Conclusions: MALDI-TOF MS-based EV analysis can support the development of protein biomarker screening tools for early diagnosis.