title: Whole protein dissociation in a quadrupole ion trap: identification of an a priori unknown modified protein.
abstract: A protein mixture derived from a whole cell lysate fraction of Saccharomyces cerevisiae, which contains roughly 19 proteins, has been analyzed to identify an a priori unknown modified protein using a quadrupole ion trap tandem mass spectrometer. Collection of the experimental data was facilitated by collision-induced dissociation and ion/ion proton-transfer reactions in multistage mass spectrometry procedures. Ion/ion reactions were used to manipulate charge states of both parent ions and product ions for the purpose of concentrating charge into the parent ion of interest and to reduce the product ion charge states for determination of product ion mass and abundance. The identification of the protein was achieved by matching the uninterpreted product ion spectrum against protein sequence databases with varying degrees of annotation, coupled with a scoring scheme weighted for the relative abundances of the experimentally observed product ions and the frequency of fragmentations occurring at preferential sites. The protein was identified to be an acetylated yeast heat shock protein, HS12_Yeast (11.6 kDa), with the initiating methionine residue removed. This constitutes the first example of the identification of an a priori unknown protein that is not present in an annotated protein database using a "top-down" approach with a quadrupole ion trap. This example illustrates the utility of relatively low cost instrumentation with modest mass analysis characteristics for the identification of modified proteins without recourse to enzymatic digestion. It also illustrates how experimental data can be used interactively with protein databases when the modified protein of interest is not initially present in the database.
authors.affiliation: Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907-2084, USA.
authors.forename: Ravi
authors.initials: R
authors.lastname: Amunugama
authors.forename: Jason M
authors.initials: JM
authors.lastname: Hogan
authors.forename: Kelly A
authors.initials: KA
authors.lastname: Newton
authors.forename: Scott A
authors.initials: SA
authors.lastname: McLuckey
mesh_terms: D000595:Amino Acid Sequence; D030562:Databases, Protein; D007477:Ions / Q000737:chemistry; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*
chemical_list: D007477:Ions; D029701:Saccharomyces cerevisiae Proteins