Rational Selection of the Optimum MALDI Matrix for Top-Down Proteomics by In-Source Decay

Abstract

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 > alpha-cyano-4-hydroxycinnamic 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 approximately 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 preparation was successfully applied to identify larger proteins 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.

Photocatalytic Redox Reactions for In‐Source Peptide Fragmentation

“…The control experiment was performed on a normal ground-steel plate with a DHB matrix in the negative-ion mode with an ion extraction delay (600 ns). Similar to the previously reported results, [26] only c n fragments were effectively obtained, as shown in Figure 2 b. It should also be mentioned that the fragment-to-parent-peak area ratio seen in Figure 2 a is much bigger than that observed in Figure 2 b, indicating a more intense dissociation.…”

“…Unlike the TiO 2 -induced peptide fragmentation, only several weak c n fragments were obtained, and due to the presence of organic matrix, it is nearly impossible to read out fragments with m/z less than 700, as shown in Figure 1 b. To further validate the unusual decay pattern, a longer peptide, oxidized b-insulin (7 pmol, FVNQHLCoxGSHLVEALYLVCoxGERGFFYTPKA), [26] was used, and the TiO 2 -induced photocatalytic peptide fragmentation was performed in the negative mode. With l-glucose as the electron donor/hole conductor and an ion extraction delay (600 ns), strong a,x decay was observed and a complete a n fragments list from a 7 to a 18 was obtained, as shown in Figure 2 a.…”

Increased encapsulated cell biodelivery of nerve growth factor in the brain by transposon-mediated gene transfer

“…The molecular mass was obtained using a-cyano-4-hydroxy-cinnamic acid as matrix, while 1.5-diaminonaphtalene was used for top --down sequencing using in-source-decay. 37 …”

A new method for analysis of disulfide-containing proteins by matrix-assisted laser desorption ionization (MALDI) mass spectrometry

“…Using this feature of 1,5-DAN, disulfide linkages in the proteins were characterized by the comparison of mass differences between the reduced, partially reduced, and nonreduced molecular ions. However, in the interpretation of the obtained data, one must exercise extreme caution because 1,5-DAN can dramatically enhance in-source decay (ISD) fragmentation [16]. Recently, a screening method that uses ISD of disulfide bonds for identification of disulfidelinked peptides in mass spectra has been developed [17,18].…”