MALDI2TOF. assisted laser desorptionπionization time of fight,maldi2tof) AAP,AI C. (neuropeptides) AAP. (abdominal ganglion)

Transcript

1 23 6 CHINA BIOTECHNOLOGY MALDI2TOF Kim Wonsunk 2 Miao Hai 2 ( Department of Chemistry and Beckman Institute University of Illinois Urbana IL 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 AAP AI CpH 615 [8 10 ], MALDI2TOF, molπl, , 52 MALDI2TOF (DHB),30min,, DHB 30s, : : (No ) 3 3 xmu. edu. cn, l MALDI2TOF,, 11213

2 80 23 Voyager DE2STR (337nm) ( ), 2300,20kV % (mπz ) Paws AAP AAP SSGVSLLTSNKDEEQRE2 LLKAISNLLD, 3 LL, Da (ph 615), AAP [M + H] +, , AAP, [M + H] 2 +, ( mΠz 1), 1, AAP,AAP,Paws AAP ( ), 1 1 AAP 1,AAP 27, LL % 1 3 L2L,AAP,AAP L2L, N C L,,L2R (R 1 L ), 1 AAP AAP ( %) SSGVSLLTSNKDEEQRELLKAISNLLD SSGVSLLTSNKDEEQRELLKAISNLL LLTSNKDEEQRELLKAIS NLLD VSLLTSNKDEEQRELLK EEQRELLKAISNL RELLKAISNLL 212 AI C AI, 9146Da [1 ] AI (prohormone) A B,CC AI AI C, 15, DTENVNDKLRG ILLN, Da, L2L 2 [ mπz : ,(M + H) ],AI C AAP,, , , [M + H] + [ 2M + H ] + [ 3M + H ] +, [M + H] + [2M - 2H] + [ 3M - 9H] +,,,, A + B = A - B, H +,, AI C,, (, COO - O - ), H +,

3 6 :MALDI2TOF 81 (1) AI C (2) AI C 2 AI C 2 AI C AI C ( %) DTENVNDKLRGILLN DTENVNDKLRGILL TENVNDKLRGILLN NVNDKLRGILLN DTENVDKLRG NDKLRGILL 2 (mπz, ) AI CAI C, , , AI CDTENVNDKLRGILLN, AI C ( 2), 2,, 5 AI C 4 L2L, L2R, AI C L2L, L2R,, AAP L2R 312 An An 10, DRVYIHPFHL, Da, 3 An AAP AI C L2L 3,An AI C,,,,, [M] + [2M] + [ 3M] + [ 4M] +, , , ,,, 3, ( [M + ]) ( [ 4M + ]), 100 % 3 %,,,,,,, 3 3,An L2R AAP AI C L2R,, ( 1 2) 3 An An ( %) DRVYIHPFHL DRVYIHPFH

4 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) : [ 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), [ 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) : [ 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) : [ 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) : [ 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) : [ 7 ] Chiu D T, Zare R N. Assaying forpeptides in individual Aplysia neurons with mass spectrometry. Proc Natl Acda Sci USA,1998,95 (7) : [ 8 ] Sithigorngul P, Saraithongkum W,Longyant S, et al. Three more

5 6 :MALDI2TOF 83 novel FMRFamide2like neuopeptide sequence from the eyestalk of the giant freshwater prawn macrobrachium rosenbergii. Peptides, 2001,22 (1) : [ 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) : [ 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) : [ 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) : [ 12 ],,,. MALDI2TOF.,2002,18 (1) : 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 ) (2 Department of Chemistry and Beckman Institute University of Illinois Urbana IL 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, , (www. cmht. com. cn) :154 (100009),:, : , , , : , : com. cn