A simple and efficient approach to improve protein identification by the peptide mass fingerprinting method: concomitant use of negative ionization

[摘要] Peptidemassfingerprinting(PMF)hasbeenwidelyusedasanefficientanalyticalstrategyforproteinidentification.Thisismostcommonlyusedwithacombinationofproteindigestionusingsequence-specificproteasesandMALDI-TOFMS.Thendatabasesearchesareperformedcomparingthepatternoftheexperimentallyobtainedmasseswiththepatternofthetheoreticalpeptidemassesofproteinsstoredinthedatabase.ThepositiveionizationmodehasbeenmainlyusedforMALDIanalyseswithafewexceptionsforphosphopeptides,oligonucleotides,etc.Therefore,nonphosphorylatedpeptidesthathavelowpIvaluescouldbemissedfromPMFusingthepositiveionizationmode.Here,weintroduceoptimalconditionsfornegativeionizationofpeptidesandthepracticaladvantagesofnegativeionizationsinPMF.AngiotensinI(pI6.9)andbovineserumalbumin(BSA)trypticdigestswereusedasmodelpeptides.Eightmatrixcandidatesandsevenadditiveswereexaminedintermsofsensitivity,robustnessandreproducibility.ThecombinationofDHBandphosphoricacidwasthebestconditionfornegativeionizationofpeptidesandwasfoundtobecompatiblewiththepositiveionizationmode.Using150mMDHB/1%phosphoricacid,thecoverage(%byaminoacidcount)ofBSAtrypticdigest(0.6pmolperspot)totaled67.2%(negative+positive).The24.1%ofpeptides(pIrange4.1–6.2)weredetectedonlybynegativeionization,whichindicatedthatacidicpeptideswereefficientlyrecoveredbythenegativeionmode.Thismethodologyhasbeensuccessfullyemployedtoanalyzeotherproteinswithoutfalsepositiveidentifications...

[学科分类] 分析化学