Data-dependent electron transfer dissociation of large peptides and medium size proteins in a QTOF instrument on a liquid chromatography timescale.
Liquid chromatography (LC) electron transfer dissociation (ETD) tandem mass spectrometry (MS/MS) of protein digests is demonstrated in a hybrid quadrupole-hexapole orthogonal time-of-flight (OTOF) mass spectrometer. Analyte ions are selected in a mass-analyzing quadrupole, accumulated in the hexapole linear ETD reaction cell and mutually stored with ETD reagent anions. Product ions are collected in an ion cooler and then analyzed by an OTOF mass analyzer. The hexapole structure of the ETD reaction cell allows for a broad fragment ion mass range distribution and a high ion storage capacity. Analytically useful ETD OTOF-MS/MS spectra could be obtained at a rate of faster than 2 Hz. When used in conjunction with LC this high speed allows for several MS and MS/MS spectra to be obtained across each LC peak. An MS scan is used to select the precursor ions. With a 1 m flight tube and single reflection, resolutions of about 10 k and a mass accuracy of 5 ppm were achieved. When analyzing a 100 fmol solution of a tryptic digest of bovine serum albumin (BSA) by LC/ETD MS/MS, 27 unique peptides were identified with a summed Mascot score of 1316 using the Swiss Prot database. In addition, we explored the capability for analyzing small proteins with the present hybrid instrument. ETD MS/MS of intact ubiquitin ([M+12H](12+)) leads to the identification of the protein with a Mascot score of 264.
Hartmer
Ralf G
RG
Bruker Daltonik, GmbH, Fahrenheitstr. 4, D-28359 Bremen, Germany. ralf.hartmer@bdal.de
Kaplan
Desmond A
DA
Stoermer
Carsten
C
Lubeck
Markus
M
Park
Melvin A
MA
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000191:economics / Q000379:methods*; D004583:Electrons; D004867:Equipment Design; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000191:economics / Q000295:instrumentation* / Q000379:methods*; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism; D025801:Ubiquitin / Q000032:analysis / Q000378:metabolism
D010455:Peptides; D011506:Proteins; D025801:Ubiquitin; D012710:Serum Albumin, Bovine; D014357:Trypsin