23 6 CHINA BIOTECHNOLOGY 2003 6 MALDI2TOF 3 1 3 3 Kim Wonsunk 2 Miao Hai 2 (1 361005 2 Department of Chemistry and Beckman Institute University of Illinois Urbana IL 61801 USA) ( Aplysia acidic peptide,aap) C ( Aplysia Insulin C,AI C ) 27 15, 6 (L),3 L2L, 2, 1 L2L (matrix2 assisted laser desorptionπionization time of fight,maldi2tof) AAP,AI C,,AAP AI CL2L L2R (R L ), AI C I(angiotensin I, AnI), AI C, H + MALDI2TOF (neuropeptides), [1 2 ] ( Aplysia) (buccal ganglion) (cerebral ganglion) (abdominal ganglion) 111 AAP AI CSweedler (pleural2abdominal connective) [2 4 ] AnI Simga 2, 52, dihydroxybenzoic acid (DHB) ICN [5 7 ] 112 MALDI2TOF 11211 AAP AI CpH 615 [8 10 ], MALDI2TOF, 1 10-4 molπl, 11212 2, 52 MALDI2TOF (DHB),30min,, DHB 30s, :2002210205 :2003203206 3 (No. 40276033) 3 3 :hqhuang @jingxian. xmu. edu. cn, 1 018 l MALDI2TOF,, 11213
80 23 Voyager DE2STR (337nm) ( ), 2300,20kV 100 99 % (mπz 296115) Paws 2 211 AAP AAP SSGVSLLTSNKDEEQRE2 LLKAISNLLD, 3 LL, 2960128Da (ph 615), AAP [M + H] +, 2961128, AAP, [M + H] 2 +, 1481189 (1480164mΠz 1), 1, AAP,AAP,Paws AAP ( ), 1 1 AAP 1,AAP 27, LL 26 1115 % 1 3 L2L,AAP,AAP L2L, 1 6 4 N C L,,L2R (R 1 L ), 1 AAP AAP ( %) 2961128 2961130 01001 SSGVSLLTSNKDEEQRELLKAISNLLD 284512 2847107 01066 SSGVSLLTSNKDEEQRELLKAISNLL 254219 2542161 01011 LLTSNKDEEQRELLKAIS NLLD 200212 2003142 01061 VSLLTSNKDEEQRELLK 154217 1543181 01072 EEQRELLKAISNL 126915 1271134 01144 RELLKAISNLL 212 AI C AI, 9146Da [1 ] AI (prohormone) A B,CC AI AI C, 15, DTENVNDKLRG ILLN, 171319Da, L2L 2 [ mπz : 1714191,(M + H) ],AI C AAP,, 1714191, 3425127 5133132, [M + H] + [ 2M + H ] + [ 3M + H ] +, [M + H] + [2M - 2H] + [ 3M - 9H] +,,,, A + B = A - B, H +,, AI C,, (, COO - O - ), H +,
6 :MALDI2TOF 81 (1) AI C (2) AI C 2 AI C 2 AI C AI C ( %) 171319 1714191 01059 DTENVNDKLRGILLN 159918 1601119 01131 DTENVNDKLRGILL 159818 1601119 01149 TENVNDKLRGILLN 136816 1369117 01041 NVNDKLRGILLN 126013 1260182 01032 DTENVDKLRG 114113 1143142 01185 NDKLRGILL 2 (mπz,1714191) AI CAI C, 1601119,1369117,1260182 1143142 AI CDTENVNDKLRGILLN, AI C ( 2), 2,, 5 AI C 4 L2L, L2R, AI C L2L, L2R,, AAP L2R 312 An An 10, DRVYIHPFHL, 129615Da, 3 An AAP AI C L2L 3,An AI C,,,,, [M] + [2M] + [ 3M] + [ 4M] +, 129615,2593138, 388915 5186,,, 3, ( [M + ]) ( [ 4M + ]), 100 % 3 %,,,,,,, 3 3,An L2R AAP AI C L2R,, ( 1 2) 3 An An ( %) 129615 1296169 01084 DRVYIHPFHL 118313 1184117 01073 DRVYIHPFH
82 23 (1) AI C,, fmol, MALDI2TOF [11 12 ],,, H +, (),,, [ XM + H] +, An,,,,AI C, [ XM + H] + [ 1 ] Floyd F D, Li L, Rubakhin S S, et al. Insulin prohormone (2) AI C 3 An,,, MALDI2TOF [11 ], ( 1 3),, processing, distribution, and relation to metabolism in Aplysia californica. The Journal of Neurosicence, 1999, 19 (18) : 7732 7741 [ 2 ] Hummon A B, Huang H Q, Sweedler J V. A Novel prohormone processing site in Aplysia californica :the Leu2Leu rule. Journal of Neurochemistry,2002,82 (6),1398 1405 [ 3 ] Li L, Floyed F D, Rubakhin S S, et al. Cerebrin prohormonr processing,distribution and action in Aplysia californica.journal of Nurochemistry,2001,77 (6) :1569 1580 [ 4 ] Fujisawa Y, Furukawa Y, Ohta S, et al. The Aplysia mytilus inhibitory peptide2related peptides : identification, cloning, processing,distribution,and action. The Journal of Neurosicence, 1999,19 (21) :9618 9634 [ 5 ] Sweedler J V. Mass spectrometric survey of peptides in cephalopods with an emphasis on the FMRFamide2related peptides. The Journal of Experimental Biology. 