IDENTIFYING PROTEINS USING MATRIX-ASSISTED-LASER-DESORPTION IONIZATION IN-SOURCE FRAGMENTATION DATA COMBINED WITH DATABASE SEARCHING/

Metastable ion decay in matrix-assisted laser desorption/ ionization ( MALDI) has become a routine method for obtaining primary structures of peptides. Significant fragmentation occurs in the MALDI ion source an d can be observed via delayed ion extraction TOF-MS. Insource decay (I SD) can provide C-and N-terminal primary sequence data for even modera te-sized peptides (<5000 Dal. The unique c(n), series fragmentation th at occurs in ISD has been exploited to obtain partial C-terminal seque nces for proteins as large as human apotransferrin (75 kDa). Two appro aches for combining this I:SD MALDI-generated partial sequence informa tion with protein database searching techniques are presented. In one approach, cyanogen bromide is used to cleave relatively large peptide fragments from a sample elf human apotransferrin. One of the larger cl eavage products (6034.84 Dal was isolated by HPLC and subjected to ISD MALDI;DI analysis. An easily identified c(n), fragment ion series all owed two noncontiguous segments of the peptide's sequence to be determ ined (about 55% of the total sequence). This partial sequence informat ion was used to search protein and oligonucleotide sequence databases. In addition to uniquely identifying human apotransferrin in a protein sequence database, an example of the use of this ISD MALDI-determined partial sequence information to search expressed sequence tag databas es is presented. Such searches have the potential for rapidly identify ing new genes that code for target proteins. An alternate approach for obtaining partial sequence information on proteins is also demonstrat ed that utilizes ISD MAI;DI fragmentation of the intact protein to gen erate partial sequence information. This approach is shown to generate about 5-7% of a protein's sequence, usually near the C-terminus of th e protein. Examples of the ISD MALDI fragmentation data obtained from intact (reduced) human apotransferrin and intact (nonreduced) bovine s erum albumin (66 kDa) proteins are presented.