A proteomics grade electron transfer dissociation-enabled hybrid linear ion trap-orbitrap mass spectrometer.
Here we detail the modification of a quadrupole linear ion trap-orbitrap hybrid (QLT-orbitrap) mass spectrometer to accommodate a negative chemical ionization (NCI) source. The NCI source is used to produce fluoranthene radical anions for imparting electron transfer dissociation (ETD). The anion beam is stable, robust, and intense so that a sufficient amount of reagents can be injected into the QLT in only 4-8 ms. Following ion/ion reaction in the QLT, ETD product ions are mass-to-charge (m/z) analyzed in either the QLT (for speed and sensitivity) or the orbitrap (for mass resolution and accuracy). Here we describe the physical layout of this device, parametric optimization of anion transport, an evaluation of relevant ETD figures of merit, and the application of this instrument to protein sequence analysis. Described proteomic applications include complex peptide mixture analysis, post-translational modification (PTM) site identification, isotope-encoded quantitation, large peptide characterization, and intact protein analysis. From these experiments, we conclude the ETD-enabled orbitrap will provide the proteomic field with several new opportunities and represents an advance in protein sequence analysis technologies.
McAlister
Graeme C
GC
Department of Chemistry and Biomolecular Chemistry, University of Wisconsin, Madison, WI 53706, USA.
Berggren
W Travis
WT
Griep-Raming
Jens
J
Horning
Stevan
S
Makarov
Alexander
A
Phanstiel
Doug
D
Stafford
George
G
Swaney
Danielle L
DL
Syka
John E P
JE
Zabrouskov
Vlad
V
Coon
Joshua J
JJ
D000595:Amino Acid Sequence; D002247:Carbon Isotopes; D002460:Cell Line; D045744:Cell Line, Tumor; D045424:Complex Mixtures / Q000032:analysis; D004583:Electrons; D053595:Embryonic Stem Cells / Q000737:chemistry; D004867:Equipment Design; D005449:Fluorenes / Q000737:chemistry; D006657:Histones / Q000032:analysis; D006801:Humans; D007477:Ions; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis; D040901:Proteomics / Q000295:instrumentation*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis; D053719:Tandem Mass Spectrometry / Q000295:instrumentation*
D002247:Carbon Isotopes; D045424:Complex Mixtures; D005449:Fluorenes; D006657:Histones; D007477:Ions; D010748:Phosphopeptides; D029701:Saccharomyces cerevisiae Proteins; C007738:fluoranthene