385 1 1 2 1 (11, 100021 ; 21, 100730) :(MALDI2TOF2MS) MALDI2TOF2MS,, MALDI2TOF2MS, MBT; IAA ;Π1 1 ; MALDI2TOF2MS :,, ; ;, Detection and Identification of Fungi by Matrix2Assisted Laser DesorptionΠIonization Time of Flight Mass Spectrometry LI Feng2qin, WU Duo2jia, WU Shu2zhen, LI Yu2wei (National Institute for Nutrition and Food Safety, Chinese CDC, Beijing 100021, China) Abstract: Objective To examine and identify fungi with important significance in food safety by matrix2assisted laser desorptionπionization time2of2flight mass spectrometry (MALDI2TOF2MS). Method Sample preparation, screening of appropriate matrix for specific fungal analysis, suitable ratio of matrix to sample, methods for loading samples on mass spectrometer sample tray and repeatability were investigated to optimize the parameters for fungal detection and identification by MALDI2TOF2MS. Results The results showed that different genus of fungi owned their proper matrix in sample preparation : MBTfor both Monascus and Fusaruim species, IAA for Aspergillus and Penicillium species as well as yeast, respectively. The optimum ratio of matrix to sample was 1 1 (vπv). Dried droplet is the best choice for applying sample on sample tray of the mass spectrometer. Conclusion MALDI2TOF2MS is of stability and repeatability characteristics in detecting and identifying fungus. Key word : Spectrometry, Mass, Matrix2Assisted Laser Desorption2Ionization ; Fungi ; Chemistry, Analytical, ( matrix2assisted,,,,,, : (30471456) ; (2006BAK02A27) :, 20 80, laser desorptionπionization time of flight mass spectrometry, MALDI2TOF2MS),,,,,, [1-5 ] MALDI2TOF2MS
386 CHINESE JOURNAL OF FOOD HYGIENE 2007 19 5 1 111 11111 ( Aspergillus species) ( A. flavus ) 314408 31951,,78-465 78-490 78-478 - 484 78-509 78-145 78-178 - 399 78-334 78-339 78-116 78-363 78-368 78-385 78-386 78-394 78-400 78-238 78-231 78-218, ( A. parasiticus) 316155 31124,, ATCC2999 ( A. sojae) 31494 ( A. ochraceus ) 311409 ( A. oryzae ) 312792 ( A. niger) 314309 ( A. carbonarius) 313558 ( Penicillium species) ( P1 verrucosum ) 314024 ( P1 expansum) 313703 ( P1 urticae) 31833, ( P1 chysogenum) ( P1 purpurogenum) ( P1 islandicum) ( P1 rugulosum) ( Fusarium species) ( F1 graminaerum) 313488 314521 ( F1 moniliforme) 311756 ( F1 tritinctum ) 314731 ( F1 proliferatum ) 314726 ( F1 oxysporum) 311781 ( F1 culmorum) 314283,7, ( Monascus species) ( Monascus ruber) SJS - 6 SJS - 8 SJS - 10 SJS - 18 SJS - 21 SJS - 22 SJS - 24 SJS - 26 SJS - 28 SJS - 33 ( Yeast) 21742 ( ), FX - 2X - 0203 ( Saccharomyces cerevisiae) 1 2, 11112 ( Malt Extract Agar, MEA) - - ( Potato Dextrose Agar, PDA) (Malt Agar, MA) ( GBΠT 4789128 2003) [6 ] 11113 MALDI2TOF2MS 2, 5 - ( 2, 5 - dihydroxybenzoic acid, DHB) 2 - - 4 - - 5 - (2 - amino - 4 - methyl - 5 - nitropyridine, 2a4m5n) - 3 - ( trans - 3 - indoleacrylic acid, IAA) 2 - ( 2 - mercaptobenzothiazole, MBT) 2 - (4 - ) [ 2 - (4 - hydroxyphenylazo) benzoic acid, HABA ] 2, 6 - ( 2, 6 - dihydroxyacetophenone, DHAP) (Sinapinic acid, 3,5 - dimenthoxy - 4 - hydroxycinnamic acid, SA) - - 4 - (- cyano - 4 - hydroxycinnamic acid, CHCA,cyano) 5 - - 2 - (5 - chloro - 2 - mercaptobenzothiazole, CMBT), Sigma 9 + 011 % ( Trifluoroacetic acid, TFA) (70 + 30, ) MALDI2TOF2MS 9, 11114 MALDI2TOF2MS ( Voyager DE TM PRO Biospectrometry TM Workstation),:,,, :20 kv, :400 ns, : 90025 000 Da, : 100, : 2010 Hz, : 9219 %, Bin size : 015 ns, Vertical scale : 500 mv, Vertical offset : 015 %, Input bandwith : 500 MHz, Guide wire :0105 %Angiotensin ( : 1 2961685 3) ACTH 18-39 (: 2 4651198 9), :35015 000 Da 112 11211 MALDI2TOF2MS SJS - 22 31124 1, MALDI2TOF2MS () SJS - 22 MEA,30 10 d ;
387 314731MA, 31124 PDA,28 7 d, 1 PDA,35 24 h ( ) 1 ml, 115 ml Eppendorf, 4 000 rπmin 5 min, 2, 20 l 110 ml Eppendorf, SJS - 22 SJS - 22 (1 + 2 1 + 1 2 + 1 4 + 1, ),1 l MALDI2 TOF2MS MALDI2TOF2MS SJS - 22 3 1 l,,1 l, MALDI2TOF2MS, 1 l, MALDI2TOF2MS, 1 l,1 l, 1 l, MALDI2TOF2MS 11212 MALDI2TOF2MS SJS - 22 3 MEA, 7 d,maldi2 TOF2MS 11213 MALDI2TOF2MS 2 211 MALDI2TOF2MS SJS - 22 9 1 + 1 MALDI2TOF2MS 1 ( mπz),1,, MBT, mπz 4 030,, MBT MALDI2 TOF2MS 212 SJS - 22 MBT 1 + 2 1 + 1 2 + 1 4 + 1 ( ) MALDI2TOF2MS, 22, 1 + 1,, 1 + 1,,1 + 1 213 SJS - 22 3 33,,mΠz 4 030 ;, ; mπz 4 030,,,MALDI2TOF2MS 214 MALDI2TOF2MS 314731 31124 + (1 + 1) 9 MALDI2TOF2MS, 4 56, 31124 IAA (mπz 3 378 mπz 6 095 mπz 6 263 mπz 6 422 ),IAA (mπz 4 431 mπz 6 527 mπz 9 326mΠz 10 221 ),MBT (mπz 1 152 mπz mπz 1 382 mπz 1 759mΠz 1 990 ),, MALDI2TOF2 MS,IAA MBT, IAA 215 MALDI2TOF2MS
388 CHINESE JOURNAL OF FOOD HYGIENE 2007 19 5 1 SJS - 22 9 SJS - 22 3 7 d MALDI2TOF2MS 7, 3 MALDI2TOF2MS,, mπz4 030,, MALDI2TOF2MS,
389 2 SJS - 22 MBT 3 SJS - 22 3 MALDI2TOF2MS,,,,, MALDI2TOF2MS,, [7 ] TFA, NH 2,,, TFA,,,,, [8 ] MALDI2TOF2
390 CHINESE JOURNAL OF FOOD HYGIENE 2007 19 5 4 31124 9 MS : ; MALDI2TOF2MS,,MALDI2TOF2MS,,,
391 5 9,, MALDI2TOF2MS MALDI2TOF2MS [7 ], 9,,,, MALDI2TOF2 MS, [8 ],,,,
392 CHINESE JOURNAL OF FOOD HYGIENE 2007 19 5 6 3147319,,,,,, MALDI2TOF2MS
393 7 SJS - 22 MEA, 1 1001 5 000, 1 + 1 ( ), Π 1 + 2 2 + 14 + 1 ( ),,,, ;, Π 1 + 1 ( ) [1 ],,. [J ].,2004, 33 (5) : 5522554. [2 ],. [J ].,2002,38 (4) : 5582562. [3 ] WELHAM KJ,DOMIN M A,JOHNSON K, et al. Characterization of fungal sporesby laser desorptionπionazation time2of2flight mass spectrometry[j ]. Rapid Common Mass Spectrom,2000,14 :3072310. [4 ] NANCYB VALENTINE, JON H WAHL, MARK T KINGSLEY, et al. Direct surface analysis of fungal species by matrix2assisted laser desorptionπionazation mass spectrometry [ J ]. Spectrom,2002,16 :135221357. Rapid Common Mass [5 ] BIJAN AMIRI2ELIASI, CATHERINE FENSELAU. Characterization of Protein Biomarkers Desorbed by MALDI from Whole Fungal Cells[J ]. Anal Chem,2001, 73 (21) :522825231. [6 ] GBΠT 4789. 28 2003. [ S]. [7 ],,. Π [J ].,2004, 32 (12) :168321688. [8 ],,,. [J ].,2002, 30 (8) :9382941. [ :2007-05 - 16 ] :R15 ;Q949132 ;O65713 :A :1004-8456 (2007) 05-0385 - 09