High precision quantitative proteomics using iTRAQ on an LTQ Orbitrap: a new mass spectrometric method combining the benefits of all.
The development of quantitative techniques in mass spectrometry has generated the ability to systematically monitor protein expression. Isobaric tags for relative and absolute quantification (iTRAQ) have become a widely used tool for the quantification of proteins. However, application of iTRAQ methodology using ion traps and hybrid mass spectrometers containing an ion trap such as the LTQ-Orbitrap was not possible until the development of pulsed Q dissociation (PQD) and higher energy C-trap dissociation (HCD). Both methods allow iTRAQ-based quantification on an LTQ-Orbitrap but are less suited for protein identification at a proteomic scale than the commonly used collisional induced dissociation (CID) fragmentation. We developed an analytical strategy combining the advantages of CID and HCD, allowing sensitive and accurate protein identification and quantitation at the same time. In a direct comparison, the novel method outperformed PQD and HCD regarding its limit of detection, the number of identified peptides and the analytical precision of quantitation. The new method was applied to study changes in protein expression in mouse hearts upon transverse aortic constriction, a model for cardiac stress.
Köcher
Thomas
T
Research Institute of Molecular Pathology (IMP), Vienna, Austria. Thomas.Koecher@imp.ac.at
Pichler
Peter
P
Schutzbier
Michael
M
Stingl
Christoph
C
Kaul
Axel
A
Teucher
Nils
N
Hasenfuss
Gerd
G
Penninger
Josef M
JM
Mechtler
Karl
K
D000818:Animals; D006352:Heart Ventricles / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D010455:Peptides / Q000032:analysis / Q000737:chemistry; D011506:Proteins / Q000032:analysis / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D012107:Research Design; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000737:chemistry; D013312:Stress, Physiological
D010455:Peptides; D011506:Proteins; D012710:Serum Albumin, Bovine