2000,203 (6) :2565 2573 [ 6 ] Phares G A,Walent J H,Niece R L,et al. Primary structure of a new neurpeptide,cerebral peptide 2,purified from cerebral ganglia of Aplysia. Biochemistry,1996,35 (10) :5921 5927 [ 7 ] Chiu D T, Zare R N. Assaying forpeptides in individual Aplysia neurons with mass spectrometry. Proc Natl Acda Sci USA,1998,95 (7) :3338 3340 [ 8 ] Sithigorngul P, Saraithongkum W,Longyant S, et al. Three more
6 :MALDI2TOF 83 novel FMRFamide2like neuopeptide sequence from the eyestalk of the giant freshwater prawn macrobrachium rosenbergii. Peptides, 2001,22 (1) :191 197 [ 9 ] Jespersen S,Chaurand P,Strien F J C,et al. Direct sequencing of neuropeptides in biological tissue by MALDI2PSD mass spectrometry. Anal Chem,1999,71 (2) :660 666 [ 10 ] Johnson S L, Schroeder M L, Sanchez J A D, et al. Axonal regeneration in the centeral nervous system of aplysia californica determined by anterograde transport of biocytin. The Journal of Comparative Neurology,1999,406 (2) :476 486 [ 11 ] Li L, Garden R W,Sweedler J V,et al. Single cell MALDI :A new tool for direct peptide profiling. Trends in Biochemistry,2000,18 (1) :151 160 [ 12 ],,,. MALDI2TOF.,2002,18 (1) :99 103 Studies on Primary Structure Characteristics of Polypeptides by a Technology of MALDI2TOF Mass Spectrometry Huang Heqing 1 Kim Wonsunk 2 Miao Hai 2 (1 Department of Biology Schools of Life Sciences Xiamen University Xiamen 361005) (2 Department of Chemistry and Beckman Institute University of Illinois Urbana IL 61801 USA) Abstract Primary structures both Aplysia acidic peptide (AAP) and Aplysia insulin C (AI C ) consisted of 27 and 15 residues of amino acid respectively. The front polypeptide had six leucine (L) residues that consisted of three pairs with L2L bond,and later one had two leucine residues that consisted of one pair with L2L bond. Stability between AAP,AI C and their decomposed produces had been studied by matrix2assisted laser desorptionπionization time of fight (MALDI2TOF) mass spectrometry under the medium condition with weak acidity. Moreover,the experimental results were found that the energy of amide bond of L2L was stronger than that of L2R (R shown all amino acid residues except L),which were decomposed hardly. AIC and Angiotensin I (AnI) were the polypeptides with single charge. With the numbers of polypeptide increment,the relative intensity of the mass spectrometry peaks of the polypeptide decreased acutely. AIC released H + while it formed the polypeptides. Key words MALDI2TOF Mass spectrometry Peptide Primary structure Aplysia Insulin Acid peptide 2003, 2003 7 25 28 2003, (www. cmht. com. cn) :154 (100009),:, : 010264033255,64011361, 64014351, :010264075052, : sdattc @sina. com,sdattc @cmht. com. cn