L M Andrade1, M A Mendes1, P Kowalski2, C A O Nascimento1. 1. Chemical Engineering Department of Polytechnic School of University of São Paulo, Av Prof Lineu Prestes 580, Bloco 21, térreo, 05508-900, São Paulo, SP, Brazil. 2. Bruker Daltonics, 40 Manning Road, Billerica, MA, 01821, USA.
Abstract
RATIONALE: Due to increases in greenhouse gas emissions, it is necessary to explore renewable sources of energy. Interesting alternatives are biofuels derived from microalgae. One challenge is the development of a detailed microalgae database compiling species identifications and characterizations that would facilitate microalgae selection for biomass production. Mass spectrometric (MS) analysis using a matrix-assisted laser desorption/ionization (MALDI) source is an advanced technique that enables advancement in this biological area. In this work a MALDI time-of-flight (TOF)MS method for the rapid identification of proteins in whole cells of selected microalgae species was studied. Furthermore, the efficiency of different matrix and solvent systems was tested. MS analyses were performed using an UltrafleXtreme MALDI-TOF mass spectrometer operating in linear positive ion mode. METHODS: Mass spectra were acquired in a mass range from 4000 to 20,000 Da with ions generated from Smartbeam laser irradiation using a frequency of 2000 Hz, a PIE 100 ns and a lens 7 kV. The voltage was 25 kV for the first ion source and 23 kV for the second. Each spectrum was generated by averaging of 10,000 laser shots and the laser irradiance was set at 95-100%. RESULTS: Similar mass spectra were obtained for all matrices (SA, HCCA, DHB and sDHB); however, the use of the sDHB matrix resulted in spectrum profiles with a greater amount number of proteins, a better signal/noise (S/N) ratio and higher intensities for the majority of microalgae analyzed. Trifluoroacetic acid (TFA) content was also studied and the best results in terms of S/N ratio, number of proteins and signal intensities were obtained with 0.1% TFA in the matrix solvent. The addition of isopropanol did not produce improvement in the quality of spectrum profiles. CONCLUSIONS: Therefore, the optimal matrix for the analysis of protein from intact microalgae cells is sDHB with TA50 as the matrix solvent and without isopropanol. These conditions allow the acquisition of high quality spectrum profiles.
RATIONALE: Due to increases in greenhouse gas emissions, it is necessary to explore renewable sources of energy. Interesting alternatives are biofuels derived from microalgae. One challenge is the development of a detailed microalgae database compiling species identifications and characterizations that would facilitate microalgae selection for biomass production. Mass spectrometric (MS) analysis using a matrix-assisted laser desorption/ionization (MALDI) source is an advanced technique that enables advancement in this biological area. In this work a MALDI time-of-flight (TOF)MS method for the rapid identification of proteins in whole cells of selected microalgae species was studied. Furthermore, the efficiency of different matrix and solvent systems was tested. MS analyses were performed using an UltrafleXtreme MALDI-TOF mass spectrometer operating in linear positive ion mode. METHODS: Mass spectra were acquired in a mass range from 4000 to 20,000 Da with ions generated from Smartbeam laser irradiation using a frequency of 2000 Hz, a PIE 100 ns and a lens 7 kV. The voltage was 25 kV for the first ion source and 23 kV for the second. Each spectrum was generated by averaging of 10,000 laser shots and the laser irradiance was set at 95-100%. RESULTS: Similar mass spectra were obtained for all matrices (SA, HCCA, DHB and sDHB); however, the use of the sDHB matrix resulted in spectrum profiles with a greater amount number of proteins, a better signal/noise (S/N) ratio and higher intensities for the majority of microalgae analyzed. Trifluoroacetic acid (TFA) content was also studied and the best results in terms of S/N ratio, number of proteins and signal intensities were obtained with 0.1% TFA in the matrix solvent. The addition of isopropanol did not produce improvement in the quality of spectrum profiles. CONCLUSIONS: Therefore, the optimal matrix for the analysis of protein from intact microalgae cells is sDHB with TA50 as the matrix solvent and without isopropanol. These conditions allow the acquisition of high quality spectrum profiles.