demeuredepauw - Rational Selection of the Optimum MALDI...

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Rational Selection of the Optimum MALDI Matrix for Top-Down Proteomics by In-Source Decay Kevin Demeure,* Loı 1 c Quinton, Vale ´ rie Gabelica, and Edwin De Pauw Laboratoire de Spectrome ´ trie de Masse, CART/GIGA-R, Institut de Chimie, Bat. B6c, Universite ´ de Lie ` ge, Lie ` ge B-4000, Belgium In-source decay (ISD) in MALDI leads to c- and z-fragment ion series enhanced by hydrogen radical donors and is a useful method for sequencing purified peptides and proteins. Until now, most efforts to improve methods using ISD concerned instrumental optimization. The most widely used ISD matrix is 2,5-dihydroxybenzoic acid (DHB). We present here a rational way to select MALDI matrixes likely to enhance ISD for top-down proteomic approaches. Starting from Takayama’s model (Takayama, M. J. Am. Soc. Mass Spectrom. 2001, 12 , 1044 - 9), according to which formation of ISD fragments (c and z) would be due to a transfer of hydrogen radical from the matrix to the analyte, we evaluated the hydrogen-donating capacities of matrixes, and thus their ISD abilities, with spirooxazines (hydrogen scavengers). The determined hydrogen-donating abilities of the matrixes are ranked as follows: picolinic acid (PA) > 1,5-diaminonaphtalene (1,5-DAN) > DHB > sinapinic acid >r -cyano-4-hydroxy- cinnamic acid. The ISD enhancement obtained by using 1,5-DAN compared to DHB was confirmed with peptides and proteins. On that basis, a matrix-enhanced ISD approach was successfully applied to sequence peptides and proteins up to 8 kDa. Although PA alone is not suitable for peptide and protein ionization, ISD signals could be further enhanced when PA was used as an additive to 1,5-DAN. The optimized matrix prepara- tion was successfully applied to identify larger pro- teins by large ISD tag researches in protein databases (BLASTp). Coupled with an adequate separation method, ISD is a promising tool to include in a top-down proteomic strategy. Matrix-assisted laser desorption/ionization (MALDI) 1,2 is a widely used ionization method for the identification of peptides and proteins. In MALDI, the analyte is cocrystallized in a large excess of a host matrix. The matrix is an aromatic compound of low molecular mass that absorbs the UV laser irradiation, preventing the analyte from direct degradation. The ionization step in MALDI is not completely understood. 3 But the actual models converge all on a two-step mechanism, the primary and the secondary ionization. 4 The primary ionization concerns the forma- tion or the separation of the ions. During the desorption/ablation in the MALDI plume, reactions of ion - molecule occur, forming the secondary ions, which will reach the detector. The secondary ionization concerns the formation of these secondary ions and becomes less controversial. In fact, it is increasingly accepted that in typical MALDI conditions the local thermal equilibrium is reached in the MALDI plume, which is generally dense or long enough. Under those conditions, conventional kinetic laws may
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This note was uploaded on 05/28/2010 for the course WE BIBI010000 taught by Professor Marnikvuylsteke during the Spring '10 term at Ghent University.

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demeuredepauw - Rational Selection of the Optimum MALDI...

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