Studies On Simple,Rapid Identification Of Cyanobacteria Bloom By Maldi-TOF MS

It has been reported that the increasing eutrophication,with consequent frequent cyanobacterial blooms are a series of problems caused by microcystis,have had long-term adverse effects and harms on the water quality security of our country.While taking control measures,it is necessary to real-time monitor cyanobacterial bloom waterbodies and to make early warning of their occurrence possibility,so to formulate timely and effectively means of governance.However,there are continues lack of rapid,simple and accurate monitoring methods for cyanobacterial blooms in eutrophic lakes.In this study,a new method for the identification of cyanobacteria was established employing ribosomal proteins as biomarkers and matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS)as a measuring tool,and the possibility was addressed to explore its application in the identification of cyanobacteria toxicity and cyanobacteria diversity and its application in daily monitoring.Firstly,three pre-treatment methods were optimized screened to analyze the feasibility of ribosomal proteins as biomarkers for the identification of cyanobacteria and to make the ribosomal subunit proteins of the cyanobacteria samples as major peaks can be detected in the spectrum within the appropriate range and strong signal,so a new standard method for direct identification of cyanobacteria by using MALDI-TOF MS with ribosomal proteins as biomarker was established on this basis.The results showed that the ribosomal subunit proteins of Microcystis aeruginosa NIES-843 were obtained by two-step pre-treatment procedure only including cell lysis and centrifugation.By comparing their theoretical m/z values,31 of the 52 ribosomal proteins were successfully identified using MALDI-TOF MS and the list of ribosomal proteins was established.Among them,13 ribosomal proteins(L32,S21,L33,L35,L33,S18,L29,L31,L28,S16,S19,S15,S20 and S14)with high frequency and high intensity were selected as the characteristic biomarkers to identify cyanobacteria.Then,55 strains of M.aeruginosa from different collection environments were analyzed using MALDI-TOF MS and proteomics approaches.At the same time,a polygenetic analysis was performed to build a dendrogram using the unweighted pair group method with arithmetic mean(UPGMA)cluster analysis and different ribosomal protein types.The result indicated that there were different types of the same ribosomal proteins in different M.aeruginosa strains and all 55 strains were classified into five major clades(?,?,?,? and ?)using UPGMA.Among them,toxic strains were primarily divided into clade ? and ?,while the other three clades contained exclusively non-toxic strains.That is,the MALDI-TOF MS method using the ribosomal protein as the biomarker could identify cyanobacteria in strain level and differentiate between toxic and non-toxic cyanobacterial strains effectively.In order to investigate the applicability of identifying the cyanobacteria in the actual water by the established MALDI-TOF MS method,firstly the sensitivity and rational sampling volume of the method were determined by measuring a series of M.aeruginosa NIES-843 samples with different cell densities,then the laboratory feasibility of the method was verified by detecting artificial mixing samples with different concentrations of four different kinds of cyanobacteria simulating the actual waterbodies.The results showed that the lowest detection limit by MALDI-TOF MS were 1.955 x106cells in pure samples,and the detection rate was 84.6%with 11 ribosomal proteins identified,while the corresponding needed volume of water samples in lakes with cyanobacterial blooms were 0.1-1L,which were reasonable in daily water quality monitoring.Meanwhile,M.aeruginosa NIES-843 could be accurately identified in the artificial mixing cyanobacteria samples using MALDI-TOF MS method.The lowest detection limit for NIES-843 were 2.88 x106cells in mixing samples,the detection rate was 76.9%with 10 ribosomal proteins identified,while the corresponding mixing ratio of NIES-843 in the mixed sample 3 was 37%.Finally,field samples collected during the summer from four selected locations in Taihu Lake were detected and analyzed by MALDI-TOF MS employing 13 characteristic ribosomal proteins as biomarkers.The results demonstrated that 9 labeled biomarker peaks were observed in Taihu sample 1 and 4,and the detection rate was 69.2%,while 11 labeled biomarker peaks were observed in Taihu sample 2 and 3,and the detection rate was 84.6%.Furthermore,different types(L32,L35,L29,L28,and S14)of M.aeruginosa strains were also detected at different locations in Taihu Lake,indicating the diversity of M.aeruginosa in Taihu Lake and the detection power of MALDI-TOF MS at the strain level.In conclusion,the new established MALDI-TOF MS method could identify M.aeruginosa simply,rapidly and reliably and is promising in routine environmental monitoring and identification of cyanobacteria in actual lake water in the future.